| anno_start anno_end anno_text entity_type sentence section | |
| 24 47 in vitro reconstitution experimental_method Structural insights and in vitro reconstitution of membrane targeting and activation of human PI4KB by the ACBD3 protein TITLE | |
| 88 93 human species Structural insights and in vitro reconstitution of membrane targeting and activation of human PI4KB by the ACBD3 protein TITLE | |
| 94 99 PI4KB protein Structural insights and in vitro reconstitution of membrane targeting and activation of human PI4KB by the ACBD3 protein TITLE | |
| 107 112 ACBD3 protein Structural insights and in vitro reconstitution of membrane targeting and activation of human PI4KB by the ACBD3 protein TITLE | |
| 0 34 Phosphatidylinositol 4-kinase beta protein Phosphatidylinositol 4-kinase beta (PI4KB) is one of four human PI4K enzymes that generate phosphatidylinositol 4-phosphate (PI4P), a minor but essential regulatory lipid found in all eukaryotic cells. ABSTRACT | |
| 36 41 PI4KB protein Phosphatidylinositol 4-kinase beta (PI4KB) is one of four human PI4K enzymes that generate phosphatidylinositol 4-phosphate (PI4P), a minor but essential regulatory lipid found in all eukaryotic cells. ABSTRACT | |
| 58 63 human species Phosphatidylinositol 4-kinase beta (PI4KB) is one of four human PI4K enzymes that generate phosphatidylinositol 4-phosphate (PI4P), a minor but essential regulatory lipid found in all eukaryotic cells. ABSTRACT | |
| 64 68 PI4K protein_type Phosphatidylinositol 4-kinase beta (PI4KB) is one of four human PI4K enzymes that generate phosphatidylinositol 4-phosphate (PI4P), a minor but essential regulatory lipid found in all eukaryotic cells. ABSTRACT | |
| 91 123 phosphatidylinositol 4-phosphate chemical Phosphatidylinositol 4-kinase beta (PI4KB) is one of four human PI4K enzymes that generate phosphatidylinositol 4-phosphate (PI4P), a minor but essential regulatory lipid found in all eukaryotic cells. ABSTRACT | |
| 125 129 PI4P chemical Phosphatidylinositol 4-kinase beta (PI4KB) is one of four human PI4K enzymes that generate phosphatidylinositol 4-phosphate (PI4P), a minor but essential regulatory lipid found in all eukaryotic cells. ABSTRACT | |
| 184 194 eukaryotic taxonomy_domain Phosphatidylinositol 4-kinase beta (PI4KB) is one of four human PI4K enzymes that generate phosphatidylinositol 4-phosphate (PI4P), a minor but essential regulatory lipid found in all eukaryotic cells. ABSTRACT | |
| 35 40 PI4Ks protein_type To convert their lipid substrates, PI4Ks must be recruited to the correct membrane compartment. ABSTRACT | |
| 0 5 PI4KB protein PI4KB is critical for the maintenance of the Golgi and trans Golgi network (TGN) PI4P pools, however, the actual targeting mechanism of PI4KB to the Golgi and TGN membranes is unknown. ABSTRACT | |
| 81 85 PI4P chemical PI4KB is critical for the maintenance of the Golgi and trans Golgi network (TGN) PI4P pools, however, the actual targeting mechanism of PI4KB to the Golgi and TGN membranes is unknown. ABSTRACT | |
| 136 141 PI4KB protein PI4KB is critical for the maintenance of the Golgi and trans Golgi network (TGN) PI4P pools, however, the actual targeting mechanism of PI4KB to the Golgi and TGN membranes is unknown. ABSTRACT | |
| 20 23 NMR experimental_method Here, we present an NMR structure of the complex of PI4KB and its interacting partner, Golgi adaptor protein acyl-coenzyme A binding domain containing protein 3 (ACBD3). ABSTRACT | |
| 24 33 structure evidence Here, we present an NMR structure of the complex of PI4KB and its interacting partner, Golgi adaptor protein acyl-coenzyme A binding domain containing protein 3 (ACBD3). ABSTRACT | |
| 52 57 PI4KB protein Here, we present an NMR structure of the complex of PI4KB and its interacting partner, Golgi adaptor protein acyl-coenzyme A binding domain containing protein 3 (ACBD3). ABSTRACT | |
| 87 108 Golgi adaptor protein protein_type Here, we present an NMR structure of the complex of PI4KB and its interacting partner, Golgi adaptor protein acyl-coenzyme A binding domain containing protein 3 (ACBD3). ABSTRACT | |
| 109 160 acyl-coenzyme A binding domain containing protein 3 protein Here, we present an NMR structure of the complex of PI4KB and its interacting partner, Golgi adaptor protein acyl-coenzyme A binding domain containing protein 3 (ACBD3). ABSTRACT | |
| 162 167 ACBD3 protein Here, we present an NMR structure of the complex of PI4KB and its interacting partner, Golgi adaptor protein acyl-coenzyme A binding domain containing protein 3 (ACBD3). ABSTRACT | |
| 13 18 ACBD3 protein We show that ACBD3 is capable of recruiting PI4KB to membranes both in vitro and in vivo, and that membrane recruitment of PI4KB by ACBD3 increases its enzymatic activity and that the ACBD3:PI4KB complex formation is essential for proper function of the Golgi. ABSTRACT | |
| 44 49 PI4KB protein We show that ACBD3 is capable of recruiting PI4KB to membranes both in vitro and in vivo, and that membrane recruitment of PI4KB by ACBD3 increases its enzymatic activity and that the ACBD3:PI4KB complex formation is essential for proper function of the Golgi. ABSTRACT | |
| 123 128 PI4KB protein We show that ACBD3 is capable of recruiting PI4KB to membranes both in vitro and in vivo, and that membrane recruitment of PI4KB by ACBD3 increases its enzymatic activity and that the ACBD3:PI4KB complex formation is essential for proper function of the Golgi. ABSTRACT | |
| 132 137 ACBD3 protein We show that ACBD3 is capable of recruiting PI4KB to membranes both in vitro and in vivo, and that membrane recruitment of PI4KB by ACBD3 increases its enzymatic activity and that the ACBD3:PI4KB complex formation is essential for proper function of the Golgi. ABSTRACT | |
| 152 170 enzymatic activity evidence We show that ACBD3 is capable of recruiting PI4KB to membranes both in vitro and in vivo, and that membrane recruitment of PI4KB by ACBD3 increases its enzymatic activity and that the ACBD3:PI4KB complex formation is essential for proper function of the Golgi. ABSTRACT | |
| 184 195 ACBD3:PI4KB complex_assembly We show that ACBD3 is capable of recruiting PI4KB to membranes both in vitro and in vivo, and that membrane recruitment of PI4KB by ACBD3 increases its enzymatic activity and that the ACBD3:PI4KB complex formation is essential for proper function of the Golgi. ABSTRACT | |
| 0 34 Phosphatidylinositol 4-kinase beta protein Phosphatidylinositol 4-kinase beta (PI4KB, also known as PI4K IIIβ) is a soluble cytosolic protein yet its function is to phosphorylate membrane lipids. INTRO | |
| 36 41 PI4KB protein Phosphatidylinositol 4-kinase beta (PI4KB, also known as PI4K IIIβ) is a soluble cytosolic protein yet its function is to phosphorylate membrane lipids. INTRO | |
| 57 66 PI4K IIIβ protein Phosphatidylinositol 4-kinase beta (PI4KB, also known as PI4K IIIβ) is a soluble cytosolic protein yet its function is to phosphorylate membrane lipids. INTRO | |
| 18 23 human species It is one of four human PI4K enzymes that phosphorylate phosphatidylinositol (PI) to generate phosphatidylinositol 4-phosphate (PI4P). INTRO | |
| 24 28 PI4K protein_type It is one of four human PI4K enzymes that phosphorylate phosphatidylinositol (PI) to generate phosphatidylinositol 4-phosphate (PI4P). INTRO | |
| 56 76 phosphatidylinositol chemical It is one of four human PI4K enzymes that phosphorylate phosphatidylinositol (PI) to generate phosphatidylinositol 4-phosphate (PI4P). INTRO | |
| 78 80 PI chemical It is one of four human PI4K enzymes that phosphorylate phosphatidylinositol (PI) to generate phosphatidylinositol 4-phosphate (PI4P). INTRO | |
| 94 126 phosphatidylinositol 4-phosphate chemical It is one of four human PI4K enzymes that phosphorylate phosphatidylinositol (PI) to generate phosphatidylinositol 4-phosphate (PI4P). INTRO | |
| 128 132 PI4P chemical It is one of four human PI4K enzymes that phosphorylate phosphatidylinositol (PI) to generate phosphatidylinositol 4-phosphate (PI4P). INTRO | |
| 0 4 PI4P chemical PI4P is an essential lipid found in various membrane compartments including the Golgi and trans-Golgi network (TGN), the plasma membrane and the endocytic compartments. INTRO | |
| 20 24 PI4P chemical In these locations, PI4P plays an important role in cell signaling and lipid transport, and serves as a precursor for higher phosphoinositides or as a docking site for clathrin adaptor or lipid transfer proteins. INTRO | |
| 125 142 phosphoinositides chemical In these locations, PI4P plays an important role in cell signaling and lipid transport, and serves as a precursor for higher phosphoinositides or as a docking site for clathrin adaptor or lipid transfer proteins. INTRO | |
| 168 176 clathrin protein_type In these locations, PI4P plays an important role in cell signaling and lipid transport, and serves as a precursor for higher phosphoinositides or as a docking site for clathrin adaptor or lipid transfer proteins. INTRO | |
| 16 58 positive-sense single-stranded RNA viruses taxonomy_domain A wide range of positive-sense single-stranded RNA viruses (+RNA viruses), including many that are important human pathogens, hijack human PI4KA or PI4KB enzymes to generate specific PI4P-enriched organelles called membranous webs or replication factories. INTRO | |
| 60 72 +RNA viruses taxonomy_domain A wide range of positive-sense single-stranded RNA viruses (+RNA viruses), including many that are important human pathogens, hijack human PI4KA or PI4KB enzymes to generate specific PI4P-enriched organelles called membranous webs or replication factories. INTRO | |
| 109 114 human species A wide range of positive-sense single-stranded RNA viruses (+RNA viruses), including many that are important human pathogens, hijack human PI4KA or PI4KB enzymes to generate specific PI4P-enriched organelles called membranous webs or replication factories. INTRO | |
| 133 138 human species A wide range of positive-sense single-stranded RNA viruses (+RNA viruses), including many that are important human pathogens, hijack human PI4KA or PI4KB enzymes to generate specific PI4P-enriched organelles called membranous webs or replication factories. INTRO | |
| 139 144 PI4KA protein A wide range of positive-sense single-stranded RNA viruses (+RNA viruses), including many that are important human pathogens, hijack human PI4KA or PI4KB enzymes to generate specific PI4P-enriched organelles called membranous webs or replication factories. INTRO | |
| 148 153 PI4KB protein A wide range of positive-sense single-stranded RNA viruses (+RNA viruses), including many that are important human pathogens, hijack human PI4KA or PI4KB enzymes to generate specific PI4P-enriched organelles called membranous webs or replication factories. INTRO | |
| 183 187 PI4P chemical A wide range of positive-sense single-stranded RNA viruses (+RNA viruses), including many that are important human pathogens, hijack human PI4KA or PI4KB enzymes to generate specific PI4P-enriched organelles called membranous webs or replication factories. INTRO | |
| 6 16 structures evidence These structures are essential for effective viral replication. INTRO | |
| 45 50 viral taxonomy_domain These structures are essential for effective viral replication. INTRO | |
| 26 31 PI4KB protein Recently, highly specific PI4KB inhibitors were developed as potential antivirals. INTRO | |
| 0 4 PI4K protein_type PI4K kinases must be recruited to the correct membrane type to fulfill their enzymatic functions. INTRO | |
| 5 12 kinases protein_type PI4K kinases must be recruited to the correct membrane type to fulfill their enzymatic functions. INTRO | |
| 0 13 Type II PI4Ks protein_type Type II PI4Ks (PI4K2A and PI4K2B) are heavily palmitoylated and thus behave as membrane proteins. INTRO | |
| 15 21 PI4K2A protein Type II PI4Ks (PI4K2A and PI4K2B) are heavily palmitoylated and thus behave as membrane proteins. INTRO | |
| 26 32 PI4K2B protein Type II PI4Ks (PI4K2A and PI4K2B) are heavily palmitoylated and thus behave as membrane proteins. INTRO | |
| 38 59 heavily palmitoylated protein_state Type II PI4Ks (PI4K2A and PI4K2B) are heavily palmitoylated and thus behave as membrane proteins. INTRO | |
| 79 96 membrane proteins protein Type II PI4Ks (PI4K2A and PI4K2B) are heavily palmitoylated and thus behave as membrane proteins. INTRO | |
| 13 27 type III PI4Ks protein_type In contrast, type III PI4Ks (PI4KA and PI4KB) are soluble cytosolic proteins that are recruited to appropriate membranes indirectly via protein-protein interactions. INTRO | |
| 29 34 PI4KA protein In contrast, type III PI4Ks (PI4KA and PI4KB) are soluble cytosolic proteins that are recruited to appropriate membranes indirectly via protein-protein interactions. INTRO | |
| 39 44 PI4KB protein In contrast, type III PI4Ks (PI4KA and PI4KB) are soluble cytosolic proteins that are recruited to appropriate membranes indirectly via protein-protein interactions. INTRO | |
| 19 24 PI4KA protein The recruitment of PI4KA to the plasma membrane by EFR3 and TTC7 is relatively well understood even at the structural level, but, the actual molecular mechanism of PI4KB recruitment to the Golgi is still poorly understood. INTRO | |
| 51 55 EFR3 protein The recruitment of PI4KA to the plasma membrane by EFR3 and TTC7 is relatively well understood even at the structural level, but, the actual molecular mechanism of PI4KB recruitment to the Golgi is still poorly understood. INTRO | |
| 60 64 TTC7 protein The recruitment of PI4KA to the plasma membrane by EFR3 and TTC7 is relatively well understood even at the structural level, but, the actual molecular mechanism of PI4KB recruitment to the Golgi is still poorly understood. INTRO | |
| 164 169 PI4KB protein The recruitment of PI4KA to the plasma membrane by EFR3 and TTC7 is relatively well understood even at the structural level, but, the actual molecular mechanism of PI4KB recruitment to the Golgi is still poorly understood. INTRO | |
| 0 51 Acyl-coenzyme A binding domain containing protein 3 protein Acyl-coenzyme A binding domain containing protein 3 (ACBD3, also known as GCP60 and PAP7) is a Golgi resident protein. INTRO | |
| 53 58 ACBD3 protein Acyl-coenzyme A binding domain containing protein 3 (ACBD3, also known as GCP60 and PAP7) is a Golgi resident protein. INTRO | |
| 74 79 GCP60 protein Acyl-coenzyme A binding domain containing protein 3 (ACBD3, also known as GCP60 and PAP7) is a Golgi resident protein. INTRO | |
| 84 88 PAP7 protein Acyl-coenzyme A binding domain containing protein 3 (ACBD3, also known as GCP60 and PAP7) is a Golgi resident protein. INTRO | |
| 89 98 golgin B1 protein Its membrane localization is mediated by the interaction with the Golgi integral protein golgin B1/giantin. INTRO | |
| 99 106 giantin protein Its membrane localization is mediated by the interaction with the Golgi integral protein golgin B1/giantin. INTRO | |
| 0 5 ACBD3 protein ACBD3 functions as an adaptor protein and signaling hub across cellular signaling pathways. INTRO | |
| 0 5 ACBD3 protein ACBD3 can interact with a number of proteins including golgin A3/golgin-160 to regulate apoptosis, Numb proteins to control asymmetric cell division and neuronal differentiation, metal transporter DMT1 and monomeric G protein Dexras1 to maintain iron homeostasis, and the lipid kinase PI4KB to regulate lipid homeostasis. INTRO | |
| 55 64 golgin A3 protein ACBD3 can interact with a number of proteins including golgin A3/golgin-160 to regulate apoptosis, Numb proteins to control asymmetric cell division and neuronal differentiation, metal transporter DMT1 and monomeric G protein Dexras1 to maintain iron homeostasis, and the lipid kinase PI4KB to regulate lipid homeostasis. INTRO | |
| 65 75 golgin-160 protein ACBD3 can interact with a number of proteins including golgin A3/golgin-160 to regulate apoptosis, Numb proteins to control asymmetric cell division and neuronal differentiation, metal transporter DMT1 and monomeric G protein Dexras1 to maintain iron homeostasis, and the lipid kinase PI4KB to regulate lipid homeostasis. INTRO | |
| 99 112 Numb proteins protein_type ACBD3 can interact with a number of proteins including golgin A3/golgin-160 to regulate apoptosis, Numb proteins to control asymmetric cell division and neuronal differentiation, metal transporter DMT1 and monomeric G protein Dexras1 to maintain iron homeostasis, and the lipid kinase PI4KB to regulate lipid homeostasis. INTRO | |
| 179 196 metal transporter protein_type ACBD3 can interact with a number of proteins including golgin A3/golgin-160 to regulate apoptosis, Numb proteins to control asymmetric cell division and neuronal differentiation, metal transporter DMT1 and monomeric G protein Dexras1 to maintain iron homeostasis, and the lipid kinase PI4KB to regulate lipid homeostasis. INTRO | |
| 197 201 DMT1 protein ACBD3 can interact with a number of proteins including golgin A3/golgin-160 to regulate apoptosis, Numb proteins to control asymmetric cell division and neuronal differentiation, metal transporter DMT1 and monomeric G protein Dexras1 to maintain iron homeostasis, and the lipid kinase PI4KB to regulate lipid homeostasis. INTRO | |
| 206 215 monomeric oligomeric_state ACBD3 can interact with a number of proteins including golgin A3/golgin-160 to regulate apoptosis, Numb proteins to control asymmetric cell division and neuronal differentiation, metal transporter DMT1 and monomeric G protein Dexras1 to maintain iron homeostasis, and the lipid kinase PI4KB to regulate lipid homeostasis. INTRO | |
| 216 225 G protein protein_type ACBD3 can interact with a number of proteins including golgin A3/golgin-160 to regulate apoptosis, Numb proteins to control asymmetric cell division and neuronal differentiation, metal transporter DMT1 and monomeric G protein Dexras1 to maintain iron homeostasis, and the lipid kinase PI4KB to regulate lipid homeostasis. INTRO | |
| 226 233 Dexras1 protein ACBD3 can interact with a number of proteins including golgin A3/golgin-160 to regulate apoptosis, Numb proteins to control asymmetric cell division and neuronal differentiation, metal transporter DMT1 and monomeric G protein Dexras1 to maintain iron homeostasis, and the lipid kinase PI4KB to regulate lipid homeostasis. INTRO | |
| 246 250 iron chemical ACBD3 can interact with a number of proteins including golgin A3/golgin-160 to regulate apoptosis, Numb proteins to control asymmetric cell division and neuronal differentiation, metal transporter DMT1 and monomeric G protein Dexras1 to maintain iron homeostasis, and the lipid kinase PI4KB to regulate lipid homeostasis. INTRO | |
| 272 284 lipid kinase protein_type ACBD3 can interact with a number of proteins including golgin A3/golgin-160 to regulate apoptosis, Numb proteins to control asymmetric cell division and neuronal differentiation, metal transporter DMT1 and monomeric G protein Dexras1 to maintain iron homeostasis, and the lipid kinase PI4KB to regulate lipid homeostasis. INTRO | |
| 285 290 PI4KB protein ACBD3 can interact with a number of proteins including golgin A3/golgin-160 to regulate apoptosis, Numb proteins to control asymmetric cell division and neuronal differentiation, metal transporter DMT1 and monomeric G protein Dexras1 to maintain iron homeostasis, and the lipid kinase PI4KB to regulate lipid homeostasis. INTRO | |
| 0 5 ACBD3 protein ACBD3 has been also implicated in the pathology of neurodegenerative diseases such as Huntington’s disease due to its interactions with a polyglutamine repeat-containing mutant huntingtin and the striatal-selective monomeric G protein Rhes/Dexras2. INTRO | |
| 138 158 polyglutamine repeat structure_element ACBD3 has been also implicated in the pathology of neurodegenerative diseases such as Huntington’s disease due to its interactions with a polyglutamine repeat-containing mutant huntingtin and the striatal-selective monomeric G protein Rhes/Dexras2. INTRO | |
| 170 176 mutant protein_state ACBD3 has been also implicated in the pathology of neurodegenerative diseases such as Huntington’s disease due to its interactions with a polyglutamine repeat-containing mutant huntingtin and the striatal-selective monomeric G protein Rhes/Dexras2. INTRO | |
| 177 187 huntingtin protein ACBD3 has been also implicated in the pathology of neurodegenerative diseases such as Huntington’s disease due to its interactions with a polyglutamine repeat-containing mutant huntingtin and the striatal-selective monomeric G protein Rhes/Dexras2. INTRO | |
| 215 224 monomeric oligomeric_state ACBD3 has been also implicated in the pathology of neurodegenerative diseases such as Huntington’s disease due to its interactions with a polyglutamine repeat-containing mutant huntingtin and the striatal-selective monomeric G protein Rhes/Dexras2. INTRO | |
| 225 234 G protein protein_type ACBD3 has been also implicated in the pathology of neurodegenerative diseases such as Huntington’s disease due to its interactions with a polyglutamine repeat-containing mutant huntingtin and the striatal-selective monomeric G protein Rhes/Dexras2. INTRO | |
| 235 239 Rhes protein ACBD3 has been also implicated in the pathology of neurodegenerative diseases such as Huntington’s disease due to its interactions with a polyglutamine repeat-containing mutant huntingtin and the striatal-selective monomeric G protein Rhes/Dexras2. INTRO | |
| 240 247 Dexras2 protein ACBD3 has been also implicated in the pathology of neurodegenerative diseases such as Huntington’s disease due to its interactions with a polyglutamine repeat-containing mutant huntingtin and the striatal-selective monomeric G protein Rhes/Dexras2. INTRO | |
| 0 5 ACBD3 protein ACBD3 is a binding partner of viral non-structural 3A proteins and a host factor of several picornaviruses including poliovirus, coxsackievirus B3, and Aichi virus. INTRO | |
| 30 35 viral taxonomy_domain ACBD3 is a binding partner of viral non-structural 3A proteins and a host factor of several picornaviruses including poliovirus, coxsackievirus B3, and Aichi virus. INTRO | |
| 36 62 non-structural 3A proteins protein_type ACBD3 is a binding partner of viral non-structural 3A proteins and a host factor of several picornaviruses including poliovirus, coxsackievirus B3, and Aichi virus. INTRO | |
| 92 106 picornaviruses taxonomy_domain ACBD3 is a binding partner of viral non-structural 3A proteins and a host factor of several picornaviruses including poliovirus, coxsackievirus B3, and Aichi virus. INTRO | |
| 117 127 poliovirus taxonomy_domain ACBD3 is a binding partner of viral non-structural 3A proteins and a host factor of several picornaviruses including poliovirus, coxsackievirus B3, and Aichi virus. INTRO | |
| 129 146 coxsackievirus B3 taxonomy_domain ACBD3 is a binding partner of viral non-structural 3A proteins and a host factor of several picornaviruses including poliovirus, coxsackievirus B3, and Aichi virus. INTRO | |
| 152 163 Aichi virus taxonomy_domain ACBD3 is a binding partner of viral non-structural 3A proteins and a host factor of several picornaviruses including poliovirus, coxsackievirus B3, and Aichi virus. INTRO | |
| 13 56 biochemical and structural characterization experimental_method We present a biochemical and structural characterization of the molecular complex composed of the ACBD3 protein and the PI4KB enzyme. INTRO | |
| 98 103 ACBD3 protein We present a biochemical and structural characterization of the molecular complex composed of the ACBD3 protein and the PI4KB enzyme. INTRO | |
| 120 125 PI4KB protein We present a biochemical and structural characterization of the molecular complex composed of the ACBD3 protein and the PI4KB enzyme. INTRO | |
| 13 18 ACBD3 protein We show that ACBD3 can recruit PI4KB to model membranes as well as redirect PI4KB to cellular membranes where it is not naturally found. INTRO | |
| 31 36 PI4KB protein We show that ACBD3 can recruit PI4KB to model membranes as well as redirect PI4KB to cellular membranes where it is not naturally found. INTRO | |
| 76 81 PI4KB protein We show that ACBD3 can recruit PI4KB to model membranes as well as redirect PI4KB to cellular membranes where it is not naturally found. INTRO | |
| 24 29 ACBD3 protein Our data also show that ACBD3 regulates the enzymatic activity of PI4KB kinase through membrane recruitment rather than allostery. INTRO | |
| 44 62 enzymatic activity evidence Our data also show that ACBD3 regulates the enzymatic activity of PI4KB kinase through membrane recruitment rather than allostery. INTRO | |
| 66 71 PI4KB protein Our data also show that ACBD3 regulates the enzymatic activity of PI4KB kinase through membrane recruitment rather than allostery. INTRO | |
| 72 78 kinase protein_type Our data also show that ACBD3 regulates the enzymatic activity of PI4KB kinase through membrane recruitment rather than allostery. INTRO | |
| 0 5 ACBD3 protein ACBD3 and PI4KB interact with 1:1 stoichiometry with submicromolar affinity RESULTS | |
| 10 15 PI4KB protein ACBD3 and PI4KB interact with 1:1 stoichiometry with submicromolar affinity RESULTS | |
| 44 49 ACBD3 protein In order to verify the interactions between ACBD3 and PI4KB we expressed and purified both proteins. RESULTS | |
| 54 59 PI4KB protein In order to verify the interactions between ACBD3 and PI4KB we expressed and purified both proteins. RESULTS | |
| 63 85 expressed and purified experimental_method In order to verify the interactions between ACBD3 and PI4KB we expressed and purified both proteins. RESULTS | |
| 22 42 bacterial expression experimental_method To increase yields of bacterial expression the intrinsically disordered region of PI4KB (residues 423–522) was removed (Fig. 1A). RESULTS | |
| 47 78 intrinsically disordered region structure_element To increase yields of bacterial expression the intrinsically disordered region of PI4KB (residues 423–522) was removed (Fig. 1A). RESULTS | |
| 82 87 PI4KB protein To increase yields of bacterial expression the intrinsically disordered region of PI4KB (residues 423–522) was removed (Fig. 1A). RESULTS | |
| 98 105 423–522 residue_range To increase yields of bacterial expression the intrinsically disordered region of PI4KB (residues 423–522) was removed (Fig. 1A). RESULTS | |
| 111 118 removed experimental_method To increase yields of bacterial expression the intrinsically disordered region of PI4KB (residues 423–522) was removed (Fig. 1A). RESULTS | |
| 14 22 deletion experimental_method This internal deletion does not significantly affect the kinase activity(SI Fig. 1A) or interaction with ACBD3 (SI Fig. 1B,C). RESULTS | |
| 57 63 kinase protein_type This internal deletion does not significantly affect the kinase activity(SI Fig. 1A) or interaction with ACBD3 (SI Fig. 1B,C). RESULTS | |
| 105 110 ACBD3 protein This internal deletion does not significantly affect the kinase activity(SI Fig. 1A) or interaction with ACBD3 (SI Fig. 1B,C). RESULTS | |
| 6 28 in vitro binding assay experimental_method In an in vitro binding assay, ACBD3 co-purified with the NiNTA-immobilized N-terminal His6GB1-tagged PI4KB (Fig. 1B, left panel), suggesting a direct interaction. RESULTS | |
| 30 35 ACBD3 protein In an in vitro binding assay, ACBD3 co-purified with the NiNTA-immobilized N-terminal His6GB1-tagged PI4KB (Fig. 1B, left panel), suggesting a direct interaction. RESULTS | |
| 36 74 co-purified with the NiNTA-immobilized experimental_method In an in vitro binding assay, ACBD3 co-purified with the NiNTA-immobilized N-terminal His6GB1-tagged PI4KB (Fig. 1B, left panel), suggesting a direct interaction. RESULTS | |
| 86 100 His6GB1-tagged protein_state In an in vitro binding assay, ACBD3 co-purified with the NiNTA-immobilized N-terminal His6GB1-tagged PI4KB (Fig. 1B, left panel), suggesting a direct interaction. RESULTS | |
| 101 106 PI4KB protein In an in vitro binding assay, ACBD3 co-purified with the NiNTA-immobilized N-terminal His6GB1-tagged PI4KB (Fig. 1B, left panel), suggesting a direct interaction. RESULTS | |
| 8 34 mammalian two-hybrid assay experimental_method Using a mammalian two-hybrid assay Greninger and colleagues localized this interaction to the Q domain of ACBD3 (named according to its high content of glutamine residues) and the N-terminal region of PI4KB preceding its helical domain. RESULTS | |
| 60 69 localized evidence Using a mammalian two-hybrid assay Greninger and colleagues localized this interaction to the Q domain of ACBD3 (named according to its high content of glutamine residues) and the N-terminal region of PI4KB preceding its helical domain. RESULTS | |
| 94 102 Q domain structure_element Using a mammalian two-hybrid assay Greninger and colleagues localized this interaction to the Q domain of ACBD3 (named according to its high content of glutamine residues) and the N-terminal region of PI4KB preceding its helical domain. RESULTS | |
| 106 111 ACBD3 protein Using a mammalian two-hybrid assay Greninger and colleagues localized this interaction to the Q domain of ACBD3 (named according to its high content of glutamine residues) and the N-terminal region of PI4KB preceding its helical domain. RESULTS | |
| 152 161 glutamine residue_name Using a mammalian two-hybrid assay Greninger and colleagues localized this interaction to the Q domain of ACBD3 (named according to its high content of glutamine residues) and the N-terminal region of PI4KB preceding its helical domain. RESULTS | |
| 180 197 N-terminal region structure_element Using a mammalian two-hybrid assay Greninger and colleagues localized this interaction to the Q domain of ACBD3 (named according to its high content of glutamine residues) and the N-terminal region of PI4KB preceding its helical domain. RESULTS | |
| 201 206 PI4KB protein Using a mammalian two-hybrid assay Greninger and colleagues localized this interaction to the Q domain of ACBD3 (named according to its high content of glutamine residues) and the N-terminal region of PI4KB preceding its helical domain. RESULTS | |
| 221 235 helical domain structure_element Using a mammalian two-hybrid assay Greninger and colleagues localized this interaction to the Q domain of ACBD3 (named according to its high content of glutamine residues) and the N-terminal region of PI4KB preceding its helical domain. RESULTS | |
| 3 12 expressed experimental_method We expressed the Q domain of ACBD3 (residues 241–308) and the N-terminal region of PI4KB (residues 1–68) in E. coli and using purified recombinant proteins, we confirmed that these two domains are sufficient to maintain the interaction (Fig. 1B, middle and right panel). RESULTS | |
| 17 25 Q domain structure_element We expressed the Q domain of ACBD3 (residues 241–308) and the N-terminal region of PI4KB (residues 1–68) in E. coli and using purified recombinant proteins, we confirmed that these two domains are sufficient to maintain the interaction (Fig. 1B, middle and right panel). RESULTS | |
| 29 34 ACBD3 protein We expressed the Q domain of ACBD3 (residues 241–308) and the N-terminal region of PI4KB (residues 1–68) in E. coli and using purified recombinant proteins, we confirmed that these two domains are sufficient to maintain the interaction (Fig. 1B, middle and right panel). RESULTS | |
| 45 52 241–308 residue_range We expressed the Q domain of ACBD3 (residues 241–308) and the N-terminal region of PI4KB (residues 1–68) in E. coli and using purified recombinant proteins, we confirmed that these two domains are sufficient to maintain the interaction (Fig. 1B, middle and right panel). RESULTS | |
| 62 79 N-terminal region structure_element We expressed the Q domain of ACBD3 (residues 241–308) and the N-terminal region of PI4KB (residues 1–68) in E. coli and using purified recombinant proteins, we confirmed that these two domains are sufficient to maintain the interaction (Fig. 1B, middle and right panel). RESULTS | |
| 83 88 PI4KB protein We expressed the Q domain of ACBD3 (residues 241–308) and the N-terminal region of PI4KB (residues 1–68) in E. coli and using purified recombinant proteins, we confirmed that these two domains are sufficient to maintain the interaction (Fig. 1B, middle and right panel). RESULTS | |
| 99 103 1–68 residue_range We expressed the Q domain of ACBD3 (residues 241–308) and the N-terminal region of PI4KB (residues 1–68) in E. coli and using purified recombinant proteins, we confirmed that these two domains are sufficient to maintain the interaction (Fig. 1B, middle and right panel). RESULTS | |
| 108 115 E. coli species We expressed the Q domain of ACBD3 (residues 241–308) and the N-terminal region of PI4KB (residues 1–68) in E. coli and using purified recombinant proteins, we confirmed that these two domains are sufficient to maintain the interaction (Fig. 1B, middle and right panel). RESULTS | |
| 34 39 ACBD3 protein Because it has been reported that ACBD3 can dimerize in a mammalian two-hybrid assay, we were interested in determining the stoichiometry of the ACBD3:PI4KB protein complex. RESULTS | |
| 44 52 dimerize oligomeric_state Because it has been reported that ACBD3 can dimerize in a mammalian two-hybrid assay, we were interested in determining the stoichiometry of the ACBD3:PI4KB protein complex. RESULTS | |
| 58 84 mammalian two-hybrid assay experimental_method Because it has been reported that ACBD3 can dimerize in a mammalian two-hybrid assay, we were interested in determining the stoichiometry of the ACBD3:PI4KB protein complex. RESULTS | |
| 145 156 ACBD3:PI4KB complex_assembly Because it has been reported that ACBD3 can dimerize in a mammalian two-hybrid assay, we were interested in determining the stoichiometry of the ACBD3:PI4KB protein complex. RESULTS | |
| 4 30 sedimentation coefficients evidence The sedimentation coefficients of ACBD3 and PI4KB alone, or ACBD3:PI4KB complex were determined by analytical ultracentrifugation and found to be 3.1 S, 4.1 S, and 5.1 S. These values correspond to molecular weights of approximately 55 kDa, 80 kDa, and 130 kDa, respectively. RESULTS | |
| 34 39 ACBD3 protein The sedimentation coefficients of ACBD3 and PI4KB alone, or ACBD3:PI4KB complex were determined by analytical ultracentrifugation and found to be 3.1 S, 4.1 S, and 5.1 S. These values correspond to molecular weights of approximately 55 kDa, 80 kDa, and 130 kDa, respectively. RESULTS | |
| 44 49 PI4KB protein The sedimentation coefficients of ACBD3 and PI4KB alone, or ACBD3:PI4KB complex were determined by analytical ultracentrifugation and found to be 3.1 S, 4.1 S, and 5.1 S. These values correspond to molecular weights of approximately 55 kDa, 80 kDa, and 130 kDa, respectively. RESULTS | |
| 50 55 alone protein_state The sedimentation coefficients of ACBD3 and PI4KB alone, or ACBD3:PI4KB complex were determined by analytical ultracentrifugation and found to be 3.1 S, 4.1 S, and 5.1 S. These values correspond to molecular weights of approximately 55 kDa, 80 kDa, and 130 kDa, respectively. RESULTS | |
| 60 71 ACBD3:PI4KB complex_assembly The sedimentation coefficients of ACBD3 and PI4KB alone, or ACBD3:PI4KB complex were determined by analytical ultracentrifugation and found to be 3.1 S, 4.1 S, and 5.1 S. These values correspond to molecular weights of approximately 55 kDa, 80 kDa, and 130 kDa, respectively. RESULTS | |
| 99 129 analytical ultracentrifugation experimental_method The sedimentation coefficients of ACBD3 and PI4KB alone, or ACBD3:PI4KB complex were determined by analytical ultracentrifugation and found to be 3.1 S, 4.1 S, and 5.1 S. These values correspond to molecular weights of approximately 55 kDa, 80 kDa, and 130 kDa, respectively. RESULTS | |
| 198 215 molecular weights evidence The sedimentation coefficients of ACBD3 and PI4KB alone, or ACBD3:PI4KB complex were determined by analytical ultracentrifugation and found to be 3.1 S, 4.1 S, and 5.1 S. These values correspond to molecular weights of approximately 55 kDa, 80 kDa, and 130 kDa, respectively. RESULTS | |
| 44 53 monomeric oligomeric_state This result suggests that both proteins are monomeric and the stoichiometry of the ACBD3: PI4KB protein complex is 1:1 (Fig. 1C, left panel). RESULTS | |
| 83 95 ACBD3: PI4KB complex_assembly This result suggests that both proteins are monomeric and the stoichiometry of the ACBD3: PI4KB protein complex is 1:1 (Fig. 1C, left panel). RESULTS | |
| 53 61 Q domain structure_element Similar results were obtained for the complex of the Q domain of ACBD3 and the N-terminal region of PI4KB (Fig. 1C, right panel). RESULTS | |
| 65 70 ACBD3 protein Similar results were obtained for the complex of the Q domain of ACBD3 and the N-terminal region of PI4KB (Fig. 1C, right panel). RESULTS | |
| 79 96 N-terminal region structure_element Similar results were obtained for the complex of the Q domain of ACBD3 and the N-terminal region of PI4KB (Fig. 1C, right panel). RESULTS | |
| 100 105 PI4KB protein Similar results were obtained for the complex of the Q domain of ACBD3 and the N-terminal region of PI4KB (Fig. 1C, right panel). RESULTS | |
| 71 82 full length protein_state We also determined the strength of the interaction between recombinant full length ACBD3 and PI4KB using surface plasmon resonance (SPR). RESULTS | |
| 83 88 ACBD3 protein We also determined the strength of the interaction between recombinant full length ACBD3 and PI4KB using surface plasmon resonance (SPR). RESULTS | |
| 93 98 PI4KB protein We also determined the strength of the interaction between recombinant full length ACBD3 and PI4KB using surface plasmon resonance (SPR). RESULTS | |
| 105 130 surface plasmon resonance experimental_method We also determined the strength of the interaction between recombinant full length ACBD3 and PI4KB using surface plasmon resonance (SPR). RESULTS | |
| 132 135 SPR experimental_method We also determined the strength of the interaction between recombinant full length ACBD3 and PI4KB using surface plasmon resonance (SPR). RESULTS | |
| 0 3 SPR experimental_method SPR measurements revealed a strong interaction with a Kd value of 320 +/−130 nM (Fig. 1D, SI Fig. 1D). RESULTS | |
| 54 56 Kd evidence SPR measurements revealed a strong interaction with a Kd value of 320 +/−130 nM (Fig. 1D, SI Fig. 1D). RESULTS | |
| 18 23 ACBD3 protein We concluded that ACBD3 and PI4KB interact directly through the Q domain of ACBD3 and the N-terminal region of PI4KB forming a 1:1 complex with a dissociation constant in the submicromolar range. RESULTS | |
| 28 33 PI4KB protein We concluded that ACBD3 and PI4KB interact directly through the Q domain of ACBD3 and the N-terminal region of PI4KB forming a 1:1 complex with a dissociation constant in the submicromolar range. RESULTS | |
| 64 72 Q domain structure_element We concluded that ACBD3 and PI4KB interact directly through the Q domain of ACBD3 and the N-terminal region of PI4KB forming a 1:1 complex with a dissociation constant in the submicromolar range. RESULTS | |
| 76 81 ACBD3 protein We concluded that ACBD3 and PI4KB interact directly through the Q domain of ACBD3 and the N-terminal region of PI4KB forming a 1:1 complex with a dissociation constant in the submicromolar range. RESULTS | |
| 90 107 N-terminal region structure_element We concluded that ACBD3 and PI4KB interact directly through the Q domain of ACBD3 and the N-terminal region of PI4KB forming a 1:1 complex with a dissociation constant in the submicromolar range. RESULTS | |
| 111 116 PI4KB protein We concluded that ACBD3 and PI4KB interact directly through the Q domain of ACBD3 and the N-terminal region of PI4KB forming a 1:1 complex with a dissociation constant in the submicromolar range. RESULTS | |
| 146 167 dissociation constant evidence We concluded that ACBD3 and PI4KB interact directly through the Q domain of ACBD3 and the N-terminal region of PI4KB forming a 1:1 complex with a dissociation constant in the submicromolar range. RESULTS | |
| 0 19 Structural analysis experimental_method Structural analysis of the ACBD3:PI4KB complex RESULTS | |
| 27 38 ACBD3:PI4KB complex_assembly Structural analysis of the ACBD3:PI4KB complex RESULTS | |
| 0 11 Full length protein_state Full length ACBD3 and PI4KB both contain large intrinsically disordered regions that impede crystallization. RESULTS | |
| 12 17 ACBD3 protein Full length ACBD3 and PI4KB both contain large intrinsically disordered regions that impede crystallization. RESULTS | |
| 22 27 PI4KB protein Full length ACBD3 and PI4KB both contain large intrinsically disordered regions that impede crystallization. RESULTS | |
| 47 79 intrinsically disordered regions structure_element Full length ACBD3 and PI4KB both contain large intrinsically disordered regions that impede crystallization. RESULTS | |
| 8 53 hydrogen-deuterium exchange mass spectrometry experimental_method We used hydrogen-deuterium exchange mass spectrometry (HDX-MS) analysis of the complex to determine which parts of the complex are well folded (SI Fig. 2). RESULTS | |
| 55 61 HDX-MS experimental_method We used hydrogen-deuterium exchange mass spectrometry (HDX-MS) analysis of the complex to determine which parts of the complex are well folded (SI Fig. 2). RESULTS | |
| 131 142 well folded protein_state We used hydrogen-deuterium exchange mass spectrometry (HDX-MS) analysis of the complex to determine which parts of the complex are well folded (SI Fig. 2). RESULTS | |
| 34 42 crystals evidence However, we were unable to obtain crystals even when using significantly truncated constructs that included only the ACBD3 Q domain and the N-terminal region of PI4KB. RESULTS | |
| 73 82 truncated protein_state However, we were unable to obtain crystals even when using significantly truncated constructs that included only the ACBD3 Q domain and the N-terminal region of PI4KB. RESULTS | |
| 117 122 ACBD3 protein However, we were unable to obtain crystals even when using significantly truncated constructs that included only the ACBD3 Q domain and the N-terminal region of PI4KB. RESULTS | |
| 123 131 Q domain structure_element However, we were unable to obtain crystals even when using significantly truncated constructs that included only the ACBD3 Q domain and the N-terminal region of PI4KB. RESULTS | |
| 140 157 N-terminal region structure_element However, we were unable to obtain crystals even when using significantly truncated constructs that included only the ACBD3 Q domain and the N-terminal region of PI4KB. RESULTS | |
| 161 166 PI4KB protein However, we were unable to obtain crystals even when using significantly truncated constructs that included only the ACBD3 Q domain and the N-terminal region of PI4KB. RESULTS | |
| 32 52 isotopically labeled protein_state For this reason, we produced an isotopically labeled ACBD3 Q domain and isotopically labeled ACBD3 Q domain:PI4KB N-terminal region protein complex and used NMR spectroscopy for structural characterization. RESULTS | |
| 53 58 ACBD3 protein For this reason, we produced an isotopically labeled ACBD3 Q domain and isotopically labeled ACBD3 Q domain:PI4KB N-terminal region protein complex and used NMR spectroscopy for structural characterization. RESULTS | |
| 59 67 Q domain structure_element For this reason, we produced an isotopically labeled ACBD3 Q domain and isotopically labeled ACBD3 Q domain:PI4KB N-terminal region protein complex and used NMR spectroscopy for structural characterization. RESULTS | |
| 72 92 isotopically labeled protein_state For this reason, we produced an isotopically labeled ACBD3 Q domain and isotopically labeled ACBD3 Q domain:PI4KB N-terminal region protein complex and used NMR spectroscopy for structural characterization. RESULTS | |
| 93 98 ACBD3 protein For this reason, we produced an isotopically labeled ACBD3 Q domain and isotopically labeled ACBD3 Q domain:PI4KB N-terminal region protein complex and used NMR spectroscopy for structural characterization. RESULTS | |
| 99 107 Q domain structure_element For this reason, we produced an isotopically labeled ACBD3 Q domain and isotopically labeled ACBD3 Q domain:PI4KB N-terminal region protein complex and used NMR spectroscopy for structural characterization. RESULTS | |
| 108 113 PI4KB protein For this reason, we produced an isotopically labeled ACBD3 Q domain and isotopically labeled ACBD3 Q domain:PI4KB N-terminal region protein complex and used NMR spectroscopy for structural characterization. RESULTS | |
| 114 131 N-terminal region structure_element For this reason, we produced an isotopically labeled ACBD3 Q domain and isotopically labeled ACBD3 Q domain:PI4KB N-terminal region protein complex and used NMR spectroscopy for structural characterization. RESULTS | |
| 157 173 NMR spectroscopy experimental_method For this reason, we produced an isotopically labeled ACBD3 Q domain and isotopically labeled ACBD3 Q domain:PI4KB N-terminal region protein complex and used NMR spectroscopy for structural characterization. RESULTS | |
| 7 24 N-terminal region structure_element As the N-terminal region protein complex was prepared by co-expression of both proteins, the samples consisted of an equimolar mixture of two uniformly 15N/13C labelled molecules. RESULTS | |
| 57 70 co-expression experimental_method As the N-terminal region protein complex was prepared by co-expression of both proteins, the samples consisted of an equimolar mixture of two uniformly 15N/13C labelled molecules. RESULTS | |
| 152 155 15N chemical As the N-terminal region protein complex was prepared by co-expression of both proteins, the samples consisted of an equimolar mixture of two uniformly 15N/13C labelled molecules. RESULTS | |
| 156 159 13C chemical As the N-terminal region protein complex was prepared by co-expression of both proteins, the samples consisted of an equimolar mixture of two uniformly 15N/13C labelled molecules. RESULTS | |
| 160 168 labelled protein_state As the N-terminal region protein complex was prepared by co-expression of both proteins, the samples consisted of an equimolar mixture of two uniformly 15N/13C labelled molecules. RESULTS | |
| 68 72 free protein_state Comprehensive backbone and side-chain resonance assignments for the free ACBD3 Q domain and the complex, as illustrated by the 2D 15N/1H HSQC spectra (SI Figs 3 and 4), were obtained using a standard combination of triple-resonance experiments, as described previously. RESULTS | |
| 73 78 ACBD3 protein Comprehensive backbone and side-chain resonance assignments for the free ACBD3 Q domain and the complex, as illustrated by the 2D 15N/1H HSQC spectra (SI Figs 3 and 4), were obtained using a standard combination of triple-resonance experiments, as described previously. RESULTS | |
| 79 87 Q domain structure_element Comprehensive backbone and side-chain resonance assignments for the free ACBD3 Q domain and the complex, as illustrated by the 2D 15N/1H HSQC spectra (SI Figs 3 and 4), were obtained using a standard combination of triple-resonance experiments, as described previously. RESULTS | |
| 127 141 2D 15N/1H HSQC experimental_method Comprehensive backbone and side-chain resonance assignments for the free ACBD3 Q domain and the complex, as illustrated by the 2D 15N/1H HSQC spectra (SI Figs 3 and 4), were obtained using a standard combination of triple-resonance experiments, as described previously. RESULTS | |
| 142 149 spectra evidence Comprehensive backbone and side-chain resonance assignments for the free ACBD3 Q domain and the complex, as illustrated by the 2D 15N/1H HSQC spectra (SI Figs 3 and 4), were obtained using a standard combination of triple-resonance experiments, as described previously. RESULTS | |
| 215 243 triple-resonance experiments experimental_method Comprehensive backbone and side-chain resonance assignments for the free ACBD3 Q domain and the complex, as illustrated by the 2D 15N/1H HSQC spectra (SI Figs 3 and 4), were obtained using a standard combination of triple-resonance experiments, as described previously. RESULTS | |
| 24 27 15N chemical Backbone amide signals (15N and 1H) for the free ACBD3 Q domain were nearly completely assigned apart from the first four N-terminal residues (Met1-Lys4) and Gln44. RESULTS | |
| 32 34 1H chemical Backbone amide signals (15N and 1H) for the free ACBD3 Q domain were nearly completely assigned apart from the first four N-terminal residues (Met1-Lys4) and Gln44. RESULTS | |
| 44 48 free protein_state Backbone amide signals (15N and 1H) for the free ACBD3 Q domain were nearly completely assigned apart from the first four N-terminal residues (Met1-Lys4) and Gln44. RESULTS | |
| 49 54 ACBD3 protein Backbone amide signals (15N and 1H) for the free ACBD3 Q domain were nearly completely assigned apart from the first four N-terminal residues (Met1-Lys4) and Gln44. RESULTS | |
| 55 63 Q domain structure_element Backbone amide signals (15N and 1H) for the free ACBD3 Q domain were nearly completely assigned apart from the first four N-terminal residues (Met1-Lys4) and Gln44. RESULTS | |
| 143 152 Met1-Lys4 residue_range Backbone amide signals (15N and 1H) for the free ACBD3 Q domain were nearly completely assigned apart from the first four N-terminal residues (Met1-Lys4) and Gln44. RESULTS | |
| 158 163 Gln44 residue_name_number Backbone amide signals (15N and 1H) for the free ACBD3 Q domain were nearly completely assigned apart from the first four N-terminal residues (Met1-Lys4) and Gln44. RESULTS | |
| 70 74 free protein_state Over 93% of non-exchangeable side-chain signals were assigned for the free ACBD3 Q domain. RESULTS | |
| 75 80 ACBD3 protein Over 93% of non-exchangeable side-chain signals were assigned for the free ACBD3 Q domain. RESULTS | |
| 81 89 Q domain structure_element Over 93% of non-exchangeable side-chain signals were assigned for the free ACBD3 Q domain. RESULTS | |
| 85 88 Gln residue_name Apart from the four N-terminal residues, the side-chain assignments were missing for Gln (Hg3), Gln (Ha/Hb/Hg), Gln44 (Ha/Hb/Hg) and Gln48 (Hg) mainly due to extensive overlaps within the spectral regions populated by highly abundant glutamine side-chain resonances. RESULTS | |
| 96 99 Gln residue_name Apart from the four N-terminal residues, the side-chain assignments were missing for Gln (Hg3), Gln (Ha/Hb/Hg), Gln44 (Ha/Hb/Hg) and Gln48 (Hg) mainly due to extensive overlaps within the spectral regions populated by highly abundant glutamine side-chain resonances. RESULTS | |
| 112 117 Gln44 residue_name_number Apart from the four N-terminal residues, the side-chain assignments were missing for Gln (Hg3), Gln (Ha/Hb/Hg), Gln44 (Ha/Hb/Hg) and Gln48 (Hg) mainly due to extensive overlaps within the spectral regions populated by highly abundant glutamine side-chain resonances. RESULTS | |
| 133 138 Gln48 residue_name_number Apart from the four N-terminal residues, the side-chain assignments were missing for Gln (Hg3), Gln (Ha/Hb/Hg), Gln44 (Ha/Hb/Hg) and Gln48 (Hg) mainly due to extensive overlaps within the spectral regions populated by highly abundant glutamine side-chain resonances. RESULTS | |
| 234 243 glutamine residue_name Apart from the four N-terminal residues, the side-chain assignments were missing for Gln (Hg3), Gln (Ha/Hb/Hg), Gln44 (Ha/Hb/Hg) and Gln48 (Hg) mainly due to extensive overlaps within the spectral regions populated by highly abundant glutamine side-chain resonances. RESULTS | |
| 53 60 spectra evidence The protein complex yielded relatively well resolved spectra (SI Fig. 4) that resulted in assignment of backbone amide signals for all residues apart from Gln (ACBD3) and Ala2 (PI4KB). RESULTS | |
| 155 158 Gln residue_name The protein complex yielded relatively well resolved spectra (SI Fig. 4) that resulted in assignment of backbone amide signals for all residues apart from Gln (ACBD3) and Ala2 (PI4KB). RESULTS | |
| 160 165 ACBD3 protein The protein complex yielded relatively well resolved spectra (SI Fig. 4) that resulted in assignment of backbone amide signals for all residues apart from Gln (ACBD3) and Ala2 (PI4KB). RESULTS | |
| 171 175 Ala2 residue_name_number The protein complex yielded relatively well resolved spectra (SI Fig. 4) that resulted in assignment of backbone amide signals for all residues apart from Gln (ACBD3) and Ala2 (PI4KB). RESULTS | |
| 177 182 PI4KB protein The protein complex yielded relatively well resolved spectra (SI Fig. 4) that resulted in assignment of backbone amide signals for all residues apart from Gln (ACBD3) and Ala2 (PI4KB). RESULTS | |
| 25 28 15N chemical The essentially complete 15N, 13C and 1H resonance assignments allowed automated assignment of the NOEs identified in the 3D 15N/1H NOESY-HSQC and 13C/1H HMQC-NOESY spectra that were subsequently used in structural calculation. RESULTS | |
| 30 33 13C chemical The essentially complete 15N, 13C and 1H resonance assignments allowed automated assignment of the NOEs identified in the 3D 15N/1H NOESY-HSQC and 13C/1H HMQC-NOESY spectra that were subsequently used in structural calculation. RESULTS | |
| 38 40 1H chemical The essentially complete 15N, 13C and 1H resonance assignments allowed automated assignment of the NOEs identified in the 3D 15N/1H NOESY-HSQC and 13C/1H HMQC-NOESY spectra that were subsequently used in structural calculation. RESULTS | |
| 99 103 NOEs evidence The essentially complete 15N, 13C and 1H resonance assignments allowed automated assignment of the NOEs identified in the 3D 15N/1H NOESY-HSQC and 13C/1H HMQC-NOESY spectra that were subsequently used in structural calculation. RESULTS | |
| 122 142 3D 15N/1H NOESY-HSQC experimental_method The essentially complete 15N, 13C and 1H resonance assignments allowed automated assignment of the NOEs identified in the 3D 15N/1H NOESY-HSQC and 13C/1H HMQC-NOESY spectra that were subsequently used in structural calculation. RESULTS | |
| 147 164 13C/1H HMQC-NOESY experimental_method The essentially complete 15N, 13C and 1H resonance assignments allowed automated assignment of the NOEs identified in the 3D 15N/1H NOESY-HSQC and 13C/1H HMQC-NOESY spectra that were subsequently used in structural calculation. RESULTS | |
| 165 172 spectra evidence The essentially complete 15N, 13C and 1H resonance assignments allowed automated assignment of the NOEs identified in the 3D 15N/1H NOESY-HSQC and 13C/1H HMQC-NOESY spectra that were subsequently used in structural calculation. RESULTS | |
| 204 226 structural calculation experimental_method The essentially complete 15N, 13C and 1H resonance assignments allowed automated assignment of the NOEs identified in the 3D 15N/1H NOESY-HSQC and 13C/1H HMQC-NOESY spectra that were subsequently used in structural calculation. RESULTS | |
| 0 21 Structural statistics evidence Structural statistics for the final water-refined sets of structures are shown in SI Table 1. RESULTS | |
| 58 68 structures evidence Structural statistics for the final water-refined sets of structures are shown in SI Table 1. RESULTS | |
| 5 14 structure evidence This structure revealed that the Q domain forms a two helix hairpin. RESULTS | |
| 33 41 Q domain structure_element This structure revealed that the Q domain forms a two helix hairpin. RESULTS | |
| 50 67 two helix hairpin structure_element This structure revealed that the Q domain forms a two helix hairpin. RESULTS | |
| 10 15 helix structure_element The first helix bends sharply over the second helix and creates a fold resembling a three helix bundle that serves as a nest for one helix of the PI4KB N-terminus (residues 44–64, from this point on referred to as the kinase helix) (Fig. 2A). RESULTS | |
| 46 51 helix structure_element The first helix bends sharply over the second helix and creates a fold resembling a three helix bundle that serves as a nest for one helix of the PI4KB N-terminus (residues 44–64, from this point on referred to as the kinase helix) (Fig. 2A). RESULTS | |
| 84 102 three helix bundle structure_element The first helix bends sharply over the second helix and creates a fold resembling a three helix bundle that serves as a nest for one helix of the PI4KB N-terminus (residues 44–64, from this point on referred to as the kinase helix) (Fig. 2A). RESULTS | |
| 133 138 helix structure_element The first helix bends sharply over the second helix and creates a fold resembling a three helix bundle that serves as a nest for one helix of the PI4KB N-terminus (residues 44–64, from this point on referred to as the kinase helix) (Fig. 2A). RESULTS | |
| 146 151 PI4KB protein The first helix bends sharply over the second helix and creates a fold resembling a three helix bundle that serves as a nest for one helix of the PI4KB N-terminus (residues 44–64, from this point on referred to as the kinase helix) (Fig. 2A). RESULTS | |
| 173 178 44–64 residue_range The first helix bends sharply over the second helix and creates a fold resembling a three helix bundle that serves as a nest for one helix of the PI4KB N-terminus (residues 44–64, from this point on referred to as the kinase helix) (Fig. 2A). RESULTS | |
| 218 230 kinase helix structure_element The first helix bends sharply over the second helix and creates a fold resembling a three helix bundle that serves as a nest for one helix of the PI4KB N-terminus (residues 44–64, from this point on referred to as the kinase helix) (Fig. 2A). RESULTS | |
| 14 26 kinase helix structure_element Preceding the kinase helix are three ordered residues (Val42, Ile43, and Asp44) that also contribute to the interaction (Fig. 2B). RESULTS | |
| 55 60 Val42 residue_name_number Preceding the kinase helix are three ordered residues (Val42, Ile43, and Asp44) that also contribute to the interaction (Fig. 2B). RESULTS | |
| 62 67 Ile43 residue_name_number Preceding the kinase helix are three ordered residues (Val42, Ile43, and Asp44) that also contribute to the interaction (Fig. 2B). RESULTS | |
| 73 78 Asp44 residue_name_number Preceding the kinase helix are three ordered residues (Val42, Ile43, and Asp44) that also contribute to the interaction (Fig. 2B). RESULTS | |
| 26 31 PI4KB protein The remaining part of the PI4KB N-termini, however, is disordered (SI Fig. 5). RESULTS | |
| 18 29 PI4KB:ACBD3 complex_assembly Almost all of the PI4KB:ACBD3 interactions are hydrophobic with the exception of hydrogen bonds between the side chains of ACBD3 Tyr261 and PI4KB His63, and between the sidechain of ACBD3 Tyr288 and the PI4KB backbone (Asp44) (Fig. 2B). RESULTS | |
| 30 58 interactions are hydrophobic bond_interaction Almost all of the PI4KB:ACBD3 interactions are hydrophobic with the exception of hydrogen bonds between the side chains of ACBD3 Tyr261 and PI4KB His63, and between the sidechain of ACBD3 Tyr288 and the PI4KB backbone (Asp44) (Fig. 2B). RESULTS | |
| 81 95 hydrogen bonds bond_interaction Almost all of the PI4KB:ACBD3 interactions are hydrophobic with the exception of hydrogen bonds between the side chains of ACBD3 Tyr261 and PI4KB His63, and between the sidechain of ACBD3 Tyr288 and the PI4KB backbone (Asp44) (Fig. 2B). RESULTS | |
| 123 128 ACBD3 protein Almost all of the PI4KB:ACBD3 interactions are hydrophobic with the exception of hydrogen bonds between the side chains of ACBD3 Tyr261 and PI4KB His63, and between the sidechain of ACBD3 Tyr288 and the PI4KB backbone (Asp44) (Fig. 2B). RESULTS | |
| 129 135 Tyr261 residue_name_number Almost all of the PI4KB:ACBD3 interactions are hydrophobic with the exception of hydrogen bonds between the side chains of ACBD3 Tyr261 and PI4KB His63, and between the sidechain of ACBD3 Tyr288 and the PI4KB backbone (Asp44) (Fig. 2B). RESULTS | |
| 140 145 PI4KB protein Almost all of the PI4KB:ACBD3 interactions are hydrophobic with the exception of hydrogen bonds between the side chains of ACBD3 Tyr261 and PI4KB His63, and between the sidechain of ACBD3 Tyr288 and the PI4KB backbone (Asp44) (Fig. 2B). RESULTS | |
| 146 151 His63 residue_name_number Almost all of the PI4KB:ACBD3 interactions are hydrophobic with the exception of hydrogen bonds between the side chains of ACBD3 Tyr261 and PI4KB His63, and between the sidechain of ACBD3 Tyr288 and the PI4KB backbone (Asp44) (Fig. 2B). RESULTS | |
| 182 187 ACBD3 protein Almost all of the PI4KB:ACBD3 interactions are hydrophobic with the exception of hydrogen bonds between the side chains of ACBD3 Tyr261 and PI4KB His63, and between the sidechain of ACBD3 Tyr288 and the PI4KB backbone (Asp44) (Fig. 2B). RESULTS | |
| 188 194 Tyr288 residue_name_number Almost all of the PI4KB:ACBD3 interactions are hydrophobic with the exception of hydrogen bonds between the side chains of ACBD3 Tyr261 and PI4KB His63, and between the sidechain of ACBD3 Tyr288 and the PI4KB backbone (Asp44) (Fig. 2B). RESULTS | |
| 203 208 PI4KB protein Almost all of the PI4KB:ACBD3 interactions are hydrophobic with the exception of hydrogen bonds between the side chains of ACBD3 Tyr261 and PI4KB His63, and between the sidechain of ACBD3 Tyr288 and the PI4KB backbone (Asp44) (Fig. 2B). RESULTS | |
| 219 224 Asp44 residue_name_number Almost all of the PI4KB:ACBD3 interactions are hydrophobic with the exception of hydrogen bonds between the side chains of ACBD3 Tyr261 and PI4KB His63, and between the sidechain of ACBD3 Tyr288 and the PI4KB backbone (Asp44) (Fig. 2B). RESULTS | |
| 33 38 PI4KB protein Interestingly, we noted that the PI4KB helix is amphipathic and its hydrophobic surface leans on the Q domain (Fig. 2C). RESULTS | |
| 39 44 helix structure_element Interestingly, we noted that the PI4KB helix is amphipathic and its hydrophobic surface leans on the Q domain (Fig. 2C). RESULTS | |
| 48 59 amphipathic protein_state Interestingly, we noted that the PI4KB helix is amphipathic and its hydrophobic surface leans on the Q domain (Fig. 2C). RESULTS | |
| 68 87 hydrophobic surface site Interestingly, we noted that the PI4KB helix is amphipathic and its hydrophobic surface leans on the Q domain (Fig. 2C). RESULTS | |
| 101 109 Q domain structure_element Interestingly, we noted that the PI4KB helix is amphipathic and its hydrophobic surface leans on the Q domain (Fig. 2C). RESULTS | |
| 19 34 structural data evidence To corroborate the structural data, we introduced a number of point mutations and validated their effect on complex formation using an in vitro pull-down assay (Fig. 2D). RESULTS | |
| 39 49 introduced experimental_method To corroborate the structural data, we introduced a number of point mutations and validated their effect on complex formation using an in vitro pull-down assay (Fig. 2D). RESULTS | |
| 62 77 point mutations experimental_method To corroborate the structural data, we introduced a number of point mutations and validated their effect on complex formation using an in vitro pull-down assay (Fig. 2D). RESULTS | |
| 135 159 in vitro pull-down assay experimental_method To corroborate the structural data, we introduced a number of point mutations and validated their effect on complex formation using an in vitro pull-down assay (Fig. 2D). RESULTS | |
| 0 9 Wild type protein_state Wild type ACBD3 protein co-purified together with the NiNTA-immobilized His6-tagged wild type PI4KB as well as with the PI4KB V42A and V47A mutants, but not with mutants within the imminent binding interface (I43A, V55A, L56A). RESULTS | |
| 10 15 ACBD3 protein Wild type ACBD3 protein co-purified together with the NiNTA-immobilized His6-tagged wild type PI4KB as well as with the PI4KB V42A and V47A mutants, but not with mutants within the imminent binding interface (I43A, V55A, L56A). RESULTS | |
| 24 35 co-purified experimental_method Wild type ACBD3 protein co-purified together with the NiNTA-immobilized His6-tagged wild type PI4KB as well as with the PI4KB V42A and V47A mutants, but not with mutants within the imminent binding interface (I43A, V55A, L56A). RESULTS | |
| 72 83 His6-tagged protein_state Wild type ACBD3 protein co-purified together with the NiNTA-immobilized His6-tagged wild type PI4KB as well as with the PI4KB V42A and V47A mutants, but not with mutants within the imminent binding interface (I43A, V55A, L56A). RESULTS | |
| 84 93 wild type protein_state Wild type ACBD3 protein co-purified together with the NiNTA-immobilized His6-tagged wild type PI4KB as well as with the PI4KB V42A and V47A mutants, but not with mutants within the imminent binding interface (I43A, V55A, L56A). RESULTS | |
| 94 99 PI4KB protein Wild type ACBD3 protein co-purified together with the NiNTA-immobilized His6-tagged wild type PI4KB as well as with the PI4KB V42A and V47A mutants, but not with mutants within the imminent binding interface (I43A, V55A, L56A). RESULTS | |
| 120 125 PI4KB protein Wild type ACBD3 protein co-purified together with the NiNTA-immobilized His6-tagged wild type PI4KB as well as with the PI4KB V42A and V47A mutants, but not with mutants within the imminent binding interface (I43A, V55A, L56A). RESULTS | |
| 126 130 V42A mutant Wild type ACBD3 protein co-purified together with the NiNTA-immobilized His6-tagged wild type PI4KB as well as with the PI4KB V42A and V47A mutants, but not with mutants within the imminent binding interface (I43A, V55A, L56A). RESULTS | |
| 135 139 V47A mutant Wild type ACBD3 protein co-purified together with the NiNTA-immobilized His6-tagged wild type PI4KB as well as with the PI4KB V42A and V47A mutants, but not with mutants within the imminent binding interface (I43A, V55A, L56A). RESULTS | |
| 140 147 mutants protein_state Wild type ACBD3 protein co-purified together with the NiNTA-immobilized His6-tagged wild type PI4KB as well as with the PI4KB V42A and V47A mutants, but not with mutants within the imminent binding interface (I43A, V55A, L56A). RESULTS | |
| 162 169 mutants protein_state Wild type ACBD3 protein co-purified together with the NiNTA-immobilized His6-tagged wild type PI4KB as well as with the PI4KB V42A and V47A mutants, but not with mutants within the imminent binding interface (I43A, V55A, L56A). RESULTS | |
| 190 207 binding interface site Wild type ACBD3 protein co-purified together with the NiNTA-immobilized His6-tagged wild type PI4KB as well as with the PI4KB V42A and V47A mutants, but not with mutants within the imminent binding interface (I43A, V55A, L56A). RESULTS | |
| 209 213 I43A mutant Wild type ACBD3 protein co-purified together with the NiNTA-immobilized His6-tagged wild type PI4KB as well as with the PI4KB V42A and V47A mutants, but not with mutants within the imminent binding interface (I43A, V55A, L56A). RESULTS | |
| 215 219 V55A mutant Wild type ACBD3 protein co-purified together with the NiNTA-immobilized His6-tagged wild type PI4KB as well as with the PI4KB V42A and V47A mutants, but not with mutants within the imminent binding interface (I43A, V55A, L56A). RESULTS | |
| 221 225 L56A mutant Wild type ACBD3 protein co-purified together with the NiNTA-immobilized His6-tagged wild type PI4KB as well as with the PI4KB V42A and V47A mutants, but not with mutants within the imminent binding interface (I43A, V55A, L56A). RESULTS | |
| 14 23 wild type protein_state As predicted, wild type PI4KB interacted with the ACBD3 Y266A mutant and slightly with the Y285A mutant, but not with the F258A, H284A, and Y288A mutants (Fig. 2D). RESULTS | |
| 24 29 PI4KB protein As predicted, wild type PI4KB interacted with the ACBD3 Y266A mutant and slightly with the Y285A mutant, but not with the F258A, H284A, and Y288A mutants (Fig. 2D). RESULTS | |
| 50 55 ACBD3 protein As predicted, wild type PI4KB interacted with the ACBD3 Y266A mutant and slightly with the Y285A mutant, but not with the F258A, H284A, and Y288A mutants (Fig. 2D). RESULTS | |
| 56 61 Y266A mutant As predicted, wild type PI4KB interacted with the ACBD3 Y266A mutant and slightly with the Y285A mutant, but not with the F258A, H284A, and Y288A mutants (Fig. 2D). RESULTS | |
| 62 68 mutant protein_state As predicted, wild type PI4KB interacted with the ACBD3 Y266A mutant and slightly with the Y285A mutant, but not with the F258A, H284A, and Y288A mutants (Fig. 2D). RESULTS | |
| 91 96 Y285A mutant As predicted, wild type PI4KB interacted with the ACBD3 Y266A mutant and slightly with the Y285A mutant, but not with the F258A, H284A, and Y288A mutants (Fig. 2D). RESULTS | |
| 97 103 mutant protein_state As predicted, wild type PI4KB interacted with the ACBD3 Y266A mutant and slightly with the Y285A mutant, but not with the F258A, H284A, and Y288A mutants (Fig. 2D). RESULTS | |
| 122 127 F258A mutant As predicted, wild type PI4KB interacted with the ACBD3 Y266A mutant and slightly with the Y285A mutant, but not with the F258A, H284A, and Y288A mutants (Fig. 2D). RESULTS | |
| 129 134 H284A mutant As predicted, wild type PI4KB interacted with the ACBD3 Y266A mutant and slightly with the Y285A mutant, but not with the F258A, H284A, and Y288A mutants (Fig. 2D). RESULTS | |
| 140 145 Y288A mutant As predicted, wild type PI4KB interacted with the ACBD3 Y266A mutant and slightly with the Y285A mutant, but not with the F258A, H284A, and Y288A mutants (Fig. 2D). RESULTS | |
| 146 153 mutants protein_state As predicted, wild type PI4KB interacted with the ACBD3 Y266A mutant and slightly with the Y285A mutant, but not with the F258A, H284A, and Y288A mutants (Fig. 2D). RESULTS | |
| 0 5 ACBD3 protein ACBD3 efficiently recruits the PI4KB enzyme to membranes RESULTS | |
| 31 36 PI4KB protein ACBD3 efficiently recruits the PI4KB enzyme to membranes RESULTS | |
| 35 46 ACBD3:PI4KB complex_assembly We next sought to determine if the ACBD3:PI4KB interaction drives membrane localization of the PI4KB enzyme. RESULTS | |
| 95 100 PI4KB protein We next sought to determine if the ACBD3:PI4KB interaction drives membrane localization of the PI4KB enzyme. RESULTS | |
| 36 72 in vitro membrane recruitment system experimental_method To do this, we first established an in vitro membrane recruitment system using Giant Unilamellar Vesicles (GUVs) containing the PI4KB substrate – the PI lipid. RESULTS | |
| 79 105 Giant Unilamellar Vesicles experimental_method To do this, we first established an in vitro membrane recruitment system using Giant Unilamellar Vesicles (GUVs) containing the PI4KB substrate – the PI lipid. RESULTS | |
| 107 111 GUVs experimental_method To do this, we first established an in vitro membrane recruitment system using Giant Unilamellar Vesicles (GUVs) containing the PI4KB substrate – the PI lipid. RESULTS | |
| 128 133 PI4KB protein To do this, we first established an in vitro membrane recruitment system using Giant Unilamellar Vesicles (GUVs) containing the PI4KB substrate – the PI lipid. RESULTS | |
| 150 152 PI chemical To do this, we first established an in vitro membrane recruitment system using Giant Unilamellar Vesicles (GUVs) containing the PI4KB substrate – the PI lipid. RESULTS | |
| 17 22 PI4KB protein We observed that PI4KB kinase was not membrane localized when added to the GUVs at 600 nM concentration, whereas non-covalent tethering of ACBD3 to the surface of the GUVs, using the His6 tag on ACBD3 and the DGS-NTA (Ni) lipid, led to efficient PI4KB membrane localization (Fig. 3A). RESULTS | |
| 23 29 kinase protein_type We observed that PI4KB kinase was not membrane localized when added to the GUVs at 600 nM concentration, whereas non-covalent tethering of ACBD3 to the surface of the GUVs, using the His6 tag on ACBD3 and the DGS-NTA (Ni) lipid, led to efficient PI4KB membrane localization (Fig. 3A). RESULTS | |
| 47 56 localized evidence We observed that PI4KB kinase was not membrane localized when added to the GUVs at 600 nM concentration, whereas non-covalent tethering of ACBD3 to the surface of the GUVs, using the His6 tag on ACBD3 and the DGS-NTA (Ni) lipid, led to efficient PI4KB membrane localization (Fig. 3A). RESULTS | |
| 75 79 GUVs experimental_method We observed that PI4KB kinase was not membrane localized when added to the GUVs at 600 nM concentration, whereas non-covalent tethering of ACBD3 to the surface of the GUVs, using the His6 tag on ACBD3 and the DGS-NTA (Ni) lipid, led to efficient PI4KB membrane localization (Fig. 3A). RESULTS | |
| 139 144 ACBD3 protein We observed that PI4KB kinase was not membrane localized when added to the GUVs at 600 nM concentration, whereas non-covalent tethering of ACBD3 to the surface of the GUVs, using the His6 tag on ACBD3 and the DGS-NTA (Ni) lipid, led to efficient PI4KB membrane localization (Fig. 3A). RESULTS | |
| 167 171 GUVs experimental_method We observed that PI4KB kinase was not membrane localized when added to the GUVs at 600 nM concentration, whereas non-covalent tethering of ACBD3 to the surface of the GUVs, using the His6 tag on ACBD3 and the DGS-NTA (Ni) lipid, led to efficient PI4KB membrane localization (Fig. 3A). RESULTS | |
| 195 200 ACBD3 protein We observed that PI4KB kinase was not membrane localized when added to the GUVs at 600 nM concentration, whereas non-covalent tethering of ACBD3 to the surface of the GUVs, using the His6 tag on ACBD3 and the DGS-NTA (Ni) lipid, led to efficient PI4KB membrane localization (Fig. 3A). RESULTS | |
| 209 227 DGS-NTA (Ni) lipid chemical We observed that PI4KB kinase was not membrane localized when added to the GUVs at 600 nM concentration, whereas non-covalent tethering of ACBD3 to the surface of the GUVs, using the His6 tag on ACBD3 and the DGS-NTA (Ni) lipid, led to efficient PI4KB membrane localization (Fig. 3A). RESULTS | |
| 246 251 PI4KB protein We observed that PI4KB kinase was not membrane localized when added to the GUVs at 600 nM concentration, whereas non-covalent tethering of ACBD3 to the surface of the GUVs, using the His6 tag on ACBD3 and the DGS-NTA (Ni) lipid, led to efficient PI4KB membrane localization (Fig. 3A). RESULTS | |
| 24 29 ACBD3 protein We hypothesized that if ACBD3 is one of the main Golgi localization signals for PI4KB, overexpression of the Q domain should decrease the amount of the endogenous kinase on the Golgi. Indeed, we observed loss for endogenous PI4KB signal on the Golgi in cells overexpressing the GFP – Q domain construct (Fig. 3B upper panel). RESULTS | |
| 55 75 localization signals evidence We hypothesized that if ACBD3 is one of the main Golgi localization signals for PI4KB, overexpression of the Q domain should decrease the amount of the endogenous kinase on the Golgi. Indeed, we observed loss for endogenous PI4KB signal on the Golgi in cells overexpressing the GFP – Q domain construct (Fig. 3B upper panel). RESULTS | |
| 80 85 PI4KB protein We hypothesized that if ACBD3 is one of the main Golgi localization signals for PI4KB, overexpression of the Q domain should decrease the amount of the endogenous kinase on the Golgi. Indeed, we observed loss for endogenous PI4KB signal on the Golgi in cells overexpressing the GFP – Q domain construct (Fig. 3B upper panel). RESULTS | |
| 87 101 overexpression experimental_method We hypothesized that if ACBD3 is one of the main Golgi localization signals for PI4KB, overexpression of the Q domain should decrease the amount of the endogenous kinase on the Golgi. Indeed, we observed loss for endogenous PI4KB signal on the Golgi in cells overexpressing the GFP – Q domain construct (Fig. 3B upper panel). RESULTS | |
| 109 117 Q domain structure_element We hypothesized that if ACBD3 is one of the main Golgi localization signals for PI4KB, overexpression of the Q domain should decrease the amount of the endogenous kinase on the Golgi. Indeed, we observed loss for endogenous PI4KB signal on the Golgi in cells overexpressing the GFP – Q domain construct (Fig. 3B upper panel). RESULTS | |
| 163 169 kinase protein_type We hypothesized that if ACBD3 is one of the main Golgi localization signals for PI4KB, overexpression of the Q domain should decrease the amount of the endogenous kinase on the Golgi. Indeed, we observed loss for endogenous PI4KB signal on the Golgi in cells overexpressing the GFP – Q domain construct (Fig. 3B upper panel). RESULTS | |
| 224 229 PI4KB protein We hypothesized that if ACBD3 is one of the main Golgi localization signals for PI4KB, overexpression of the Q domain should decrease the amount of the endogenous kinase on the Golgi. Indeed, we observed loss for endogenous PI4KB signal on the Golgi in cells overexpressing the GFP – Q domain construct (Fig. 3B upper panel). RESULTS | |
| 259 273 overexpressing experimental_method We hypothesized that if ACBD3 is one of the main Golgi localization signals for PI4KB, overexpression of the Q domain should decrease the amount of the endogenous kinase on the Golgi. Indeed, we observed loss for endogenous PI4KB signal on the Golgi in cells overexpressing the GFP – Q domain construct (Fig. 3B upper panel). RESULTS | |
| 278 281 GFP experimental_method We hypothesized that if ACBD3 is one of the main Golgi localization signals for PI4KB, overexpression of the Q domain should decrease the amount of the endogenous kinase on the Golgi. Indeed, we observed loss for endogenous PI4KB signal on the Golgi in cells overexpressing the GFP – Q domain construct (Fig. 3B upper panel). RESULTS | |
| 284 292 Q domain structure_element We hypothesized that if ACBD3 is one of the main Golgi localization signals for PI4KB, overexpression of the Q domain should decrease the amount of the endogenous kinase on the Golgi. Indeed, we observed loss for endogenous PI4KB signal on the Golgi in cells overexpressing the GFP – Q domain construct (Fig. 3B upper panel). RESULTS | |
| 25 31 signal evidence We attribute the loss of signal to the immunostaining protocol-the kinase that is not bound to Golgi is lost during the permeabilization step and hence the “disappearance” of the signal because overexpression of GFP alone or a non-binding Q domain mutant has no effect on the localization of the endogenous PI4KB (Fig. 3B). RESULTS | |
| 67 73 kinase protein_type We attribute the loss of signal to the immunostaining protocol-the kinase that is not bound to Golgi is lost during the permeabilization step and hence the “disappearance” of the signal because overexpression of GFP alone or a non-binding Q domain mutant has no effect on the localization of the endogenous PI4KB (Fig. 3B). RESULTS | |
| 179 185 signal evidence We attribute the loss of signal to the immunostaining protocol-the kinase that is not bound to Golgi is lost during the permeabilization step and hence the “disappearance” of the signal because overexpression of GFP alone or a non-binding Q domain mutant has no effect on the localization of the endogenous PI4KB (Fig. 3B). RESULTS | |
| 194 208 overexpression experimental_method We attribute the loss of signal to the immunostaining protocol-the kinase that is not bound to Golgi is lost during the permeabilization step and hence the “disappearance” of the signal because overexpression of GFP alone or a non-binding Q domain mutant has no effect on the localization of the endogenous PI4KB (Fig. 3B). RESULTS | |
| 212 215 GFP experimental_method We attribute the loss of signal to the immunostaining protocol-the kinase that is not bound to Golgi is lost during the permeabilization step and hence the “disappearance” of the signal because overexpression of GFP alone or a non-binding Q domain mutant has no effect on the localization of the endogenous PI4KB (Fig. 3B). RESULTS | |
| 227 238 non-binding protein_state We attribute the loss of signal to the immunostaining protocol-the kinase that is not bound to Golgi is lost during the permeabilization step and hence the “disappearance” of the signal because overexpression of GFP alone or a non-binding Q domain mutant has no effect on the localization of the endogenous PI4KB (Fig. 3B). RESULTS | |
| 239 247 Q domain structure_element We attribute the loss of signal to the immunostaining protocol-the kinase that is not bound to Golgi is lost during the permeabilization step and hence the “disappearance” of the signal because overexpression of GFP alone or a non-binding Q domain mutant has no effect on the localization of the endogenous PI4KB (Fig. 3B). RESULTS | |
| 248 254 mutant protein_state We attribute the loss of signal to the immunostaining protocol-the kinase that is not bound to Golgi is lost during the permeabilization step and hence the “disappearance” of the signal because overexpression of GFP alone or a non-binding Q domain mutant has no effect on the localization of the endogenous PI4KB (Fig. 3B). RESULTS | |
| 276 288 localization evidence We attribute the loss of signal to the immunostaining protocol-the kinase that is not bound to Golgi is lost during the permeabilization step and hence the “disappearance” of the signal because overexpression of GFP alone or a non-binding Q domain mutant has no effect on the localization of the endogenous PI4KB (Fig. 3B). RESULTS | |
| 307 312 PI4KB protein We attribute the loss of signal to the immunostaining protocol-the kinase that is not bound to Golgi is lost during the permeabilization step and hence the “disappearance” of the signal because overexpression of GFP alone or a non-binding Q domain mutant has no effect on the localization of the endogenous PI4KB (Fig. 3B). RESULTS | |
| 19 33 overexpression experimental_method Given this result, overexpression of the Q domain should also interfere with the PI4KB dependent Golgi functions. RESULTS | |
| 41 49 Q domain structure_element Given this result, overexpression of the Q domain should also interfere with the PI4KB dependent Golgi functions. RESULTS | |
| 81 86 PI4KB protein Given this result, overexpression of the Q domain should also interfere with the PI4KB dependent Golgi functions. RESULTS | |
| 0 8 Ceramide chemical Ceramide transport and accumulation in Golgi is a well-known PI4KB dependent process. RESULTS | |
| 61 66 PI4KB protein Ceramide transport and accumulation in Golgi is a well-known PI4KB dependent process. RESULTS | |
| 13 34 fluorescently labeled protein_state We have used fluorescently labeled ceramide and analyzed its trafficking in non-transfected cells and cell overexpressing the Q domain. RESULTS | |
| 35 43 ceramide chemical We have used fluorescently labeled ceramide and analyzed its trafficking in non-transfected cells and cell overexpressing the Q domain. RESULTS | |
| 107 121 overexpressing experimental_method We have used fluorescently labeled ceramide and analyzed its trafficking in non-transfected cells and cell overexpressing the Q domain. RESULTS | |
| 126 134 Q domain structure_element We have used fluorescently labeled ceramide and analyzed its trafficking in non-transfected cells and cell overexpressing the Q domain. RESULTS | |
| 39 47 ceramide chemical As expected, the Golgi accumulation of ceramide was not observed in cells expressing the wt Q domain while cells expressing RFP or the mutant Q domain accumulated ceramide normally (Fig. 3C) suggesting that ACBD3:PI4KB complex formation is crucial for the normal function of Golgi. RESULTS | |
| 74 84 expressing experimental_method As expected, the Golgi accumulation of ceramide was not observed in cells expressing the wt Q domain while cells expressing RFP or the mutant Q domain accumulated ceramide normally (Fig. 3C) suggesting that ACBD3:PI4KB complex formation is crucial for the normal function of Golgi. RESULTS | |
| 89 91 wt protein_state As expected, the Golgi accumulation of ceramide was not observed in cells expressing the wt Q domain while cells expressing RFP or the mutant Q domain accumulated ceramide normally (Fig. 3C) suggesting that ACBD3:PI4KB complex formation is crucial for the normal function of Golgi. RESULTS | |
| 92 100 Q domain structure_element As expected, the Golgi accumulation of ceramide was not observed in cells expressing the wt Q domain while cells expressing RFP or the mutant Q domain accumulated ceramide normally (Fig. 3C) suggesting that ACBD3:PI4KB complex formation is crucial for the normal function of Golgi. RESULTS | |
| 124 127 RFP experimental_method As expected, the Golgi accumulation of ceramide was not observed in cells expressing the wt Q domain while cells expressing RFP or the mutant Q domain accumulated ceramide normally (Fig. 3C) suggesting that ACBD3:PI4KB complex formation is crucial for the normal function of Golgi. RESULTS | |
| 135 141 mutant protein_state As expected, the Golgi accumulation of ceramide was not observed in cells expressing the wt Q domain while cells expressing RFP or the mutant Q domain accumulated ceramide normally (Fig. 3C) suggesting that ACBD3:PI4KB complex formation is crucial for the normal function of Golgi. RESULTS | |
| 142 150 Q domain structure_element As expected, the Golgi accumulation of ceramide was not observed in cells expressing the wt Q domain while cells expressing RFP or the mutant Q domain accumulated ceramide normally (Fig. 3C) suggesting that ACBD3:PI4KB complex formation is crucial for the normal function of Golgi. RESULTS | |
| 163 171 ceramide chemical As expected, the Golgi accumulation of ceramide was not observed in cells expressing the wt Q domain while cells expressing RFP or the mutant Q domain accumulated ceramide normally (Fig. 3C) suggesting that ACBD3:PI4KB complex formation is crucial for the normal function of Golgi. RESULTS | |
| 207 218 ACBD3:PI4KB complex_assembly As expected, the Golgi accumulation of ceramide was not observed in cells expressing the wt Q domain while cells expressing RFP or the mutant Q domain accumulated ceramide normally (Fig. 3C) suggesting that ACBD3:PI4KB complex formation is crucial for the normal function of Golgi. RESULTS | |
| 36 47 ACBD3:PI4KB complex_assembly We further analyzed the function of ACBD3:PI4KB interaction in membrane recruitment of PI4KB in living cells using fluorescently tagged proteins. RESULTS | |
| 87 92 PI4KB protein We further analyzed the function of ACBD3:PI4KB interaction in membrane recruitment of PI4KB in living cells using fluorescently tagged proteins. RESULTS | |
| 115 135 fluorescently tagged protein_state We further analyzed the function of ACBD3:PI4KB interaction in membrane recruitment of PI4KB in living cells using fluorescently tagged proteins. RESULTS | |
| 12 21 rapamycin chemical We used the rapamycin-inducible heteromerization of FKBP12 (FK506 binding protein 12) and FRB (fragment of mTOR that binds rapamycin) system. RESULTS | |
| 52 58 FKBP12 protein We used the rapamycin-inducible heteromerization of FKBP12 (FK506 binding protein 12) and FRB (fragment of mTOR that binds rapamycin) system. RESULTS | |
| 60 84 FK506 binding protein 12 protein We used the rapamycin-inducible heteromerization of FKBP12 (FK506 binding protein 12) and FRB (fragment of mTOR that binds rapamycin) system. RESULTS | |
| 90 93 FRB structure_element We used the rapamycin-inducible heteromerization of FKBP12 (FK506 binding protein 12) and FRB (fragment of mTOR that binds rapamycin) system. RESULTS | |
| 95 103 fragment structure_element We used the rapamycin-inducible heteromerization of FKBP12 (FK506 binding protein 12) and FRB (fragment of mTOR that binds rapamycin) system. RESULTS | |
| 107 111 mTOR protein We used the rapamycin-inducible heteromerization of FKBP12 (FK506 binding protein 12) and FRB (fragment of mTOR that binds rapamycin) system. RESULTS | |
| 123 132 rapamycin chemical We used the rapamycin-inducible heteromerization of FKBP12 (FK506 binding protein 12) and FRB (fragment of mTOR that binds rapamycin) system. RESULTS | |
| 3 8 fused experimental_method We fused the FRB to residues 34–63 of the mitochondrial localization signal from mitochondrial A-kinase anchor protein 1 (AKAP1) and CFP. RESULTS | |
| 13 16 FRB structure_element We fused the FRB to residues 34–63 of the mitochondrial localization signal from mitochondrial A-kinase anchor protein 1 (AKAP1) and CFP. RESULTS | |
| 29 34 34–63 residue_range We fused the FRB to residues 34–63 of the mitochondrial localization signal from mitochondrial A-kinase anchor protein 1 (AKAP1) and CFP. RESULTS | |
| 42 75 mitochondrial localization signal structure_element We fused the FRB to residues 34–63 of the mitochondrial localization signal from mitochondrial A-kinase anchor protein 1 (AKAP1) and CFP. RESULTS | |
| 81 120 mitochondrial A-kinase anchor protein 1 protein We fused the FRB to residues 34–63 of the mitochondrial localization signal from mitochondrial A-kinase anchor protein 1 (AKAP1) and CFP. RESULTS | |
| 122 127 AKAP1 protein We fused the FRB to residues 34–63 of the mitochondrial localization signal from mitochondrial A-kinase anchor protein 1 (AKAP1) and CFP. RESULTS | |
| 133 136 CFP experimental_method We fused the FRB to residues 34–63 of the mitochondrial localization signal from mitochondrial A-kinase anchor protein 1 (AKAP1) and CFP. RESULTS | |
| 4 9 ACBD3 protein The ACBD3 Q domain was then fused to FKBP12 and mRFP (Fig. 3D). RESULTS | |
| 10 18 Q domain structure_element The ACBD3 Q domain was then fused to FKBP12 and mRFP (Fig. 3D). RESULTS | |
| 28 36 fused to experimental_method The ACBD3 Q domain was then fused to FKBP12 and mRFP (Fig. 3D). RESULTS | |
| 37 43 FKBP12 protein The ACBD3 Q domain was then fused to FKBP12 and mRFP (Fig. 3D). RESULTS | |
| 48 52 mRFP experimental_method The ACBD3 Q domain was then fused to FKBP12 and mRFP (Fig. 3D). RESULTS | |
| 12 24 localization evidence We analyzed localization of the ACBD3 Q domain and GFP – PI4KB before and after the addition of rapamycin. RESULTS | |
| 32 37 ACBD3 protein We analyzed localization of the ACBD3 Q domain and GFP – PI4KB before and after the addition of rapamycin. RESULTS | |
| 38 46 Q domain structure_element We analyzed localization of the ACBD3 Q domain and GFP – PI4KB before and after the addition of rapamycin. RESULTS | |
| 51 54 GFP experimental_method We analyzed localization of the ACBD3 Q domain and GFP – PI4KB before and after the addition of rapamycin. RESULTS | |
| 57 62 PI4KB protein We analyzed localization of the ACBD3 Q domain and GFP – PI4KB before and after the addition of rapamycin. RESULTS | |
| 96 105 rapamycin chemical We analyzed localization of the ACBD3 Q domain and GFP – PI4KB before and after the addition of rapamycin. RESULTS | |
| 21 26 H284A mutant As a control we used H284A mutant of the ACBD3 Q domain that does not significantly bind PI4KB kinase. RESULTS | |
| 27 33 mutant protein_state As a control we used H284A mutant of the ACBD3 Q domain that does not significantly bind PI4KB kinase. RESULTS | |
| 41 46 ACBD3 protein As a control we used H284A mutant of the ACBD3 Q domain that does not significantly bind PI4KB kinase. RESULTS | |
| 47 55 Q domain structure_element As a control we used H284A mutant of the ACBD3 Q domain that does not significantly bind PI4KB kinase. RESULTS | |
| 89 94 PI4KB protein As a control we used H284A mutant of the ACBD3 Q domain that does not significantly bind PI4KB kinase. RESULTS | |
| 95 101 kinase protein_type As a control we used H284A mutant of the ACBD3 Q domain that does not significantly bind PI4KB kinase. RESULTS | |
| 18 23 ACDB3 protein In every case the ACDB3 Q domain was rapidly (within 5 minutes) recruited to the mitochondrial membrane upon addition of rapamycin, but only the wild-type protein effectively directed the kinase to the mitochondria (Fig. 3E, Movie 1 and 2). RESULTS | |
| 24 32 Q domain structure_element In every case the ACDB3 Q domain was rapidly (within 5 minutes) recruited to the mitochondrial membrane upon addition of rapamycin, but only the wild-type protein effectively directed the kinase to the mitochondria (Fig. 3E, Movie 1 and 2). RESULTS | |
| 121 130 rapamycin chemical In every case the ACDB3 Q domain was rapidly (within 5 minutes) recruited to the mitochondrial membrane upon addition of rapamycin, but only the wild-type protein effectively directed the kinase to the mitochondria (Fig. 3E, Movie 1 and 2). RESULTS | |
| 145 154 wild-type protein_state In every case the ACDB3 Q domain was rapidly (within 5 minutes) recruited to the mitochondrial membrane upon addition of rapamycin, but only the wild-type protein effectively directed the kinase to the mitochondria (Fig. 3E, Movie 1 and 2). RESULTS | |
| 188 194 kinase protein_type In every case the ACDB3 Q domain was rapidly (within 5 minutes) recruited to the mitochondrial membrane upon addition of rapamycin, but only the wild-type protein effectively directed the kinase to the mitochondria (Fig. 3E, Movie 1 and 2). RESULTS | |
| 35 38 GFP experimental_method Notably, we observed that when the GFP-PI4KB kinase is co-expressed with the wild-type ACDB3 Q domain it loses its typical Golgi localization (Fig. 3E upper panel). RESULTS | |
| 39 44 PI4KB protein Notably, we observed that when the GFP-PI4KB kinase is co-expressed with the wild-type ACDB3 Q domain it loses its typical Golgi localization (Fig. 3E upper panel). RESULTS | |
| 45 51 kinase protein_type Notably, we observed that when the GFP-PI4KB kinase is co-expressed with the wild-type ACDB3 Q domain it loses its typical Golgi localization (Fig. 3E upper panel). RESULTS | |
| 55 67 co-expressed experimental_method Notably, we observed that when the GFP-PI4KB kinase is co-expressed with the wild-type ACDB3 Q domain it loses its typical Golgi localization (Fig. 3E upper panel). RESULTS | |
| 77 86 wild-type protein_state Notably, we observed that when the GFP-PI4KB kinase is co-expressed with the wild-type ACDB3 Q domain it loses its typical Golgi localization (Fig. 3E upper panel). RESULTS | |
| 87 92 ACDB3 protein Notably, we observed that when the GFP-PI4KB kinase is co-expressed with the wild-type ACDB3 Q domain it loses its typical Golgi localization (Fig. 3E upper panel). RESULTS | |
| 93 101 Q domain structure_element Notably, we observed that when the GFP-PI4KB kinase is co-expressed with the wild-type ACDB3 Q domain it loses its typical Golgi localization (Fig. 3E upper panel). RESULTS | |
| 129 141 localization evidence Notably, we observed that when the GFP-PI4KB kinase is co-expressed with the wild-type ACDB3 Q domain it loses its typical Golgi localization (Fig. 3E upper panel). RESULTS | |
| 9 14 PI4KB protein However, PI4KB retains it Golgi localization when co-expressed with the non-interacting Q domain mutant (Fig. 3E lower panel). RESULTS | |
| 32 44 localization evidence However, PI4KB retains it Golgi localization when co-expressed with the non-interacting Q domain mutant (Fig. 3E lower panel). RESULTS | |
| 50 62 co-expressed experimental_method However, PI4KB retains it Golgi localization when co-expressed with the non-interacting Q domain mutant (Fig. 3E lower panel). RESULTS | |
| 72 87 non-interacting protein_state However, PI4KB retains it Golgi localization when co-expressed with the non-interacting Q domain mutant (Fig. 3E lower panel). RESULTS | |
| 88 96 Q domain structure_element However, PI4KB retains it Golgi localization when co-expressed with the non-interacting Q domain mutant (Fig. 3E lower panel). RESULTS | |
| 97 103 mutant protein_state However, PI4KB retains it Golgi localization when co-expressed with the non-interacting Q domain mutant (Fig. 3E lower panel). RESULTS | |
| 0 5 ACBD3 protein ACBD3 increases PI4KB enzymatic activity by recruiting PI4KB to close vicinity of its substrate RESULTS | |
| 16 21 PI4KB protein ACBD3 increases PI4KB enzymatic activity by recruiting PI4KB to close vicinity of its substrate RESULTS | |
| 22 40 enzymatic activity evidence ACBD3 increases PI4KB enzymatic activity by recruiting PI4KB to close vicinity of its substrate RESULTS | |
| 55 60 PI4KB protein ACBD3 increases PI4KB enzymatic activity by recruiting PI4KB to close vicinity of its substrate RESULTS | |
| 16 21 ACBD3 protein To test whether ACBD3 can stimulate PI4KB kinase enzymatic activity we performed a standard luminescent kinase assay using PI-containing micelles as the substrate. RESULTS | |
| 36 41 PI4KB protein To test whether ACBD3 can stimulate PI4KB kinase enzymatic activity we performed a standard luminescent kinase assay using PI-containing micelles as the substrate. RESULTS | |
| 42 48 kinase protein_type To test whether ACBD3 can stimulate PI4KB kinase enzymatic activity we performed a standard luminescent kinase assay using PI-containing micelles as the substrate. RESULTS | |
| 49 67 enzymatic activity evidence To test whether ACBD3 can stimulate PI4KB kinase enzymatic activity we performed a standard luminescent kinase assay using PI-containing micelles as the substrate. RESULTS | |
| 92 116 luminescent kinase assay experimental_method To test whether ACBD3 can stimulate PI4KB kinase enzymatic activity we performed a standard luminescent kinase assay using PI-containing micelles as the substrate. RESULTS | |
| 123 125 PI chemical To test whether ACBD3 can stimulate PI4KB kinase enzymatic activity we performed a standard luminescent kinase assay using PI-containing micelles as the substrate. RESULTS | |
| 29 35 kinase protein_type We observed no effect on the kinase activity of PI4KB (Fig. 4A) suggesting that ACBD3 does not directly affect the enzyme (e.g. induction of a conformation change). RESULTS | |
| 48 53 PI4KB protein We observed no effect on the kinase activity of PI4KB (Fig. 4A) suggesting that ACBD3 does not directly affect the enzyme (e.g. induction of a conformation change). RESULTS | |
| 80 85 ACBD3 protein We observed no effect on the kinase activity of PI4KB (Fig. 4A) suggesting that ACBD3 does not directly affect the enzyme (e.g. induction of a conformation change). RESULTS | |
| 17 22 ACBD3 protein However, in vivo ACBD3 is located at the Golgi membranes, whereas in this experiment, ACBD3 was located in the solution and PI is provided as micelles. RESULTS | |
| 86 91 ACBD3 protein However, in vivo ACBD3 is located at the Golgi membranes, whereas in this experiment, ACBD3 was located in the solution and PI is provided as micelles. RESULTS | |
| 124 126 PI chemical However, in vivo ACBD3 is located at the Golgi membranes, whereas in this experiment, ACBD3 was located in the solution and PI is provided as micelles. RESULTS | |
| 33 36 GUV experimental_method For this, we again turned to the GUV system with ACBD3 localized to the GUV membrane. RESULTS | |
| 49 54 ACBD3 protein For this, we again turned to the GUV system with ACBD3 localized to the GUV membrane. RESULTS | |
| 55 64 localized evidence For this, we again turned to the GUV system with ACBD3 localized to the GUV membrane. RESULTS | |
| 72 75 GUV experimental_method For this, we again turned to the GUV system with ACBD3 localized to the GUV membrane. RESULTS | |
| 4 8 GUVs experimental_method The GUVs contained 10% PI to serve as a substrate for PI4KB kinase. RESULTS | |
| 23 25 PI chemical The GUVs contained 10% PI to serve as a substrate for PI4KB kinase. RESULTS | |
| 54 59 PI4KB protein The GUVs contained 10% PI to serve as a substrate for PI4KB kinase. RESULTS | |
| 60 66 kinase protein_type The GUVs contained 10% PI to serve as a substrate for PI4KB kinase. RESULTS | |
| 26 29 CFP experimental_method The buffer also contained CFP-SidC, which binds to PI4P with nanomolar affinity. RESULTS | |
| 30 34 SidC protein The buffer also contained CFP-SidC, which binds to PI4P with nanomolar affinity. RESULTS | |
| 51 55 PI4P chemical The buffer also contained CFP-SidC, which binds to PI4P with nanomolar affinity. RESULTS | |
| 34 40 kinase protein_type This enabled visualization of the kinase reaction using a confocal microscope. RESULTS | |
| 58 77 confocal microscope experimental_method This enabled visualization of the kinase reaction using a confocal microscope. RESULTS | |
| 34 49 phosphorylation ptm We compared the efficiency of the phosphorylation reaction of the kinase alone with that of kinase recruited to the surface of the GUVs by ACBD3. RESULTS | |
| 66 72 kinase protein_type We compared the efficiency of the phosphorylation reaction of the kinase alone with that of kinase recruited to the surface of the GUVs by ACBD3. RESULTS | |
| 73 78 alone protein_state We compared the efficiency of the phosphorylation reaction of the kinase alone with that of kinase recruited to the surface of the GUVs by ACBD3. RESULTS | |
| 92 98 kinase protein_type We compared the efficiency of the phosphorylation reaction of the kinase alone with that of kinase recruited to the surface of the GUVs by ACBD3. RESULTS | |
| 131 135 GUVs experimental_method We compared the efficiency of the phosphorylation reaction of the kinase alone with that of kinase recruited to the surface of the GUVs by ACBD3. RESULTS | |
| 139 144 ACBD3 protein We compared the efficiency of the phosphorylation reaction of the kinase alone with that of kinase recruited to the surface of the GUVs by ACBD3. RESULTS | |
| 35 45 absence of protein_state Reaction was also performed in the absence of ATP as a negative control (Fig. 4B). RESULTS | |
| 46 49 ATP chemical Reaction was also performed in the absence of ATP as a negative control (Fig. 4B). RESULTS | |
| 30 35 PI4KB protein These experiments showed that PI4KB enzymatic activity increases when ACBD3 is membrane localized (Fig. 4C, SI Fig. 6). RESULTS | |
| 36 54 enzymatic activity evidence These experiments showed that PI4KB enzymatic activity increases when ACBD3 is membrane localized (Fig. 4C, SI Fig. 6). RESULTS | |
| 70 75 ACBD3 protein These experiments showed that PI4KB enzymatic activity increases when ACBD3 is membrane localized (Fig. 4C, SI Fig. 6). RESULTS | |
| 24 29 PI4KB protein Membrane recruitment of PI4KB enzyme is crucial to ensure its proper function at the Golgi and TGN. DISCUSS | |
| 58 63 PI4KB protein However, the molecular mechanism and structural basis for PI4KB interaction with the membrane is poorly understood. DISCUSS | |
| 45 50 PI4KB protein In principle, any of the binding partners of PI4KB could play a role in membrane recruitment. DISCUSS | |
| 17 22 PI4KB protein To date, several PI4KB interacting proteins have been reported, including the small GTPases Rab11 and Arf1, the Golgi resident acyl-CoA binding domain containing 3 (ACBD3) protein, neuronal calcium sensor-1 (NCS-1 also known as frequenin in yeast) and the 14-3-3 proteins. DISCUSS | |
| 78 91 small GTPases protein_type To date, several PI4KB interacting proteins have been reported, including the small GTPases Rab11 and Arf1, the Golgi resident acyl-CoA binding domain containing 3 (ACBD3) protein, neuronal calcium sensor-1 (NCS-1 also known as frequenin in yeast) and the 14-3-3 proteins. DISCUSS | |
| 92 97 Rab11 protein To date, several PI4KB interacting proteins have been reported, including the small GTPases Rab11 and Arf1, the Golgi resident acyl-CoA binding domain containing 3 (ACBD3) protein, neuronal calcium sensor-1 (NCS-1 also known as frequenin in yeast) and the 14-3-3 proteins. DISCUSS | |
| 102 106 Arf1 protein To date, several PI4KB interacting proteins have been reported, including the small GTPases Rab11 and Arf1, the Golgi resident acyl-CoA binding domain containing 3 (ACBD3) protein, neuronal calcium sensor-1 (NCS-1 also known as frequenin in yeast) and the 14-3-3 proteins. DISCUSS | |
| 127 163 acyl-CoA binding domain containing 3 protein To date, several PI4KB interacting proteins have been reported, including the small GTPases Rab11 and Arf1, the Golgi resident acyl-CoA binding domain containing 3 (ACBD3) protein, neuronal calcium sensor-1 (NCS-1 also known as frequenin in yeast) and the 14-3-3 proteins. DISCUSS | |
| 165 170 ACBD3 protein To date, several PI4KB interacting proteins have been reported, including the small GTPases Rab11 and Arf1, the Golgi resident acyl-CoA binding domain containing 3 (ACBD3) protein, neuronal calcium sensor-1 (NCS-1 also known as frequenin in yeast) and the 14-3-3 proteins. DISCUSS | |
| 181 206 neuronal calcium sensor-1 protein To date, several PI4KB interacting proteins have been reported, including the small GTPases Rab11 and Arf1, the Golgi resident acyl-CoA binding domain containing 3 (ACBD3) protein, neuronal calcium sensor-1 (NCS-1 also known as frequenin in yeast) and the 14-3-3 proteins. DISCUSS | |
| 208 213 NCS-1 protein To date, several PI4KB interacting proteins have been reported, including the small GTPases Rab11 and Arf1, the Golgi resident acyl-CoA binding domain containing 3 (ACBD3) protein, neuronal calcium sensor-1 (NCS-1 also known as frequenin in yeast) and the 14-3-3 proteins. DISCUSS | |
| 228 237 frequenin protein To date, several PI4KB interacting proteins have been reported, including the small GTPases Rab11 and Arf1, the Golgi resident acyl-CoA binding domain containing 3 (ACBD3) protein, neuronal calcium sensor-1 (NCS-1 also known as frequenin in yeast) and the 14-3-3 proteins. DISCUSS | |
| 241 246 yeast taxonomy_domain To date, several PI4KB interacting proteins have been reported, including the small GTPases Rab11 and Arf1, the Golgi resident acyl-CoA binding domain containing 3 (ACBD3) protein, neuronal calcium sensor-1 (NCS-1 also known as frequenin in yeast) and the 14-3-3 proteins. DISCUSS | |
| 256 271 14-3-3 proteins protein_type To date, several PI4KB interacting proteins have been reported, including the small GTPases Rab11 and Arf1, the Golgi resident acyl-CoA binding domain containing 3 (ACBD3) protein, neuronal calcium sensor-1 (NCS-1 also known as frequenin in yeast) and the 14-3-3 proteins. DISCUSS | |
| 4 13 monomeric oligomeric_state The monomeric G protein Rab11 binds mammalian PI4KB through the helical domain of the kinase. DISCUSS | |
| 14 23 G protein protein_type The monomeric G protein Rab11 binds mammalian PI4KB through the helical domain of the kinase. DISCUSS | |
| 24 29 Rab11 protein The monomeric G protein Rab11 binds mammalian PI4KB through the helical domain of the kinase. DISCUSS | |
| 36 45 mammalian taxonomy_domain The monomeric G protein Rab11 binds mammalian PI4KB through the helical domain of the kinase. DISCUSS | |
| 46 51 PI4KB protein The monomeric G protein Rab11 binds mammalian PI4KB through the helical domain of the kinase. DISCUSS | |
| 64 78 helical domain structure_element The monomeric G protein Rab11 binds mammalian PI4KB through the helical domain of the kinase. DISCUSS | |
| 86 92 kinase protein_type The monomeric G protein Rab11 binds mammalian PI4KB through the helical domain of the kinase. DISCUSS | |
| 9 14 Rab11 protein Although Rab11 does not appear to be required for recruitment of PI4KB to the Golgi, PI4KB is required for Golgi recruitment of Rab11. DISCUSS | |
| 65 70 PI4KB protein Although Rab11 does not appear to be required for recruitment of PI4KB to the Golgi, PI4KB is required for Golgi recruitment of Rab11. DISCUSS | |
| 85 90 PI4KB protein Although Rab11 does not appear to be required for recruitment of PI4KB to the Golgi, PI4KB is required for Golgi recruitment of Rab11. DISCUSS | |
| 128 133 Rab11 protein Although Rab11 does not appear to be required for recruitment of PI4KB to the Golgi, PI4KB is required for Golgi recruitment of Rab11. DISCUSS | |
| 0 4 Arf1 protein Arf1, the other small GTP binding protein, is known to influence the activity and localization of PI4KB, but it does not appear to interact directly with PI4KB (our unpublished data). DISCUSS | |
| 16 41 small GTP binding protein protein_type Arf1, the other small GTP binding protein, is known to influence the activity and localization of PI4KB, but it does not appear to interact directly with PI4KB (our unpublished data). DISCUSS | |
| 98 103 PI4KB protein Arf1, the other small GTP binding protein, is known to influence the activity and localization of PI4KB, but it does not appear to interact directly with PI4KB (our unpublished data). DISCUSS | |
| 154 159 PI4KB protein Arf1, the other small GTP binding protein, is known to influence the activity and localization of PI4KB, but it does not appear to interact directly with PI4KB (our unpublished data). DISCUSS | |
| 4 9 yeast taxonomy_domain The yeast homologue of NCS1 called frequenin has been shown to interact with Pik1p, the yeast orthologue of PI4KB and regulate its activity and perhaps its membrane association, but the role of NCS-1 in PI4KB recruitment in mammalian cells is unclear. DISCUSS | |
| 23 27 NCS1 protein The yeast homologue of NCS1 called frequenin has been shown to interact with Pik1p, the yeast orthologue of PI4KB and regulate its activity and perhaps its membrane association, but the role of NCS-1 in PI4KB recruitment in mammalian cells is unclear. DISCUSS | |
| 35 44 frequenin protein The yeast homologue of NCS1 called frequenin has been shown to interact with Pik1p, the yeast orthologue of PI4KB and regulate its activity and perhaps its membrane association, but the role of NCS-1 in PI4KB recruitment in mammalian cells is unclear. DISCUSS | |
| 77 82 Pik1p protein The yeast homologue of NCS1 called frequenin has been shown to interact with Pik1p, the yeast orthologue of PI4KB and regulate its activity and perhaps its membrane association, but the role of NCS-1 in PI4KB recruitment in mammalian cells is unclear. DISCUSS | |
| 88 93 yeast taxonomy_domain The yeast homologue of NCS1 called frequenin has been shown to interact with Pik1p, the yeast orthologue of PI4KB and regulate its activity and perhaps its membrane association, but the role of NCS-1 in PI4KB recruitment in mammalian cells is unclear. DISCUSS | |
| 108 113 PI4KB protein The yeast homologue of NCS1 called frequenin has been shown to interact with Pik1p, the yeast orthologue of PI4KB and regulate its activity and perhaps its membrane association, but the role of NCS-1 in PI4KB recruitment in mammalian cells is unclear. DISCUSS | |
| 194 199 NCS-1 protein The yeast homologue of NCS1 called frequenin has been shown to interact with Pik1p, the yeast orthologue of PI4KB and regulate its activity and perhaps its membrane association, but the role of NCS-1 in PI4KB recruitment in mammalian cells is unclear. DISCUSS | |
| 203 208 PI4KB protein The yeast homologue of NCS1 called frequenin has been shown to interact with Pik1p, the yeast orthologue of PI4KB and regulate its activity and perhaps its membrane association, but the role of NCS-1 in PI4KB recruitment in mammalian cells is unclear. DISCUSS | |
| 224 233 mammalian taxonomy_domain The yeast homologue of NCS1 called frequenin has been shown to interact with Pik1p, the yeast orthologue of PI4KB and regulate its activity and perhaps its membrane association, but the role of NCS-1 in PI4KB recruitment in mammalian cells is unclear. DISCUSS | |
| 0 5 NCS-1 protein NCS-1 is an N-terminally myristoylated protein that participates in exocytosis. DISCUSS | |
| 25 38 myristoylated protein_state NCS-1 is an N-terminally myristoylated protein that participates in exocytosis. DISCUSS | |
| 81 86 PI4KB protein It is expressed only in certain cell types, suggesting that if it contributes to PI4KB membrane recruitment, it does so in a tissues specific manner. DISCUSS | |
| 19 24 PI4KB protein The interaction of PI4KB with 14-3-3 proteins, promoted by phosphorylation of PI4KB by protein kinase D, influences the activity of PI4KB by stabilizing its active conformation. DISCUSS | |
| 30 45 14-3-3 proteins protein_type The interaction of PI4KB with 14-3-3 proteins, promoted by phosphorylation of PI4KB by protein kinase D, influences the activity of PI4KB by stabilizing its active conformation. DISCUSS | |
| 59 74 phosphorylation ptm The interaction of PI4KB with 14-3-3 proteins, promoted by phosphorylation of PI4KB by protein kinase D, influences the activity of PI4KB by stabilizing its active conformation. DISCUSS | |
| 78 83 PI4KB protein The interaction of PI4KB with 14-3-3 proteins, promoted by phosphorylation of PI4KB by protein kinase D, influences the activity of PI4KB by stabilizing its active conformation. DISCUSS | |
| 87 103 protein kinase D protein The interaction of PI4KB with 14-3-3 proteins, promoted by phosphorylation of PI4KB by protein kinase D, influences the activity of PI4KB by stabilizing its active conformation. DISCUSS | |
| 132 137 PI4KB protein The interaction of PI4KB with 14-3-3 proteins, promoted by phosphorylation of PI4KB by protein kinase D, influences the activity of PI4KB by stabilizing its active conformation. DISCUSS | |
| 157 163 active protein_state The interaction of PI4KB with 14-3-3 proteins, promoted by phosphorylation of PI4KB by protein kinase D, influences the activity of PI4KB by stabilizing its active conformation. DISCUSS | |
| 9 24 14-3-3 proteins protein_type However, 14-3-3 proteins do not appear to interfere with membrane recruitment of this kinase. DISCUSS | |
| 86 92 kinase protein_type However, 14-3-3 proteins do not appear to interfere with membrane recruitment of this kinase. DISCUSS | |
| 0 5 ACBD3 protein ACBD3 is a Golgi resident protein, conserved among vertebrates (SI Fig. 7), that interacts directly with PI4KB (see also SI Fig. 8 and SI Discussion), and whose genetic inactivation interferes with the Golgi localization of the kinase. DISCUSS | |
| 35 44 conserved protein_state ACBD3 is a Golgi resident protein, conserved among vertebrates (SI Fig. 7), that interacts directly with PI4KB (see also SI Fig. 8 and SI Discussion), and whose genetic inactivation interferes with the Golgi localization of the kinase. DISCUSS | |
| 51 62 vertebrates taxonomy_domain ACBD3 is a Golgi resident protein, conserved among vertebrates (SI Fig. 7), that interacts directly with PI4KB (see also SI Fig. 8 and SI Discussion), and whose genetic inactivation interferes with the Golgi localization of the kinase. DISCUSS | |
| 105 110 PI4KB protein ACBD3 is a Golgi resident protein, conserved among vertebrates (SI Fig. 7), that interacts directly with PI4KB (see also SI Fig. 8 and SI Discussion), and whose genetic inactivation interferes with the Golgi localization of the kinase. DISCUSS | |
| 228 234 kinase protein_type ACBD3 is a Golgi resident protein, conserved among vertebrates (SI Fig. 7), that interacts directly with PI4KB (see also SI Fig. 8 and SI Discussion), and whose genetic inactivation interferes with the Golgi localization of the kinase. DISCUSS | |
| 55 60 PI4KB protein For these reasons we focused on the interaction of the PI4KB enzyme with the Golgi resident ACBD3 protein in this study. DISCUSS | |
| 92 97 ACBD3 protein For these reasons we focused on the interaction of the PI4KB enzyme with the Golgi resident ACBD3 protein in this study. DISCUSS | |
| 58 63 PI4KB protein Here we present the mechanism for membrane recruitment of PI4KB by the Golgi resident ACBD3 protein. DISCUSS | |
| 86 91 ACBD3 protein Here we present the mechanism for membrane recruitment of PI4KB by the Golgi resident ACBD3 protein. DISCUSS | |
| 53 55 Kd evidence We show that these proteins interact directly with a Kd value in the submicromolar range. DISCUSS | |
| 41 46 PI4KB protein The interaction is sufficient to recruit PI4KB to model membranes in vitro as well as to the mitochondria where PI4KB is never naturally found. DISCUSS | |
| 112 117 PI4KB protein The interaction is sufficient to recruit PI4KB to model membranes in vitro as well as to the mitochondria where PI4KB is never naturally found. DISCUSS | |
| 50 56 solved experimental_method To understand this process at the atomic level we solved the solution structure of ACBD3:PI4KB sub complex (Fig. 1A) and found that the PI4KB N-terminal region contains a short amphipatic helix (residues 44–64) that binds the ACBD3 Q domain. DISCUSS | |
| 61 79 solution structure evidence To understand this process at the atomic level we solved the solution structure of ACBD3:PI4KB sub complex (Fig. 1A) and found that the PI4KB N-terminal region contains a short amphipatic helix (residues 44–64) that binds the ACBD3 Q domain. DISCUSS | |
| 83 94 ACBD3:PI4KB complex_assembly To understand this process at the atomic level we solved the solution structure of ACBD3:PI4KB sub complex (Fig. 1A) and found that the PI4KB N-terminal region contains a short amphipatic helix (residues 44–64) that binds the ACBD3 Q domain. DISCUSS | |
| 136 141 PI4KB protein To understand this process at the atomic level we solved the solution structure of ACBD3:PI4KB sub complex (Fig. 1A) and found that the PI4KB N-terminal region contains a short amphipatic helix (residues 44–64) that binds the ACBD3 Q domain. DISCUSS | |
| 142 159 N-terminal region structure_element To understand this process at the atomic level we solved the solution structure of ACBD3:PI4KB sub complex (Fig. 1A) and found that the PI4KB N-terminal region contains a short amphipatic helix (residues 44–64) that binds the ACBD3 Q domain. DISCUSS | |
| 171 193 short amphipatic helix structure_element To understand this process at the atomic level we solved the solution structure of ACBD3:PI4KB sub complex (Fig. 1A) and found that the PI4KB N-terminal region contains a short amphipatic helix (residues 44–64) that binds the ACBD3 Q domain. DISCUSS | |
| 204 209 44–64 residue_range To understand this process at the atomic level we solved the solution structure of ACBD3:PI4KB sub complex (Fig. 1A) and found that the PI4KB N-terminal region contains a short amphipatic helix (residues 44–64) that binds the ACBD3 Q domain. DISCUSS | |
| 226 231 ACBD3 protein To understand this process at the atomic level we solved the solution structure of ACBD3:PI4KB sub complex (Fig. 1A) and found that the PI4KB N-terminal region contains a short amphipatic helix (residues 44–64) that binds the ACBD3 Q domain. DISCUSS | |
| 232 240 Q domain structure_element To understand this process at the atomic level we solved the solution structure of ACBD3:PI4KB sub complex (Fig. 1A) and found that the PI4KB N-terminal region contains a short amphipatic helix (residues 44–64) that binds the ACBD3 Q domain. DISCUSS | |
| 4 12 Q domain structure_element The Q domain adopts a helical hairpin fold that is further stabilized upon binding the kinase helix (Fig. 2A). DISCUSS | |
| 22 42 helical hairpin fold structure_element The Q domain adopts a helical hairpin fold that is further stabilized upon binding the kinase helix (Fig. 2A). DISCUSS | |
| 87 99 kinase helix structure_element The Q domain adopts a helical hairpin fold that is further stabilized upon binding the kinase helix (Fig. 2A). DISCUSS | |
| 115 121 kinase protein_type Our data strongly suggest that formation of the complex does not directly influence the catalytic abilities of the kinase but experiments with model membranes revealed that ACBD3 enhances catalytic activity of the kinase by a recruitment based mechanism; it recruits the kinase to the membrane and thus increases the local concentration of the substrate in the vicinity of the kinase. DISCUSS | |
| 173 178 ACBD3 protein Our data strongly suggest that formation of the complex does not directly influence the catalytic abilities of the kinase but experiments with model membranes revealed that ACBD3 enhances catalytic activity of the kinase by a recruitment based mechanism; it recruits the kinase to the membrane and thus increases the local concentration of the substrate in the vicinity of the kinase. DISCUSS | |
| 214 220 kinase protein_type Our data strongly suggest that formation of the complex does not directly influence the catalytic abilities of the kinase but experiments with model membranes revealed that ACBD3 enhances catalytic activity of the kinase by a recruitment based mechanism; it recruits the kinase to the membrane and thus increases the local concentration of the substrate in the vicinity of the kinase. DISCUSS | |
| 271 277 kinase protein_type Our data strongly suggest that formation of the complex does not directly influence the catalytic abilities of the kinase but experiments with model membranes revealed that ACBD3 enhances catalytic activity of the kinase by a recruitment based mechanism; it recruits the kinase to the membrane and thus increases the local concentration of the substrate in the vicinity of the kinase. DISCUSS | |
| 377 383 kinase protein_type Our data strongly suggest that formation of the complex does not directly influence the catalytic abilities of the kinase but experiments with model membranes revealed that ACBD3 enhances catalytic activity of the kinase by a recruitment based mechanism; it recruits the kinase to the membrane and thus increases the local concentration of the substrate in the vicinity of the kinase. DISCUSS | |
| 38 48 structures evidence Based on our and previously published structures we built a pseudoatomic model of PI4KB multi-protein assembly on the membrane (Fig. 5) that illustrates how the enzyme is recruited and positioned towards its lipidic substrate and how it in turn recruits Rab11. DISCUSS | |
| 60 78 pseudoatomic model evidence Based on our and previously published structures we built a pseudoatomic model of PI4KB multi-protein assembly on the membrane (Fig. 5) that illustrates how the enzyme is recruited and positioned towards its lipidic substrate and how it in turn recruits Rab11. DISCUSS | |
| 82 87 PI4KB protein Based on our and previously published structures we built a pseudoatomic model of PI4KB multi-protein assembly on the membrane (Fig. 5) that illustrates how the enzyme is recruited and positioned towards its lipidic substrate and how it in turn recruits Rab11. DISCUSS | |
| 254 259 Rab11 protein Based on our and previously published structures we built a pseudoatomic model of PI4KB multi-protein assembly on the membrane (Fig. 5) that illustrates how the enzyme is recruited and positioned towards its lipidic substrate and how it in turn recruits Rab11. DISCUSS | |
| 0 12 +RNA viruses taxonomy_domain +RNA viruses replicate at specific PI4P-enriched membranous compartments. DISCUSS | |
| 35 39 PI4P chemical +RNA viruses replicate at specific PI4P-enriched membranous compartments. DISCUSS | |
| 61 66 viral taxonomy_domain These are called replication factories (because they enhance viral replication) or membranous webs (because of their appearance under the electron microscope). DISCUSS | |
| 35 42 viruses taxonomy_domain To generate replication factories, viruses hijack several host factors including the PI4K kinases to secure high content of the PI4P lipid. DISCUSS | |
| 85 89 PI4K protein_type To generate replication factories, viruses hijack several host factors including the PI4K kinases to secure high content of the PI4P lipid. DISCUSS | |
| 90 97 kinases protein_type To generate replication factories, viruses hijack several host factors including the PI4K kinases to secure high content of the PI4P lipid. DISCUSS | |
| 128 132 PI4P chemical To generate replication factories, viruses hijack several host factors including the PI4K kinases to secure high content of the PI4P lipid. DISCUSS | |
| 133 138 lipid chemical To generate replication factories, viruses hijack several host factors including the PI4K kinases to secure high content of the PI4P lipid. DISCUSS | |
| 0 26 Non-structural 3A proteins protein_type Non-structural 3A proteins from many picornaviruses from the Enterovirus (e.g. poliovirus, coxsackievirus-B3, rhinovirus-14) and Kobuvirus (e.g. Aichi virus-1) genera directly interact with ACBD3. DISCUSS | |
| 37 51 picornaviruses taxonomy_domain Non-structural 3A proteins from many picornaviruses from the Enterovirus (e.g. poliovirus, coxsackievirus-B3, rhinovirus-14) and Kobuvirus (e.g. Aichi virus-1) genera directly interact with ACBD3. DISCUSS | |
| 61 72 Enterovirus taxonomy_domain Non-structural 3A proteins from many picornaviruses from the Enterovirus (e.g. poliovirus, coxsackievirus-B3, rhinovirus-14) and Kobuvirus (e.g. Aichi virus-1) genera directly interact with ACBD3. DISCUSS | |
| 79 89 poliovirus species Non-structural 3A proteins from many picornaviruses from the Enterovirus (e.g. poliovirus, coxsackievirus-B3, rhinovirus-14) and Kobuvirus (e.g. Aichi virus-1) genera directly interact with ACBD3. DISCUSS | |
| 91 108 coxsackievirus-B3 species Non-structural 3A proteins from many picornaviruses from the Enterovirus (e.g. poliovirus, coxsackievirus-B3, rhinovirus-14) and Kobuvirus (e.g. Aichi virus-1) genera directly interact with ACBD3. DISCUSS | |
| 110 123 rhinovirus-14 species Non-structural 3A proteins from many picornaviruses from the Enterovirus (e.g. poliovirus, coxsackievirus-B3, rhinovirus-14) and Kobuvirus (e.g. Aichi virus-1) genera directly interact with ACBD3. DISCUSS | |
| 129 138 Kobuvirus taxonomy_domain Non-structural 3A proteins from many picornaviruses from the Enterovirus (e.g. poliovirus, coxsackievirus-B3, rhinovirus-14) and Kobuvirus (e.g. Aichi virus-1) genera directly interact with ACBD3. DISCUSS | |
| 145 158 Aichi virus-1 species Non-structural 3A proteins from many picornaviruses from the Enterovirus (e.g. poliovirus, coxsackievirus-B3, rhinovirus-14) and Kobuvirus (e.g. Aichi virus-1) genera directly interact with ACBD3. DISCUSS | |
| 190 195 ACBD3 protein Non-structural 3A proteins from many picornaviruses from the Enterovirus (e.g. poliovirus, coxsackievirus-B3, rhinovirus-14) and Kobuvirus (e.g. Aichi virus-1) genera directly interact with ACBD3. DISCUSS | |
| 45 59 3A:ACBD3:PI4KB complex_assembly Our data suggest that they could do this via 3A:ACBD3:PI4KB complex formation. DISCUSS | |
| 4 13 structure evidence The structure of the ACBD3 Q domain and the kinase helix described here provides a novel opportunity for further research on the role of ACBD3, PI4KB, and the ACBD3:PI4KB interaction in picornaviral replication. DISCUSS | |
| 21 26 ACBD3 protein The structure of the ACBD3 Q domain and the kinase helix described here provides a novel opportunity for further research on the role of ACBD3, PI4KB, and the ACBD3:PI4KB interaction in picornaviral replication. DISCUSS | |
| 27 35 Q domain structure_element The structure of the ACBD3 Q domain and the kinase helix described here provides a novel opportunity for further research on the role of ACBD3, PI4KB, and the ACBD3:PI4KB interaction in picornaviral replication. DISCUSS | |
| 44 56 kinase helix structure_element The structure of the ACBD3 Q domain and the kinase helix described here provides a novel opportunity for further research on the role of ACBD3, PI4KB, and the ACBD3:PI4KB interaction in picornaviral replication. DISCUSS | |
| 137 142 ACBD3 protein The structure of the ACBD3 Q domain and the kinase helix described here provides a novel opportunity for further research on the role of ACBD3, PI4KB, and the ACBD3:PI4KB interaction in picornaviral replication. DISCUSS | |
| 144 149 PI4KB protein The structure of the ACBD3 Q domain and the kinase helix described here provides a novel opportunity for further research on the role of ACBD3, PI4KB, and the ACBD3:PI4KB interaction in picornaviral replication. DISCUSS | |
| 159 170 ACBD3:PI4KB complex_assembly The structure of the ACBD3 Q domain and the kinase helix described here provides a novel opportunity for further research on the role of ACBD3, PI4KB, and the ACBD3:PI4KB interaction in picornaviral replication. DISCUSS | |
| 186 198 picornaviral taxonomy_domain The structure of the ACBD3 Q domain and the kinase helix described here provides a novel opportunity for further research on the role of ACBD3, PI4KB, and the ACBD3:PI4KB interaction in picornaviral replication. DISCUSS | |
| 79 93 picornaviruses taxonomy_domain This could eventually have implications for therapeutic intervention to combat picornaviruses-mediated diseases ranging from polio to the common cold. DISCUSS | |
| 0 28 Biochemical characterization experimental_method Biochemical characterization of the ACBD3:PI4KB complex. FIG | |
| 36 47 ACBD3:PI4KB complex_assembly Biochemical characterization of the ACBD3:PI4KB complex. FIG | |
| 36 41 ACBD3 protein (A) Schematic representation of the ACBD3 and PI4KB constructs used for the experiments. FIG | |
| 46 51 PI4KB protein (A) Schematic representation of the ACBD3 and PI4KB constructs used for the experiments. FIG | |
| 0 5 ACBD3 protein ACBD3 contains the acyl-CoA binding domain (ACBD), charged amino acids region (CAR), glutamine rich region (Q), and Golgi dynamics domain (GOLD). FIG | |
| 19 42 acyl-CoA binding domain structure_element ACBD3 contains the acyl-CoA binding domain (ACBD), charged amino acids region (CAR), glutamine rich region (Q), and Golgi dynamics domain (GOLD). FIG | |
| 44 48 ACBD structure_element ACBD3 contains the acyl-CoA binding domain (ACBD), charged amino acids region (CAR), glutamine rich region (Q), and Golgi dynamics domain (GOLD). FIG | |
| 51 77 charged amino acids region structure_element ACBD3 contains the acyl-CoA binding domain (ACBD), charged amino acids region (CAR), glutamine rich region (Q), and Golgi dynamics domain (GOLD). FIG | |
| 79 82 CAR structure_element ACBD3 contains the acyl-CoA binding domain (ACBD), charged amino acids region (CAR), glutamine rich region (Q), and Golgi dynamics domain (GOLD). FIG | |
| 85 106 glutamine rich region structure_element ACBD3 contains the acyl-CoA binding domain (ACBD), charged amino acids region (CAR), glutamine rich region (Q), and Golgi dynamics domain (GOLD). FIG | |
| 108 109 Q structure_element ACBD3 contains the acyl-CoA binding domain (ACBD), charged amino acids region (CAR), glutamine rich region (Q), and Golgi dynamics domain (GOLD). FIG | |
| 116 137 Golgi dynamics domain structure_element ACBD3 contains the acyl-CoA binding domain (ACBD), charged amino acids region (CAR), glutamine rich region (Q), and Golgi dynamics domain (GOLD). FIG | |
| 139 143 GOLD structure_element ACBD3 contains the acyl-CoA binding domain (ACBD), charged amino acids region (CAR), glutamine rich region (Q), and Golgi dynamics domain (GOLD). FIG | |
| 0 5 PI4KB protein PI4KB is composed of the N-terminal region, helical domain, and kinase domain which can be divided into N- and C-terminal lobes. FIG | |
| 25 42 N-terminal region structure_element PI4KB is composed of the N-terminal region, helical domain, and kinase domain which can be divided into N- and C-terminal lobes. FIG | |
| 44 58 helical domain structure_element PI4KB is composed of the N-terminal region, helical domain, and kinase domain which can be divided into N- and C-terminal lobes. FIG | |
| 64 77 kinase domain structure_element PI4KB is composed of the N-terminal region, helical domain, and kinase domain which can be divided into N- and C-terminal lobes. FIG | |
| 104 127 N- and C-terminal lobes structure_element PI4KB is composed of the N-terminal region, helical domain, and kinase domain which can be divided into N- and C-terminal lobes. FIG | |
| 4 28 In vitro pull-down assay experimental_method (B) In vitro pull-down assay. FIG | |
| 0 16 Pull-down assays experimental_method Pull-down assays were performed using NiNTA-immobilized N-terminal His6GB1-tagged proteins as indicated and untagged full-length PI4KB or ACBD3. FIG | |
| 67 81 His6GB1-tagged protein_state Pull-down assays were performed using NiNTA-immobilized N-terminal His6GB1-tagged proteins as indicated and untagged full-length PI4KB or ACBD3. FIG | |
| 108 116 untagged protein_state Pull-down assays were performed using NiNTA-immobilized N-terminal His6GB1-tagged proteins as indicated and untagged full-length PI4KB or ACBD3. FIG | |
| 117 128 full-length protein_state Pull-down assays were performed using NiNTA-immobilized N-terminal His6GB1-tagged proteins as indicated and untagged full-length PI4KB or ACBD3. FIG | |
| 129 134 PI4KB protein Pull-down assays were performed using NiNTA-immobilized N-terminal His6GB1-tagged proteins as indicated and untagged full-length PI4KB or ACBD3. FIG | |
| 138 143 ACBD3 protein Pull-down assays were performed using NiNTA-immobilized N-terminal His6GB1-tagged proteins as indicated and untagged full-length PI4KB or ACBD3. FIG | |
| 47 55 SDS gels experimental_method The inputs and bound proteins were analyzed on SDS gels stained with Coomassie Blue. FIG | |
| 35 46 full-length protein_state Please, see SI Fig. 9 for original full-length gels. (C) Analytical Ultracentrifugation. FIG | |
| 57 87 Analytical Ultracentrifugation experimental_method Please, see SI Fig. 9 for original full-length gels. (C) Analytical Ultracentrifugation. FIG | |
| 0 3 AUC experimental_method AUC analysis of the ACBD3:PI4KB full-length complex at the concentration of 5 μM (both proteins, left panel) and ACBD3 Q domain: PI4KB N terminal region complex at the concentration of 35 μM (both proteins, right panel). (D) Surface plasmon resonance. FIG | |
| 20 31 ACBD3:PI4KB complex_assembly AUC analysis of the ACBD3:PI4KB full-length complex at the concentration of 5 μM (both proteins, left panel) and ACBD3 Q domain: PI4KB N terminal region complex at the concentration of 35 μM (both proteins, right panel). (D) Surface plasmon resonance. FIG | |
| 32 43 full-length protein_state AUC analysis of the ACBD3:PI4KB full-length complex at the concentration of 5 μM (both proteins, left panel) and ACBD3 Q domain: PI4KB N terminal region complex at the concentration of 35 μM (both proteins, right panel). (D) Surface plasmon resonance. FIG | |
| 113 152 ACBD3 Q domain: PI4KB N terminal region complex_assembly AUC analysis of the ACBD3:PI4KB full-length complex at the concentration of 5 μM (both proteins, left panel) and ACBD3 Q domain: PI4KB N terminal region complex at the concentration of 35 μM (both proteins, right panel). (D) Surface plasmon resonance. FIG | |
| 225 250 Surface plasmon resonance experimental_method AUC analysis of the ACBD3:PI4KB full-length complex at the concentration of 5 μM (both proteins, left panel) and ACBD3 Q domain: PI4KB N terminal region complex at the concentration of 35 μM (both proteins, right panel). (D) Surface plasmon resonance. FIG | |
| 0 3 SPR experimental_method SPR analysis of the PI4KB binding to immobilized ACBD3. FIG | |
| 20 25 PI4KB protein SPR analysis of the PI4KB binding to immobilized ACBD3. FIG | |
| 49 54 ACBD3 protein SPR analysis of the PI4KB binding to immobilized ACBD3. FIG | |
| 0 11 Sensorgrams evidence Sensorgrams for four concentrations of PI4KB are shown. FIG | |
| 39 44 PI4KB protein Sensorgrams for four concentrations of PI4KB are shown. FIG | |
| 0 19 Structural analysis experimental_method Structural analysis of the ACBD3:PI4KB complex. FIG | |
| 27 38 ACBD3:PI4KB complex_assembly Structural analysis of the ACBD3:PI4KB complex. FIG | |
| 12 21 structure evidence (A) Overall structure of the ACBD3 Q domain by itself and in complex with the PI4KB N-terminal region. FIG | |
| 29 34 ACBD3 protein (A) Overall structure of the ACBD3 Q domain by itself and in complex with the PI4KB N-terminal region. FIG | |
| 35 43 Q domain structure_element (A) Overall structure of the ACBD3 Q domain by itself and in complex with the PI4KB N-terminal region. FIG | |
| 58 73 in complex with protein_state (A) Overall structure of the ACBD3 Q domain by itself and in complex with the PI4KB N-terminal region. FIG | |
| 78 83 PI4KB protein (A) Overall structure of the ACBD3 Q domain by itself and in complex with the PI4KB N-terminal region. FIG | |
| 84 101 N-terminal region structure_element (A) Overall structure of the ACBD3 Q domain by itself and in complex with the PI4KB N-terminal region. FIG | |
| 0 13 Superposition experimental_method Superposition of the 30 converged structures obtained for the Q domain (top) and the 45 converged structures obtained for the complex (bottom), with only the folded part of PI4KB shown (see SI Fig. 2 for the complete view). (B) Detailed view of the complex. FIG | |
| 34 44 structures evidence Superposition of the 30 converged structures obtained for the Q domain (top) and the 45 converged structures obtained for the complex (bottom), with only the folded part of PI4KB shown (see SI Fig. 2 for the complete view). (B) Detailed view of the complex. FIG | |
| 62 70 Q domain structure_element Superposition of the 30 converged structures obtained for the Q domain (top) and the 45 converged structures obtained for the complex (bottom), with only the folded part of PI4KB shown (see SI Fig. 2 for the complete view). (B) Detailed view of the complex. FIG | |
| 98 108 structures evidence Superposition of the 30 converged structures obtained for the Q domain (top) and the 45 converged structures obtained for the complex (bottom), with only the folded part of PI4KB shown (see SI Fig. 2 for the complete view). (B) Detailed view of the complex. FIG | |
| 158 164 folded protein_state Superposition of the 30 converged structures obtained for the Q domain (top) and the 45 converged structures obtained for the complex (bottom), with only the folded part of PI4KB shown (see SI Fig. 2 for the complete view). (B) Detailed view of the complex. FIG | |
| 173 178 PI4KB protein Superposition of the 30 converged structures obtained for the Q domain (top) and the 45 converged structures obtained for the complex (bottom), with only the folded part of PI4KB shown (see SI Fig. 2 for the complete view). (B) Detailed view of the complex. FIG | |
| 43 57 hydrogen bonds bond_interaction The interaction is facilitated by only two hydrogen bonds (ACBD3 Tyr261: PI4KB His63 and ACBD3 Tyr288: PI4KB Asp44), while the hydrophobic surface of the kinase helix nests in the ACBD3 Q domain. FIG | |
| 59 64 ACBD3 protein The interaction is facilitated by only two hydrogen bonds (ACBD3 Tyr261: PI4KB His63 and ACBD3 Tyr288: PI4KB Asp44), while the hydrophobic surface of the kinase helix nests in the ACBD3 Q domain. FIG | |
| 65 71 Tyr261 residue_name_number The interaction is facilitated by only two hydrogen bonds (ACBD3 Tyr261: PI4KB His63 and ACBD3 Tyr288: PI4KB Asp44), while the hydrophobic surface of the kinase helix nests in the ACBD3 Q domain. FIG | |
| 73 78 PI4KB protein The interaction is facilitated by only two hydrogen bonds (ACBD3 Tyr261: PI4KB His63 and ACBD3 Tyr288: PI4KB Asp44), while the hydrophobic surface of the kinase helix nests in the ACBD3 Q domain. FIG | |
| 79 84 His63 residue_name_number The interaction is facilitated by only two hydrogen bonds (ACBD3 Tyr261: PI4KB His63 and ACBD3 Tyr288: PI4KB Asp44), while the hydrophobic surface of the kinase helix nests in the ACBD3 Q domain. FIG | |
| 89 94 ACBD3 protein The interaction is facilitated by only two hydrogen bonds (ACBD3 Tyr261: PI4KB His63 and ACBD3 Tyr288: PI4KB Asp44), while the hydrophobic surface of the kinase helix nests in the ACBD3 Q domain. FIG | |
| 95 101 Tyr288 residue_name_number The interaction is facilitated by only two hydrogen bonds (ACBD3 Tyr261: PI4KB His63 and ACBD3 Tyr288: PI4KB Asp44), while the hydrophobic surface of the kinase helix nests in the ACBD3 Q domain. FIG | |
| 103 108 PI4KB protein The interaction is facilitated by only two hydrogen bonds (ACBD3 Tyr261: PI4KB His63 and ACBD3 Tyr288: PI4KB Asp44), while the hydrophobic surface of the kinase helix nests in the ACBD3 Q domain. FIG | |
| 109 114 Asp44 residue_name_number The interaction is facilitated by only two hydrogen bonds (ACBD3 Tyr261: PI4KB His63 and ACBD3 Tyr288: PI4KB Asp44), while the hydrophobic surface of the kinase helix nests in the ACBD3 Q domain. FIG | |
| 127 146 hydrophobic surface site The interaction is facilitated by only two hydrogen bonds (ACBD3 Tyr261: PI4KB His63 and ACBD3 Tyr288: PI4KB Asp44), while the hydrophobic surface of the kinase helix nests in the ACBD3 Q domain. FIG | |
| 154 166 kinase helix structure_element The interaction is facilitated by only two hydrogen bonds (ACBD3 Tyr261: PI4KB His63 and ACBD3 Tyr288: PI4KB Asp44), while the hydrophobic surface of the kinase helix nests in the ACBD3 Q domain. FIG | |
| 180 185 ACBD3 protein The interaction is facilitated by only two hydrogen bonds (ACBD3 Tyr261: PI4KB His63 and ACBD3 Tyr288: PI4KB Asp44), while the hydrophobic surface of the kinase helix nests in the ACBD3 Q domain. FIG | |
| 186 194 Q domain structure_element The interaction is facilitated by only two hydrogen bonds (ACBD3 Tyr261: PI4KB His63 and ACBD3 Tyr288: PI4KB Asp44), while the hydrophobic surface of the kinase helix nests in the ACBD3 Q domain. FIG | |
| 0 5 ACBD3 protein ACBD3 is shown in magenta and PI4KB in orange. FIG | |
| 30 35 PI4KB protein ACBD3 is shown in magenta and PI4KB in orange. FIG | |
| 20 32 kinase helix structure_element (C) Top view of the kinase helix. FIG | |
| 4 16 kinase helix structure_element The kinase helix is amphipathic and its hydrophobic surface overlaps with the ACBD3 binding surface (shown in magenta). FIG | |
| 20 31 amphipathic protein_state The kinase helix is amphipathic and its hydrophobic surface overlaps with the ACBD3 binding surface (shown in magenta). FIG | |
| 40 59 hydrophobic surface site The kinase helix is amphipathic and its hydrophobic surface overlaps with the ACBD3 binding surface (shown in magenta). FIG | |
| 78 83 ACBD3 protein The kinase helix is amphipathic and its hydrophobic surface overlaps with the ACBD3 binding surface (shown in magenta). FIG | |
| 84 99 binding surface site The kinase helix is amphipathic and its hydrophobic surface overlaps with the ACBD3 binding surface (shown in magenta). FIG | |
| 160 175 Pull-down assay experimental_method Strong and weak hydrophobes are in green and cyan respectively, basic residues in blue, acidic residues in red and nonpolar hydrophilic residues in orange. (D) Pull-down assay with a NiNTA-immobilized N-terminally His6GB1-tagged PI4KB kinase and untagged ACBD3 protein. FIG | |
| 214 228 His6GB1-tagged protein_state Strong and weak hydrophobes are in green and cyan respectively, basic residues in blue, acidic residues in red and nonpolar hydrophilic residues in orange. (D) Pull-down assay with a NiNTA-immobilized N-terminally His6GB1-tagged PI4KB kinase and untagged ACBD3 protein. FIG | |
| 229 234 PI4KB protein Strong and weak hydrophobes are in green and cyan respectively, basic residues in blue, acidic residues in red and nonpolar hydrophilic residues in orange. (D) Pull-down assay with a NiNTA-immobilized N-terminally His6GB1-tagged PI4KB kinase and untagged ACBD3 protein. FIG | |
| 235 241 kinase protein_type Strong and weak hydrophobes are in green and cyan respectively, basic residues in blue, acidic residues in red and nonpolar hydrophilic residues in orange. (D) Pull-down assay with a NiNTA-immobilized N-terminally His6GB1-tagged PI4KB kinase and untagged ACBD3 protein. FIG | |
| 246 254 untagged protein_state Strong and weak hydrophobes are in green and cyan respectively, basic residues in blue, acidic residues in red and nonpolar hydrophilic residues in orange. (D) Pull-down assay with a NiNTA-immobilized N-terminally His6GB1-tagged PI4KB kinase and untagged ACBD3 protein. FIG | |
| 255 260 ACBD3 protein Strong and weak hydrophobes are in green and cyan respectively, basic residues in blue, acidic residues in red and nonpolar hydrophilic residues in orange. (D) Pull-down assay with a NiNTA-immobilized N-terminally His6GB1-tagged PI4KB kinase and untagged ACBD3 protein. FIG | |
| 0 9 Wild type protein_state Wild type proteins and selected point mutants of both PI4KB and ACBD3 were used. FIG | |
| 38 45 mutants protein_state Wild type proteins and selected point mutants of both PI4KB and ACBD3 were used. FIG | |
| 54 59 PI4KB protein Wild type proteins and selected point mutants of both PI4KB and ACBD3 were used. FIG | |
| 64 69 ACBD3 protein Wild type proteins and selected point mutants of both PI4KB and ACBD3 were used. FIG | |
| 35 46 full-length protein_state Please, see SI Fig. 9 for original full-length gels. FIG | |
| 0 5 ACBD3 protein ACBD3 is sufficient to recruit the PI4KB kinase to membranes. FIG | |
| 35 40 PI4KB protein ACBD3 is sufficient to recruit the PI4KB kinase to membranes. FIG | |
| 41 47 kinase protein_type ACBD3 is sufficient to recruit the PI4KB kinase to membranes. FIG | |
| 4 26 GUVs recruitment assay experimental_method (A) GUVs recruitment assay. FIG | |
| 34 40 kinase protein_type Top – Virtually no membrane bound kinase was observed when 600 nM PI4KB was added to the GUVs. FIG | |
| 66 71 PI4KB protein Top – Virtually no membrane bound kinase was observed when 600 nM PI4KB was added to the GUVs. FIG | |
| 89 93 GUVs experimental_method Top – Virtually no membrane bound kinase was observed when 600 nM PI4KB was added to the GUVs. FIG | |
| 16 27 presence of protein_state Bottom – in the presence of 600 nM GUV tethered ACBD3 a significant signal of the kinase is detected on the surface of GUVs. FIG | |
| 35 47 GUV tethered protein_state Bottom – in the presence of 600 nM GUV tethered ACBD3 a significant signal of the kinase is detected on the surface of GUVs. FIG | |
| 48 53 ACBD3 protein Bottom – in the presence of 600 nM GUV tethered ACBD3 a significant signal of the kinase is detected on the surface of GUVs. FIG | |
| 82 88 kinase protein_type Bottom – in the presence of 600 nM GUV tethered ACBD3 a significant signal of the kinase is detected on the surface of GUVs. FIG | |
| 119 123 GUVs experimental_method Bottom – in the presence of 600 nM GUV tethered ACBD3 a significant signal of the kinase is detected on the surface of GUVs. FIG | |
| 4 33 Golgi displacement experiment experimental_method (B) Golgi displacement experiment. FIG | |
| 13 18 ACBD3 protein Upper panel: ACBD3 Q domain fused to GFP was overexpressed and the endogenous PI4KB was immunostained. FIG | |
| 19 27 Q domain structure_element Upper panel: ACBD3 Q domain fused to GFP was overexpressed and the endogenous PI4KB was immunostained. FIG | |
| 37 40 GFP experimental_method Upper panel: ACBD3 Q domain fused to GFP was overexpressed and the endogenous PI4KB was immunostained. FIG | |
| 45 58 overexpressed experimental_method Upper panel: ACBD3 Q domain fused to GFP was overexpressed and the endogenous PI4KB was immunostained. FIG | |
| 78 83 PI4KB protein Upper panel: ACBD3 Q domain fused to GFP was overexpressed and the endogenous PI4KB was immunostained. FIG | |
| 88 101 immunostained experimental_method Upper panel: ACBD3 Q domain fused to GFP was overexpressed and the endogenous PI4KB was immunostained. FIG | |
| 49 52 GFP experimental_method Middle panel: The same experiment performed with GFP alone. FIG | |
| 48 54 mutant protein_state Lower panel: The same experiment performed with mutant Q domain (F258A, H284A, Y288A) that does not bind the PI4KB. (C) ACBD3 Q domain overexpression inhibits ceramide transport to Golgi – COS-7 cells transfected with wild-type ACBD3 Q domain-FKBP-mRFP were loaded with 0.05 μM Bodipy FL-Ceramide for 20 min, then washed and depicted after 20 min. FIG | |
| 55 63 Q domain structure_element Lower panel: The same experiment performed with mutant Q domain (F258A, H284A, Y288A) that does not bind the PI4KB. (C) ACBD3 Q domain overexpression inhibits ceramide transport to Golgi – COS-7 cells transfected with wild-type ACBD3 Q domain-FKBP-mRFP were loaded with 0.05 μM Bodipy FL-Ceramide for 20 min, then washed and depicted after 20 min. FIG | |
| 65 70 F258A mutant Lower panel: The same experiment performed with mutant Q domain (F258A, H284A, Y288A) that does not bind the PI4KB. (C) ACBD3 Q domain overexpression inhibits ceramide transport to Golgi – COS-7 cells transfected with wild-type ACBD3 Q domain-FKBP-mRFP were loaded with 0.05 μM Bodipy FL-Ceramide for 20 min, then washed and depicted after 20 min. FIG | |
| 72 77 H284A mutant Lower panel: The same experiment performed with mutant Q domain (F258A, H284A, Y288A) that does not bind the PI4KB. (C) ACBD3 Q domain overexpression inhibits ceramide transport to Golgi – COS-7 cells transfected with wild-type ACBD3 Q domain-FKBP-mRFP were loaded with 0.05 μM Bodipy FL-Ceramide for 20 min, then washed and depicted after 20 min. FIG | |
| 79 84 Y288A mutant Lower panel: The same experiment performed with mutant Q domain (F258A, H284A, Y288A) that does not bind the PI4KB. (C) ACBD3 Q domain overexpression inhibits ceramide transport to Golgi – COS-7 cells transfected with wild-type ACBD3 Q domain-FKBP-mRFP were loaded with 0.05 μM Bodipy FL-Ceramide for 20 min, then washed and depicted after 20 min. FIG | |
| 109 114 PI4KB protein Lower panel: The same experiment performed with mutant Q domain (F258A, H284A, Y288A) that does not bind the PI4KB. (C) ACBD3 Q domain overexpression inhibits ceramide transport to Golgi – COS-7 cells transfected with wild-type ACBD3 Q domain-FKBP-mRFP were loaded with 0.05 μM Bodipy FL-Ceramide for 20 min, then washed and depicted after 20 min. FIG | |
| 120 125 ACBD3 protein Lower panel: The same experiment performed with mutant Q domain (F258A, H284A, Y288A) that does not bind the PI4KB. (C) ACBD3 Q domain overexpression inhibits ceramide transport to Golgi – COS-7 cells transfected with wild-type ACBD3 Q domain-FKBP-mRFP were loaded with 0.05 μM Bodipy FL-Ceramide for 20 min, then washed and depicted after 20 min. FIG | |
| 126 134 Q domain structure_element Lower panel: The same experiment performed with mutant Q domain (F258A, H284A, Y288A) that does not bind the PI4KB. (C) ACBD3 Q domain overexpression inhibits ceramide transport to Golgi – COS-7 cells transfected with wild-type ACBD3 Q domain-FKBP-mRFP were loaded with 0.05 μM Bodipy FL-Ceramide for 20 min, then washed and depicted after 20 min. FIG | |
| 135 149 overexpression experimental_method Lower panel: The same experiment performed with mutant Q domain (F258A, H284A, Y288A) that does not bind the PI4KB. (C) ACBD3 Q domain overexpression inhibits ceramide transport to Golgi – COS-7 cells transfected with wild-type ACBD3 Q domain-FKBP-mRFP were loaded with 0.05 μM Bodipy FL-Ceramide for 20 min, then washed and depicted after 20 min. FIG | |
| 159 167 ceramide chemical Lower panel: The same experiment performed with mutant Q domain (F258A, H284A, Y288A) that does not bind the PI4KB. (C) ACBD3 Q domain overexpression inhibits ceramide transport to Golgi – COS-7 cells transfected with wild-type ACBD3 Q domain-FKBP-mRFP were loaded with 0.05 μM Bodipy FL-Ceramide for 20 min, then washed and depicted after 20 min. FIG | |
| 218 227 wild-type protein_state Lower panel: The same experiment performed with mutant Q domain (F258A, H284A, Y288A) that does not bind the PI4KB. (C) ACBD3 Q domain overexpression inhibits ceramide transport to Golgi – COS-7 cells transfected with wild-type ACBD3 Q domain-FKBP-mRFP were loaded with 0.05 μM Bodipy FL-Ceramide for 20 min, then washed and depicted after 20 min. FIG | |
| 228 233 ACBD3 protein Lower panel: The same experiment performed with mutant Q domain (F258A, H284A, Y288A) that does not bind the PI4KB. (C) ACBD3 Q domain overexpression inhibits ceramide transport to Golgi – COS-7 cells transfected with wild-type ACBD3 Q domain-FKBP-mRFP were loaded with 0.05 μM Bodipy FL-Ceramide for 20 min, then washed and depicted after 20 min. FIG | |
| 234 242 Q domain structure_element Lower panel: The same experiment performed with mutant Q domain (F258A, H284A, Y288A) that does not bind the PI4KB. (C) ACBD3 Q domain overexpression inhibits ceramide transport to Golgi – COS-7 cells transfected with wild-type ACBD3 Q domain-FKBP-mRFP were loaded with 0.05 μM Bodipy FL-Ceramide for 20 min, then washed and depicted after 20 min. FIG | |
| 243 247 FKBP protein Lower panel: The same experiment performed with mutant Q domain (F258A, H284A, Y288A) that does not bind the PI4KB. (C) ACBD3 Q domain overexpression inhibits ceramide transport to Golgi – COS-7 cells transfected with wild-type ACBD3 Q domain-FKBP-mRFP were loaded with 0.05 μM Bodipy FL-Ceramide for 20 min, then washed and depicted after 20 min. FIG | |
| 248 252 mRFP experimental_method Lower panel: The same experiment performed with mutant Q domain (F258A, H284A, Y288A) that does not bind the PI4KB. (C) ACBD3 Q domain overexpression inhibits ceramide transport to Golgi – COS-7 cells transfected with wild-type ACBD3 Q domain-FKBP-mRFP were loaded with 0.05 μM Bodipy FL-Ceramide for 20 min, then washed and depicted after 20 min. FIG | |
| 278 296 Bodipy FL-Ceramide chemical Lower panel: The same experiment performed with mutant Q domain (F258A, H284A, Y288A) that does not bind the PI4KB. (C) ACBD3 Q domain overexpression inhibits ceramide transport to Golgi – COS-7 cells transfected with wild-type ACBD3 Q domain-FKBP-mRFP were loaded with 0.05 μM Bodipy FL-Ceramide for 20 min, then washed and depicted after 20 min. FIG | |
| 50 54 mRFP experimental_method Middle panel – The same experiment performed with mRFP-FKBP alone. FIG | |
| 55 59 FKBP protein Middle panel – The same experiment performed with mRFP-FKBP alone. FIG | |
| 49 55 mutant protein_state Lower panel – The same experiment performed with mutant Q domain (F258A, H284A, Y288A) that does not bind the PI4KB. (D) Scheme of the mitochondria recruitment experiment. FIG | |
| 56 64 Q domain structure_element Lower panel – The same experiment performed with mutant Q domain (F258A, H284A, Y288A) that does not bind the PI4KB. (D) Scheme of the mitochondria recruitment experiment. FIG | |
| 66 71 F258A mutant Lower panel – The same experiment performed with mutant Q domain (F258A, H284A, Y288A) that does not bind the PI4KB. (D) Scheme of the mitochondria recruitment experiment. FIG | |
| 73 78 H284A mutant Lower panel – The same experiment performed with mutant Q domain (F258A, H284A, Y288A) that does not bind the PI4KB. (D) Scheme of the mitochondria recruitment experiment. FIG | |
| 80 85 Y288A mutant Lower panel – The same experiment performed with mutant Q domain (F258A, H284A, Y288A) that does not bind the PI4KB. (D) Scheme of the mitochondria recruitment experiment. FIG | |
| 110 115 PI4KB protein Lower panel – The same experiment performed with mutant Q domain (F258A, H284A, Y288A) that does not bind the PI4KB. (D) Scheme of the mitochondria recruitment experiment. FIG | |
| 135 170 mitochondria recruitment experiment experimental_method Lower panel – The same experiment performed with mutant Q domain (F258A, H284A, Y288A) that does not bind the PI4KB. (D) Scheme of the mitochondria recruitment experiment. FIG | |
| 6 11 AKAP1 protein – The AKAP1-FRB-CFP construct is localized at the outer mitochondrial membrane, while the GFP-PI4KB and Q domain-FKBP-mRFP constructs are localized in the cytoplasm where they can form a complex. FIG | |
| 12 15 FRB structure_element – The AKAP1-FRB-CFP construct is localized at the outer mitochondrial membrane, while the GFP-PI4KB and Q domain-FKBP-mRFP constructs are localized in the cytoplasm where they can form a complex. FIG | |
| 16 19 CFP experimental_method – The AKAP1-FRB-CFP construct is localized at the outer mitochondrial membrane, while the GFP-PI4KB and Q domain-FKBP-mRFP constructs are localized in the cytoplasm where they can form a complex. FIG | |
| 33 42 localized evidence – The AKAP1-FRB-CFP construct is localized at the outer mitochondrial membrane, while the GFP-PI4KB and Q domain-FKBP-mRFP constructs are localized in the cytoplasm where they can form a complex. FIG | |
| 90 93 GFP experimental_method – The AKAP1-FRB-CFP construct is localized at the outer mitochondrial membrane, while the GFP-PI4KB and Q domain-FKBP-mRFP constructs are localized in the cytoplasm where they can form a complex. FIG | |
| 94 99 PI4KB protein – The AKAP1-FRB-CFP construct is localized at the outer mitochondrial membrane, while the GFP-PI4KB and Q domain-FKBP-mRFP constructs are localized in the cytoplasm where they can form a complex. FIG | |
| 104 112 Q domain structure_element – The AKAP1-FRB-CFP construct is localized at the outer mitochondrial membrane, while the GFP-PI4KB and Q domain-FKBP-mRFP constructs are localized in the cytoplasm where they can form a complex. FIG | |
| 113 117 FKBP protein – The AKAP1-FRB-CFP construct is localized at the outer mitochondrial membrane, while the GFP-PI4KB and Q domain-FKBP-mRFP constructs are localized in the cytoplasm where they can form a complex. FIG | |
| 118 122 mRFP experimental_method – The AKAP1-FRB-CFP construct is localized at the outer mitochondrial membrane, while the GFP-PI4KB and Q domain-FKBP-mRFP constructs are localized in the cytoplasm where they can form a complex. FIG | |
| 138 147 localized evidence – The AKAP1-FRB-CFP construct is localized at the outer mitochondrial membrane, while the GFP-PI4KB and Q domain-FKBP-mRFP constructs are localized in the cytoplasm where they can form a complex. FIG | |
| 17 26 rapamycin chemical Upon addition of rapamycin the Q domain-FKBP-mRFP construct translocates to the mitochondria and takes GFP-PI4KB with it. (E) Mitochondria recruitment experiment. FIG | |
| 31 39 Q domain structure_element Upon addition of rapamycin the Q domain-FKBP-mRFP construct translocates to the mitochondria and takes GFP-PI4KB with it. (E) Mitochondria recruitment experiment. FIG | |
| 40 44 FKBP protein Upon addition of rapamycin the Q domain-FKBP-mRFP construct translocates to the mitochondria and takes GFP-PI4KB with it. (E) Mitochondria recruitment experiment. FIG | |
| 45 49 mRFP experimental_method Upon addition of rapamycin the Q domain-FKBP-mRFP construct translocates to the mitochondria and takes GFP-PI4KB with it. (E) Mitochondria recruitment experiment. FIG | |
| 103 106 GFP experimental_method Upon addition of rapamycin the Q domain-FKBP-mRFP construct translocates to the mitochondria and takes GFP-PI4KB with it. (E) Mitochondria recruitment experiment. FIG | |
| 107 112 PI4KB protein Upon addition of rapamycin the Q domain-FKBP-mRFP construct translocates to the mitochondria and takes GFP-PI4KB with it. (E) Mitochondria recruitment experiment. FIG | |
| 126 161 Mitochondria recruitment experiment experimental_method Upon addition of rapamycin the Q domain-FKBP-mRFP construct translocates to the mitochondria and takes GFP-PI4KB with it. (E) Mitochondria recruitment experiment. FIG | |
| 30 35 AKAP1 protein Left – cells transfected with AKAP1-FRB-CFP, GFP-PI4KB and wild-type Q domain-FKBP-mRFP constructs before and five minutes after addition of rapamycin. FIG | |
| 36 39 FRB structure_element Left – cells transfected with AKAP1-FRB-CFP, GFP-PI4KB and wild-type Q domain-FKBP-mRFP constructs before and five minutes after addition of rapamycin. FIG | |
| 40 43 CFP experimental_method Left – cells transfected with AKAP1-FRB-CFP, GFP-PI4KB and wild-type Q domain-FKBP-mRFP constructs before and five minutes after addition of rapamycin. FIG | |
| 45 48 GFP experimental_method Left – cells transfected with AKAP1-FRB-CFP, GFP-PI4KB and wild-type Q domain-FKBP-mRFP constructs before and five minutes after addition of rapamycin. FIG | |
| 49 54 PI4KB protein Left – cells transfected with AKAP1-FRB-CFP, GFP-PI4KB and wild-type Q domain-FKBP-mRFP constructs before and five minutes after addition of rapamycin. FIG | |
| 59 68 wild-type protein_state Left – cells transfected with AKAP1-FRB-CFP, GFP-PI4KB and wild-type Q domain-FKBP-mRFP constructs before and five minutes after addition of rapamycin. FIG | |
| 69 77 Q domain structure_element Left – cells transfected with AKAP1-FRB-CFP, GFP-PI4KB and wild-type Q domain-FKBP-mRFP constructs before and five minutes after addition of rapamycin. FIG | |
| 78 82 FKBP protein Left – cells transfected with AKAP1-FRB-CFP, GFP-PI4KB and wild-type Q domain-FKBP-mRFP constructs before and five minutes after addition of rapamycin. FIG | |
| 83 87 mRFP experimental_method Left – cells transfected with AKAP1-FRB-CFP, GFP-PI4KB and wild-type Q domain-FKBP-mRFP constructs before and five minutes after addition of rapamycin. FIG | |
| 141 150 rapamycin chemical Left – cells transfected with AKAP1-FRB-CFP, GFP-PI4KB and wild-type Q domain-FKBP-mRFP constructs before and five minutes after addition of rapamycin. FIG | |
| 48 53 H264A mutant Right – The same experiment performed using the H264A Q domain mutant. FIG | |
| 54 62 Q domain structure_element Right – The same experiment performed using the H264A Q domain mutant. FIG | |
| 63 69 mutant protein_state Right – The same experiment performed using the H264A Q domain mutant. FIG | |
| 0 5 ACBD3 protein ACBD3 indirectly increases the activity of PI4KB. FIG | |
| 43 48 PI4KB protein ACBD3 indirectly increases the activity of PI4KB. FIG | |
| 4 31 Micelles-based kinase assay experimental_method (A) Micelles-based kinase assay – PI in TX100 micelles was used in a luminescent kinase assay and the production of PI4P was measured. FIG | |
| 34 36 PI chemical (A) Micelles-based kinase assay – PI in TX100 micelles was used in a luminescent kinase assay and the production of PI4P was measured. FIG | |
| 69 93 luminescent kinase assay experimental_method (A) Micelles-based kinase assay – PI in TX100 micelles was used in a luminescent kinase assay and the production of PI4P was measured. FIG | |
| 116 120 PI4P chemical (A) Micelles-based kinase assay – PI in TX100 micelles was used in a luminescent kinase assay and the production of PI4P was measured. FIG | |
| 38 42 PI4P chemical Bar graph presents the mean values of PI4P generated in the presence of the proteins as indicated, normalized to the amount of PI4P generated by PI4KB alone. FIG | |
| 60 71 presence of protein_state Bar graph presents the mean values of PI4P generated in the presence of the proteins as indicated, normalized to the amount of PI4P generated by PI4KB alone. FIG | |
| 127 131 PI4P chemical Bar graph presents the mean values of PI4P generated in the presence of the proteins as indicated, normalized to the amount of PI4P generated by PI4KB alone. FIG | |
| 145 150 PI4KB protein Bar graph presents the mean values of PI4P generated in the presence of the proteins as indicated, normalized to the amount of PI4P generated by PI4KB alone. FIG | |
| 15 42 standard errors of the mean evidence Error bars are standard errors of the mean (SEM) based on three independent experiments. (B) GUV-based phosphorylation assay – GUVs containing 10% PI were used as a substrate and the production of PI4P was measured using the CFP-SidC biosensor. FIG | |
| 44 47 SEM evidence Error bars are standard errors of the mean (SEM) based on three independent experiments. (B) GUV-based phosphorylation assay – GUVs containing 10% PI were used as a substrate and the production of PI4P was measured using the CFP-SidC biosensor. FIG | |
| 93 124 GUV-based phosphorylation assay experimental_method Error bars are standard errors of the mean (SEM) based on three independent experiments. (B) GUV-based phosphorylation assay – GUVs containing 10% PI were used as a substrate and the production of PI4P was measured using the CFP-SidC biosensor. FIG | |
| 127 131 GUVs experimental_method Error bars are standard errors of the mean (SEM) based on three independent experiments. (B) GUV-based phosphorylation assay – GUVs containing 10% PI were used as a substrate and the production of PI4P was measured using the CFP-SidC biosensor. FIG | |
| 147 149 PI chemical Error bars are standard errors of the mean (SEM) based on three independent experiments. (B) GUV-based phosphorylation assay – GUVs containing 10% PI were used as a substrate and the production of PI4P was measured using the CFP-SidC biosensor. FIG | |
| 197 201 PI4P chemical Error bars are standard errors of the mean (SEM) based on three independent experiments. (B) GUV-based phosphorylation assay – GUVs containing 10% PI were used as a substrate and the production of PI4P was measured using the CFP-SidC biosensor. FIG | |
| 225 243 CFP-SidC biosensor experimental_method Error bars are standard errors of the mean (SEM) based on three independent experiments. (B) GUV-based phosphorylation assay – GUVs containing 10% PI were used as a substrate and the production of PI4P was measured using the CFP-SidC biosensor. FIG | |
| 26 51 GUV phosphorylation assay experimental_method (C)–Quantification of the GUV phosphorylation assay – Mean membrane fluorescence intensity of the PI4P reporter (SidC-label) under different protein/ATP conditions. FIG | |
| 54 90 Mean membrane fluorescence intensity evidence (C)–Quantification of the GUV phosphorylation assay – Mean membrane fluorescence intensity of the PI4P reporter (SidC-label) under different protein/ATP conditions. FIG | |
| 98 102 PI4P chemical (C)–Quantification of the GUV phosphorylation assay – Mean membrane fluorescence intensity of the PI4P reporter (SidC-label) under different protein/ATP conditions. FIG | |
| 113 117 SidC protein (C)–Quantification of the GUV phosphorylation assay – Mean membrane fluorescence intensity of the PI4P reporter (SidC-label) under different protein/ATP conditions. FIG | |
| 149 152 ATP chemical (C)–Quantification of the GUV phosphorylation assay – Mean membrane fluorescence intensity of the PI4P reporter (SidC-label) under different protein/ATP conditions. FIG | |
| 4 27 mean membrane intensity evidence The mean membrane intensity value is relative to the background signal and the difference between the membrane and background signal in the reference system lacking ATP. FIG | |
| 165 168 ATP chemical The mean membrane intensity value is relative to the background signal and the difference between the membrane and background signal in the reference system lacking ATP. FIG | |
| 25 28 SEM evidence The error bars stand for SEM based on three independent experiments (also SI Fig. 6). FIG | |
| 0 18 Pseudoatomic model evidence Pseudoatomic model of the PI4KB multiprotein complex assembly. FIG | |
| 26 31 PI4KB protein Pseudoatomic model of the PI4KB multiprotein complex assembly. FIG | |
| 0 5 PI4KB protein PI4KB in orange, Rab11 in purple, ACBD3 in blue. FIG | |
| 17 22 Rab11 protein PI4KB in orange, Rab11 in purple, ACBD3 in blue. FIG | |
| 34 39 ACBD3 protein PI4KB in orange, Rab11 in purple, ACBD3 in blue. FIG | |
| 26 29 NMR experimental_method The model is based on our NMR structure and a previously published crystal structure of PI4KB:Rab11 complex (PDB code 4D0L), ACBD and GOLD domain were homology modeled based on high sequence identity structures produced by the Phyre2 web server. FIG | |
| 30 39 structure evidence The model is based on our NMR structure and a previously published crystal structure of PI4KB:Rab11 complex (PDB code 4D0L), ACBD and GOLD domain were homology modeled based on high sequence identity structures produced by the Phyre2 web server. FIG | |
| 67 84 crystal structure evidence The model is based on our NMR structure and a previously published crystal structure of PI4KB:Rab11 complex (PDB code 4D0L), ACBD and GOLD domain were homology modeled based on high sequence identity structures produced by the Phyre2 web server. FIG | |
| 88 99 PI4KB:Rab11 complex_assembly The model is based on our NMR structure and a previously published crystal structure of PI4KB:Rab11 complex (PDB code 4D0L), ACBD and GOLD domain were homology modeled based on high sequence identity structures produced by the Phyre2 web server. FIG | |
| 125 129 ACBD structure_element The model is based on our NMR structure and a previously published crystal structure of PI4KB:Rab11 complex (PDB code 4D0L), ACBD and GOLD domain were homology modeled based on high sequence identity structures produced by the Phyre2 web server. FIG | |
| 134 138 GOLD structure_element The model is based on our NMR structure and a previously published crystal structure of PI4KB:Rab11 complex (PDB code 4D0L), ACBD and GOLD domain were homology modeled based on high sequence identity structures produced by the Phyre2 web server. FIG | |
| 151 167 homology modeled experimental_method The model is based on our NMR structure and a previously published crystal structure of PI4KB:Rab11 complex (PDB code 4D0L), ACBD and GOLD domain were homology modeled based on high sequence identity structures produced by the Phyre2 web server. FIG | |
| 200 210 structures evidence The model is based on our NMR structure and a previously published crystal structure of PI4KB:Rab11 complex (PDB code 4D0L), ACBD and GOLD domain were homology modeled based on high sequence identity structures produced by the Phyre2 web server. FIG | |
| 227 233 Phyre2 experimental_method The model is based on our NMR structure and a previously published crystal structure of PI4KB:Rab11 complex (PDB code 4D0L), ACBD and GOLD domain were homology modeled based on high sequence identity structures produced by the Phyre2 web server. FIG | |
| 4 8 GOLD structure_element The GOLD domain is tethered to the membrane by GolginB1 (also known as Giantin) which is not shown for clarity. FIG | |
| 47 55 GolginB1 protein The GOLD domain is tethered to the membrane by GolginB1 (also known as Giantin) which is not shown for clarity. FIG | |
| 71 78 Giantin protein The GOLD domain is tethered to the membrane by GolginB1 (also known as Giantin) which is not shown for clarity. FIG | |
| 0 32 Intrinsically disordered linkers structure_element Intrinsically disordered linkers are modeled in an arbitrary but physically plausible conformation. FIG | |