The emergence of a primordial ribosome from the RNA world would have required access to aminoacylated RNA substrates. The spontaneous generation of such substrates without enzymes is inefficient, and it remains unclear how they could be selected for in a prebiotic milieu. In our study, we identify a possible role for aminoacylated RNA in ribozyme assembly, a longstanding problem in the origin-of-life research. We show that aminoacylation of short RNAs greatly accelerates their assembly into functional ribozymes by forming amino acid bridges in the phosphodiester backbone. Our work therefore addresses two key challenges within the origin-of-life field: we demonstrate assembly of functional ribozymes, and we identify a potential evolutionary benefit for RNA aminoacylation that is independent of coded peptide translation. Aminoacylated transfer RNAs, which harbor a covalent linkage between amino acids and RNA, are a universally conserved feature of life. Because they are essential substrates for ribosomal translation, aminoacylated oligonucleotides must have been present in the RNA world prior to the evolution of the ribosome. One possibility we are exploring is that the aminoacyl ester linkage served another function before being recruited for ribosomal protein synthesis. The nonenzymatic assembly of ribozymes from short RNA oligomers under realistic conditions remains a key challenge in demonstrating a plausible pathway from prebiotic chemistry to the RNA world. Here, we show that aminoacylated RNAs can undergo template-directed assembly into chimeric amino acid–RNA polymers that are active ribozymes. We demonstrate that such chimeric polymers can retain the enzymatic function of their all-RNA counterparts by generating chimeric hammerhead, RNA ligase, and aminoacyl transferase ribozymes. Amino acids with diverse side chains form linkages that are well tolerated within the RNA backbone and, in the case of an aminoacyl transferase, even in its catalytic center, potentially bringing novel functionalities to ribozyme catalysis. Our work suggests that aminoacylation chemistry may have played a role in primordial ribozyme assembly. Increasing the efficiency of this process provides an evolutionary rationale for the emergence of sequence and amino acid–specific aminoacyl-RNA synthetase ribozymes, which could then have generated the substrates for ribosomal protein synthesis. In t1216otocells . Carbodiotocells  or replaotocells –28, but 2+, conditions that are compatible with fatty acid–based vesicles . Upon activation with 2-methylimidazole followed by aminoacylation with Gly and addition of an RNA template, we observed rapid ligation of the glycylated oligonucleotides to yield the full-length 37-nt chimeric hammerhead, which was detectable as early as 5 min . In contrast, nonaminoacylated RNA oligomers yielded only trace amounts of full-length product after 24 h. While this result suggested that multiple ligation reactions with aminoacylated RNA could be leveraged to assemble chimeric ribozymes, the formation of a stable duplex between the RNA template and the ribozyme RNA prevented the quantitative measurement of ribozyme activity.Having previously demonstrated the enhanced template-directed ligation of aminoacylated RNAs , we askeSI Appendix, Fig. S1C). Although not prebiotically relevant, this approach permitted a systematic assessment of the activities of chimeric ribozymes. Using this method, we assembled four chimeric hammerhead ribozymes using four different amino acids with diverse side chains . All four chimeric ribozymes cleaved the native hammerhead substrate under single-turnover conditions, albeit at rates that were 20- to 80-fold lower than the all-RNA hammerhead ribozyme . This decrease in cleavage rates was not due to the hydrolysis of the aminoacyl ester linkages over the course of the reaction . Additionally, no cleavage was observed with unligated oligonucleotides under the same reaction conditions . The l-Lys–linked chimeric hammerhead exhibited the fastest cleavage of the four chimeric ribozymes, demonstrating that bulky, positively charged amino acid side chains can be tolerated within the phosphodiester backbone. Finally, replacing each amino acid bridge with a single U nucleotide led to slower cleavage rates, suggesting that amino acids in the backbone impose a lesser penalty on the ribozyme than additional nucleotides .To assess the activity of the chimeric hammerhead ribozyme, we modified the assembly process by using a DNA, rather than an RNA, template complementary to the full ribozyme sequence. We have previously found that DNase digestion of similar templates can be used to release pyrophosphate-linked  and aminSI Appendix, Fig. S5A). To accommodate flexizyme-catalyzed aminoacylation of individual oligomeric building blocks, we screened variants of this truncated sequence containing CA dinucleotides in positions that would enable us to assemble the entire sequence from shorter 3′-aminoacylated, 5′-(2-methylimidazole)–activated RNA oligonucleotides . While some variants lost ligase activity, we were able to identify a suitable ribozyme sequence that could be assembled from four oligonucleotides ranging in length from 11 to 18 nt.Having shown that amino acid–bridged RNAs can exhibit catalytic phosphodiester bond cleavage, we set out to design and assemble a chimeric ribozyme that can catalyze phosphodiester bond formation. As a starting point, we chose an in vitro–selected ligase ribozyme that catalyzes the ligation of imidazole-activated oligonucleotides . First, l-Lys because aminoacyl-RNA ligation with this amino acid resulted in the highest yields  . Performing the reaction with unligated oligonucleotides or with single U nucleotides in place of each l-Lys bridge led to the almost complete loss of ligase activity . A similarly assembled Gly-bridged ligase was also functional; however, due to the lower yield of full-length product with Gly aminoacylated oligonucleotides, we could not quantitate its activity .We assembled the chimeric ligase ribozyme using RNA oligonucleotides aminoacylated with Fig. S6) . Althougribozyme B and C. ribozyme B and C. l-Lys–aminoacylated oligonucleotides. The wild-type flexizyme sequence contains two CA sequences at positions that allow the assembly of the complete ribozyme from three oligonucleotides . Although both CA sequences are in the catalytic center of the ribozyme, we found that both Gly- and l-Lys–bridged flexizymes were functional . The decrease in the aminoacylation yield was not due to aminoacyl ester hydrolysis, as the chimeric l-Lys–bridged flexizyme displayed a half-life of 158 h under the reaction conditions . Mutating the flexizyme to reposition the CA sequences and Gly bridges outside the catalytic center did not improve the activity of the chimeric ribozyme . Given that we initially used the all-RNA flexizyme to aminoacylate the oligonucleotides that were then assembled into the chimeric flexizyme, it seemed conceivable that contamination of the purified chimeric flexizyme with traces of the all-RNA flexizyme could have contributed to the observed aminoacylating activity . The loss of activity after the hydrolysis of aminoacyl ester linkages also points to the inability of the unligated oligonucleotides to noncovalently assemble into an active ribozyme. Similar to the hammerhead and ligase ribozymes, the l-Lys–bridged chimeric flexizyme was the most active among those tested . The l-Lys–bridged ribozyme aminoacylated 10% of its RNA substrate after 22 h, demonstrating that enzymatic aminoacylation can emerge from the nonenzymatic ligation of aminoacylated oligonucleotides. Finally, replacing each l-Lys bridge with a single U nucleotide led to a drastic decrease in the aminoacylation yield, with only 2% of the primer being glycylated after 22 h .The ligation of aminoacylated oligonucleotides to generate a chimeric aminoacyl-RNA synthetase ribozyme could in principle result in an autocatalytic system in which the aminoacylating ribozyme synthesizes its own precursors, which then spontaneously assemble into more of the aminoacylating ribozyme. Even an inefficient initial chemical aminoacylation could potentially be sufficient to initiate such a self-amplifying cascade. As a first step toward such a cascade, we assembled a chimeric aminoacylating ribozyme (flexizyme) from Gly- and SI Appendix, Fig. S12A). Splint-assisted assembly has been shown to obviate the need for long template strands . To facilitate splint release and substrate binding, we added the hammerhead substrate and heated the reaction at 95 °C for 2 min before cooling it to the reaction temperature. Performing the subsequent cleavage reaction at 42 °C, conditions that release the ribozyme from the splints but leave substrate binding unaffected, we observed the expected cleavage of the substrate . Importantly, performing the reaction at 25 °C, conditions that allow both splint and substrate binding, resulted in similar levels of substrate cleavage, indicating that the chimeric hammerhead is functional despite the presence of splints .The chimeric ribozyme assembly method described above involves the use of a DNA template followed by DNase digestion to liberate the product ribozyme. Because neither the long DNA template nor the enzymatic digestion are prebiotically relevant, we sought to assemble a chimeric ribozyme using the splint-assisted assembly strategy . For faster transformations, such as the hammerhead cleavage, which uses 3 mM Mg2+, or reactions that occur at low temperatures, such as the flexizyme aminoacylation, aminoacyl ester hydrolysis is negligible on the reaction timescale but imposes a limit on the overall lifetime of the chimeric ribozyme . We suggest that the disassembly of a ribozyme into oligonucleotide components, which become reusable for activation and ligation after phosphoramidate hydrolysis in mild acid , desalted using Waters Sep-Pak C18 cartridges, and characterized by high-resolution mass spectrometry on an Agilent 6230 time-of-flight (TOF) mass spectrometer.Oligonucleotides were either purchased from Integrated DNA Technologies or synthesized in-house on an Expedite 8909 solid-phase oligonucleotide synthesizer. Phosphoramidites and reagents for the Expedite synthesizer were purchased from either Glen Research or Chemgenes. Cleavage of synthesized oligonucleotides from the solid support was performed using 1 mL of AMA (1:1 mixture of 28% aqueous ammonium hydroxide and 40% aqueous methylamine) for 30 min at room temperature, while deprotection was done in the same solution for 20 min at 65 °C. Deprotected oligonucleotides were lyophilized, resuspended in 100 µL of dimethylsulfoxide (DMSO), and 125 µL of triethylamine (TEA) trihydrofluoride and heated at 65 °C for 2.5 h to remove 4 in acetone and 1 mL of acetone for 30 min on dry ice. The pellet was washed with 1 mL of 1:1 acetone:diethylether twice. The products were resolved and purified by high-performance liquid chromatography (HPLC) on an Agilent ZORBAX analytical column  at a flow rate of 1 mL/min. The gradient constituted the following: aqueous 20 mM triethylammonium bicarbonate, pH 8.0 (A), and acetonitrile (B), from 7 to 12% B over 12 min.Oligonucleotides phosphorylated at the 5′-OH were activated with 2-methylimidazole, as previously reported , with th2-Aminoimidazole (2AI)–activated substrates for ligase ribozyme reactions were generated by reacting corresponding 5′-monophosphorylated oligonucleotides with 0.2 M 1-ethyl-3-(3 dimethylaminopropyl) carbodiimide  and 0.6 M 2AI  at room temperature for 2 h. The reaction was desalted by exchanging the reaction buffer with water in Amicon Ultra 0.5-mL 3K centrifugal filters and purified by HPLC, as described above.+-HEPES pH 8.0, 10 mM MgCl2, 10 µM oligonucleotide, 10 µM flexizyme, and 5 mM aa-DBE . Reactions were allowed to proceed for 16 to 18 h at 0 °C before being used in subsequent steps. To quantify the aminoacylation yields, 1-µL aliquots were quenched in acidic gel buffer  and resolved by a 20% acidic polyacrylamide gel (19:1 with 7 M urea and 0.1 M NaOAc pH 5.0). The gel was run for 2 h at 300 V at 4 °C and visualized on a Typhoon 9410 imager. A typical aminoacylation reaction yielded 20 to 60% product, measured by band densities in ImageQuant TL 8.1 software. Gels with unlabeled oligonucleotides were stained with SYBR Gold, according to the manufacturer’s instructions, and band intensities were quantified as described above.3,5-Dinitrobenzyl esters of amino acids (aa-DBEs), synthesized as described previously , were diOligonucleotides used in the assembly of the chimeric hammerhead ribozyme were used without purification, because gel purification of the full-length chimeric hammerhead was sufficient to remove excess flexizyme. The full-length chimeric ligase could not be resolved from excess flexizyme, and the aminoacylated oligonucleotides were purified away from the flexizyme by acidic preparative gel electrophoresis prior to the assembly reaction. Similarly, aminoacylated oligonucleotides that were used to generate the chimeric flexizyme were gel purified prior to the assembly reaction. Oligonucleotide bands were visualized by ultraviolet shadowing, the bands of interest were cut, crushed, and tumbled in 1 mL of 50 mM NaOAc and 5 mM EDTA, pH 5.0, for 3 h at 4 °C. The extracted oligonucleotides were filtered, concentrated, and desalted using Amicon Ultra 0.5-mL 3K centrifugal filters. Aliquots (1 µL) of the purified aminoacylated oligonucleotides were analyzed by acidic gel electrophoresis to quantify the aminoacylation yields. Due to the hydrolysis of 2-methylimidazole under acidic conditions, additional bands were present in the gel. The identities of oligonucleotides and relative percentages in each mixture were confirmed by liquid chromatography–mass spectrometry.l-Lys–DBE, l-Leu–DBE, and l-Asp–DBE as described above. After the 18-h incubation at 0 °C, each reaction was precipitated by the addition of 100 µL of 3 M NaOAc, pH 5.5, and 3.9 mL of 100% ethanol for 20 min on dry ice. Pellets were washed twice with 80% ethanol, dried under vacuum, and resuspended in 100 µL of nuclease-free water. Accurate concentration of the mixture of aminoacylated and nonaminoacylated oligonucleotides could not be determined due to the presence of flexizyme and was assumed to be 100 µM. For the RNA control reaction, an identical procedure was followed, except no flexizyme was used during the aminoacylation reaction. Oligonucleotides prepared in this way were used in the assembly reactions described below.Aminoacylation reactions on a 1-mL scale were performed on oligonucleotides 1 and 2 using Gly-DBE, 2, and 200 mM Na+-HEPES, pH 8.0, in a final volume of 60 µL. Reaction aliquots (1 µL) were quenched in 14 µL of quench buffer (50 mM EDTA and 9 µM reverse complement of the template in 90% [vol/vol] formamide) at the indicated time points, heated at 92 °C for 2 min, and resolved by 20% polyacrylamide gel electrophoresis (19:1 with 7 M urea). The ligated full-length product was quantified for each time point by integrating and normalizing band intensity in each gel lane in ImageQuant TL 8.1 software. For the glycylated reaction, the fraction of the full-length product was estimated from the fraction of glycylation quantified by acidic gel electrophoresis, as described above. Because the percentage of glycylated oligonucleotides 1 and 2 from optimized reactions was 21% and 20%, respectively, the raw full-length product yield was divided by 0.042 to obtain the adjusted product yield.Reactions were set up in triplicate at room temperature by mixing 1.25 µM oligonucleotides 1 to 3, 1.25 µM hammerhead RNA template, 2.5 mM MgCl+-HEPES, pH 8.0. After 3 h, the reactions were concentrated down to 50 µL by using Amicon Ultra 0.5-mL 3K centrifugal filters. TurboDNase (100 U) and 1× TurboDNase buffer were added to the concentrated reactions to a final volume of 500 µL and incubated for 15 min at 37 °C. Aliquots (1 µL) of the digested reactions were quenched in 14 µL of quenching buffer (50 mM EDTA in 90% [vol/vol] formamide), and the full-length products were resolved and quantified as above. The digested reactions were concentrated down to 50 µL by using Amicon Ultra 0.5-mL 3K centrifugal filters, diluted with 60 µL of 10 mM EDTA in 95% [vol/vol] formamide, purified by preparative 20% polyacrylamide gel electrophoresis (19:1 with 7 M urea), extracted in 1 mL of 50 mM NaOAc and 5 mM EDTA, pH 5.0, for 3 h at 4 °C, and desalted with a Zymo Clean and Concentrator kit. The concentration of each chimeric hammerhead was determined in triplicate by serially diluting a known concentration of the all-RNA standard, resolving the dilutions by denaturing gel electrophoresis, quantifying the band intensities, and generating a standard curve.Reactions (500 µL) were set up at room temperature by mixing 20 µM oligonucleotides 1 to 3, 20 µM hammerhead DNA template, 10 mM EDTA, and 200 mM Na2, and 200 mM Na+-HEPES, pH 8.0, at 0 °C. Reaction aliquots (0.5 µL) were quenched in 19.5 µL of quenching buffer (50 mM EDTA in 90% [vol/vol] formamide) at the indicated time points, heated at 92 °C for 1 min, and resolved by 20% polyacrylamide gel electrophoresis (19:1 with 7 M urea). After 43 h, the assembly reactions were directly used in the activity assay described below.Reactions (30 µL) were set up by mixing 25 µM oligonucleotides 1 to 3, 25 µM splints (either DNA or RNA), 100 mM NaCl, 5 mM MgCl2. To test cleavage activity of the chimeric hammerhead ribozyme assembled on DNA or RNA splints, the 3′-end of the ribozyme was extended by 6 nt, and the corresponding 5′-end of the substrate was extended by 4 nt, resulting in an 11-base pair stem in the ribozyme–substrate complex instead of the usual 5-base pair stem. This was done to favor the formation of a catalytically active hammerhead ribozyme–substrate complex over the inactive ribozyme–splint complex resulting from aminoacylated oligonucleotide ligation. To facilitate the separation of the chimeric ribozyme product from splint oligonucleotides, the ribozyme–splint complex was heated at 95 °C for 2 min followed by incubation at either 25 °C or 42 °C for 5 min in the presence of 100 mM Tris-Cl (pH 8) and 0.1 µM Cy5-labeled substrate. Cleavage was initiated by adding 3 mM MgCl2, and the reactions were incubated at either 25 °C or 42 °C. Aliquots (1.5 µL) were quenched in 5 µL of loading buffer  at various time points and stored on dry ice. Cleaved products were separated from uncleaved substrates by 20% polyacrylamide gel electrophoresis (19:1 with 7 M urea). Bands were quantified with ImageQuant IQTL 8.1 software. Kinetic plots were nonlinearly fitted in GraphPad Prism 9 to the modified first order rate equation y = A (1 – e−kx), where A represents the fraction of active complex, k is the first-order rate constant, x is time, and y is the fraction of cleaved product.Hammerhead cleavage was assayed in 10-µL reactions, which contained 0.12 µM ribozyme (or unligated oligonucleotides), 0.1 µM 6-carboxyfluorescein (FAM)/Cy5–labeled substrates, 100 mM Tris-Cl (pH 8), and 3 mM MgCll-Lys–DBE and Gly-DBE as described above. After the 18-h incubation at 0 °C, each reaction was precipitated by the addition of 400 µL of 3 M NaOAc, pH 5.5, and 15.6 mL of 100% ethanol for 20 min on dry ice. Pellets were washed twice with 80% ethanol, dried under vacuum, and dissolved in 100 µL of 10 mM EDTA in 95% (vol/vol) formamide. The oligonucleotides were then purified using preparative acidic gel electrophoresis, as described above.Aminoacylation reactions on a 4-mL scale were performed on oligonucleotides 1 to 3 with +-HEPES, pH 8.0, at room temperature . The reaction was allowed to proceed for 3 h before being concentrated, digested, purified, and desalted, as described for the hammerhead ribozyme assembly. Concentrations were determined as in the case of the chimeric hammerhead, except that an all-RNA ligase standard was used to generate the standard concentration curve.The assembly reaction was set up by mixing 16.7 µM oligonucleotides 1 to 4, 16.7 µM ligase RNA–DNA hybrid template, 5 mM EDTA, and 100 mM NaOAc, pH 5.0, in a final volume of 300 µL. The reaction was annealed by heating it to 70 °C for 2 min and slowly cooling to 4 °C at a rate of 0.1 °C/s. The annealed reaction was diluted to 500 µL with Na2. Aliquots (1 µL) were quenched in 5 µL of loading buffer (7 M urea and 100 mM EDTA in 1× TBE) at various times points and stored on dry ice. Ligated products were separated from unligated precursors by 10% polyacrylamide gel electrophoresis (19:1 with 7 M urea). Bands were quantified with ImageQuant TL 8.1 software. Kinetic plots were nonlinearly fitted in GraphPad Prism 9 to the modified first-order rate equation y = A(1 – e−kx), where A represents the fraction of active complex, k is the first-order rate constant, x is time, and y is the fraction of cleaved product.2AI-ligase activity was assayed in 5-µL reactions, which contained 0.1 µM ribozyme (or unligated oligonucleotides), 0.12 µM RNA template, 0.2 µM 2AI substrate, 100 mM Tris-Cl (pH 8), 250 mM NaCl, and 10 mM MgCll-Lys–DBE and Gly-DBE were performed and purified as described in the ligase assembly section. The assembly reaction was set up at room temperature by mixing 15.4 µM oligonucleotides 1 to 3, 15.4 µM Flexi DNA template, 3 mM EDTA, and 200 mM Na+-HEPES, pH 8.0, in a final volume of 500 µL. The reaction was allowed to proceed for 3 h before being concentrated, digested, and purified, as described above. Concentrations were determined as for the chimeric hammerhead, except that an all-RNA flexizyme standard was used to generate the standard concentration curve.Aminoacylation reactions on a 4-mL scale with +-HEPES, pH 8.0, 5 mM Gly-DBE (20% DMSO), 10 µM substrate oligonucleotide, and 10 mM MgCl2 and incubated at 0 °C. Reaction aliquots (0.5 µL) were quenched in 4.5 µL of acidic quenching buffer  at the indicated time points and resolved on a 20% acidic polyacrylamide gel . The gel was run for 2 h at 300 V at 4 °C and visualized with a Typhoon 9410 imager. Aminoacylation percentages were quantified by measuring band densities in ImageQuant TL 8.1 software. The remaining aminoacylation reactions at 22 h were desalted using Zip-Tip C18 columns and characterized by high-resolution mass spectrometry on an Agilent 6230 TOF mass spectrometer.Flexizyme activity was assayed as follows. Two microliters of 7.5 µM chimeric or all-RNA flexizyme was treated with 200 mM NaOH for 30 s at room temperature before being quenched with equimolar HCl . In parallel, 7.5 µM chimeric or all-RNA flexizyme was diluted with water to 5 µM and incubated for 30 s at room temperature in triplicate. The reactions were diluted to 2.5 µM flexizyme with 50 mM NaSupplementary File


Bacillus subtilis YugJ as an atypical AAOR that reduces furan aldehyde. YugJ displayed high substrate specificity toward 5-hydroxymethylfurfural (HMF), a furan aldehyde, in an NADPH- and Ni2+-dependent manner. YugJ folds into a two-domain structure consisting of a Rossmann-like domain and an α-helical domain. YugJ interacts with NADP and Ni2+ using the interdomain cleft of YugJ. A comparative analysis of three YugJ structures indicated that NADP(H) binding plays a key role in modulating the interdomain dynamics of YugJ. Noticeably, a nitrate ion was found in proximity to the nicotinamide ring of NADP in the YugJ structure, and the HMF-reducing activity of YugJ was inhibited by nitrate, providing insights into the substrate-binding mode of YugJ. These findings contribute to the characterization of the YugJ-mediated furan aldehyde reduction mechanism and to the rational design of improved furan aldehyde reductases for the biofuel industry.NAD(H)/NADP(H)-dependent aldehyde/alcohol oxidoreductase (AAOR) participates in a wide range of physiologically important cellular processes by reducing aldehydes or oxidizing alcohols. Among AAOR substrates, furan aldehyde is highly toxic to microorganisms. To counteract the toxic effect of furan aldehyde, some bacteria have evolved AAOR that converts furan aldehyde into a less toxic alcohol. Based on biochemical and structural analyses, we identified  NAD(H)/NADP(H)-dependent aldehyde/alcohol oxidoreductase (AAOR) catalyzes aldehyde reduction or alcohol oxidation . AAOR is2+ metallic cofactor using highly conserved histidine and aspartic residues. However, some group III AAORs require other divalent metal ions, such as Zn2+, Ni2+, Mg2+, Cu2+, Co2+, and Mn2+, for oxidoreduction activity . Y. Y2+ and2+ ions (YugJNi) at a 2.15 Å resolution in space group P212121 by molecular replacement  and a C-terminal domain  (YugJ184–387) a. The NT184–387) b. In conE. coli YqhD (PDB ID 1OJ7), Klebsiella pneumoniae 1,3-propanediol dehydrogenase (PDB ID 3BFJ), and Zymomonas mobilis alcohol dehydrogenase 2 (PDB ID 3OWO) .YugJ shows structural similarity to other group III AAORs, including ID 3OWO)  . Ho. HoNADP-Ni structure adopt open and closed conformations, respectively, which differ in interdomain angle by ~10°  adopts a closed conformation with a smaller interdomain angle, similar to that of YugJNi chain B  in addition to the YugJ α9–α10 loop. In the YugJrate ion . On the tructure . Thus, w2+ ion  was amplified by PCR from the genomic DNA of E. coli BL21 (DE3) cells for YugJ overexpression. Transformant cells were grown at 37 °C in LB medium until the optical density at 600 nm reached 0.6–0.8. The YugJ protein was overexpressed in the presence of 1 mM isopropyl-β-D-1-thiogalactopyranoside at 18 °C overnight. The E. coli cells were harvested by centrifugation and lysed by sonication in a solution containing 50 mM Tris, pH 8.0, 200 mM NaCl, 5 mM β-mercaptoethanol, and 1 mM phenylmethanesulfonyl fluoride. The cell lysate was cleared by centrifugation, and the resultant supernatant was incubated with Ni-NTA resin  to purify the His6-tagged YugJ protein via affinity chromatography. The YugJ protein was eluted from Ni-NTA resin using 250 mM imidazole and dialyzed against 20 mM Tris, pH 8.0, and 5 mM β-mercaptoethanol. The resulting protein was incubated with thrombin at 18 °C for 24 h to remove the N-terminal His6 tag. The tag-free YugJ protein was loaded onto a Mono Q 10/100 GL column  for further purification by anion-exchange chromatography and eluted from the Mono Q resin using a 0–500 mM NaCl gradient. The oligomeric state of the purified YugJ protein was analyzed by gel-filtration chromatography using a Superdex 200 10/300 column  in 20 mM Tris, pH 8.0, 150 mM NaCl, and 5 mM β-mercaptoethanol.The YugJ expression plasmid was transformed into Ni crystals were generated by mixing 0.5 μL of YugJ protein (12.7 mg/mL) with 0.5 μL of a well solution containing 20% PEG 6000 and 0.1 M Tris, pH 8.0, and then equilibrating the resultant 1-μL drop against 500 μL of the well solution. To obtain YugJNADP-NO3 crystals, YugJ protein (18.3 mg/mL) was mixed with NADP at a molar ratio of 1:3, and the resulting mixture was equilibrated to a well solution containing 18% PEG 3350, 0.1 M MES, pH 6.5, and 0.3 M ammonium nitrate. To generate YugJNADP-Ni crystals, YugJ crystals were first obtained in a drop containing 0.5 μL of YugJ protein (13.6 mg/mL) and 0.5 μL of a well solution  and were then soaked in a solution containing 3 mM NADP and 3 mM HMF.YugJ crystallization conditions were screened with JCSG Core Suites  and optimized by the sitting-drop vapor-diffusion method at 18 °C using YugJ protein in 20 mM Tris, pH 8.0, 200 mM NaCl, and 5 mM β-mercaptoethanol. YugJ2+ ions in the YugJ crystal, the X-ray fluorescence emission spectrum was obtained from the YugJ crystal. Data collection statistics are summarized in A YugJ crystal was briefly soaked in a cryoprotectant solution containing 25% glycerol or 25% ethylene glycol and flash-cooled under a nitrogen cryostream. X-ray diffraction was performed at the Pohang Accelerator Laboratory , and diffraction data were reduced and scaled using the HKL2000 package . To veriNi structure was determined by molecular replacement with the Phaser program using the structure of T. maritima butanol dehydrogenase A (PDB ID 1VLJ) as a search model [Ni [NADP-NO3 and YugJNADP-Ni structures were solved by molecular replacement using the YugJNi structure as a search model. The final YugJNADP-Ni and YugJNADP-NO3 structures were obtained via iterative cycles of model building and refinement [Ni, YugJNADP-Ni, and YugJNADP-NO3 structures  have been deposited in the Protein Data Bank.The YugJch model . Model bodel [Ni ,51. The finement ,51. The NADP-Ni structure has two Ramachandran outliers at the Ala141 residues from chains B and D. The two YugJNADP-Ni residues are located at the boundary between the favored region and outlier region of the Ramachandran plot, as observed for the Ala141 residues from chains A and C. Moreover, these four residues exhibit similar conformations. Notably, the YugJNi structure displays a higher average B-factor (48.7 Å2) than the YugJNADP-Ni (29.4 Å2) and YugJNADP-NO3 (22.2 Å2) structures, presumably due to lower resolution and a more disordered crystal lattice. However, the quality of the data collected for YugJNi is similar to those of YugJNADP-Ni and YugJNADP-NO3.Structure refinement statistics indicate that the three structures are overall of high quality . HoweverThe enzymatic activity of YugJ was evaluated using demetallized YugJ protein. To obtain metal-free YugJ protein, the purified recombinant YugJ protein was treated with a solution containing 1 mM EDTA and 100 mM sodium acetate, pH 5.0, at 4 °C for 2 h and subsequently diluted ~90-fold using a solution containing 20 mM Tris, pH 8.0, and 150 mM NaCl . The met2+-bound YugJ protein was reacted with 3 mM aldehyde substrate candidate  in the presence of 0.2 mM NADPH in 200 μL of a reaction solution, containing 50 mM HEPES, pH 7.4, and 150 mM NaCl, at 23 °C. The catalytic activity of YugJ was also assessed for 2,5-bis(hydroxymethyl)furan alcohol in the presence of 0.2 mM NADP in the reaction solution (pH 7.4) at 23 °C. The pH dependence of YugJ-mediated HMF reduction was determined by incubating 1 μg of Ni2+-bound YugJ with 3 mM HMF and 0.2 mM NADPH in a 200-μL solution, containing 150 mM NaCl and 50 mM buffer at different pH values  at 23 °C. To evaluate the metal specificity of YugJ, 1 μg of the YugJ protein preincubated with different metal ions was reacted with 3 mM HMF and 0.2 mM NADPH in 200 μL of the reaction solution (pH 7.4) at 23 °C. To determine the temperature dependence of YugJ-mediated HMF reduction, 1 μg of the Ni2+-bound YugJ protein was reacted with 3 mM HMF and 0.2 mM NADPH at temperatures between 16 and 58 °C in 200 μL of the reaction solution (pH 7.4). The reducing cofactor dependence of YugJ-mediated HMF reduction was determined by mixing 1 μg of the Ni2+-bound YugJ protein with 0.2 mM NADPH or NADH in the presence of 3 mM HMF in the reaction solution (pH 7.4) at 23 °C. The inhibition of YugJ-mediated HMF reduction by nitrate was addressed by incubating 360 mM lithium nitrate or lithium sulfate with 1 μg of Ni2+-bound YugJ, 3 mM HMF, and 0.2 mM NADPH in 200 μL of the reaction solution (pH 7.4) at 23 °C. The catalytic activity of YugJ was determined by measuring the absorbance at 340 nm using an EPOCH spectrophotometer .To investigate the substrate specificity of YugJ, 1 μg of the Ni2+-bound YugJ protein was reacted with serially diluted HMF (0.3–40 mM) in the presence of 0.2 mM NADPH in 200 μL of the reaction solution (pH 7.4) at 23 °C. The absorbance at 340 nm was measured in a continuous manner. The enzymatic data were fitted to the Michaelis–Menten equation using the Prism 5 program .To derive the kinetic parameters of YugJ, 1 μg of the NiThe structure of YugJ in complex with HMF was modeled via in silico molecular docking. The coordinates of HMF were obtained in the SDF format from the PubChem database . The coo


The structure was determined by single-crystal X-ray diffraction. It is isotypic to the K3Sb5O14, Rb3Sb5O14 and Cs3Sb5O14 analogues with orthorhombic Pbam symmetry and cell parameters a = 24.2899 (9) Å, b = 7.1931 (3) Å, c = 7.4182 (3) Å. The Sb atoms form irregular [SbO6] octahedra, which are linked via edges and corners into a triperiodic network. The Tl+ ions are located in distinct channels of the network extending along [010] and [001].Single crystals of Tl M[SbF6] compounds , precursors in the form of MSbO3 were synthesized. Whereas the chosen conditions  yielded the expected product for LiSbO3 and NaSbO3, the Tl-poor title compound Tl3Sb5O14 was inadvertently obtained in the case of Tl. TlSbO3 was later successfully synthesized at 1073 K. In fact, prior syntheses of TlSbO3 were performed at even lower temperatures  at 1273 K.Hong 1974 noted un3Sb5O14 crystallizes in the space group Pbam and is isotypic to M3Sb5O14 . Two different settings of the Pbam space group were used to describe the structures: a > b by Hong  =  operation is an element of the affine normalizer of the Pbam space group. Herein we use the original setting and atom labeling of Hong  to 103.83 (13)° and trans angles up to 150.66 (16)°. As noted by Hirschle et al.  was performed using the COMPSTRU , S = 0.0048 (M = Rb) and S = 0.0262 (M = Cs). This shows that the K, Rb and Tl compounds feature very similar cell parameters, with the volume increasing slightly according to K > Rb > Tl  and M = Rb (Δ = 0.035) compounds with Tl3Sb5O14, whereas the atomic positions in Cs3Sb5O14 differ distinctly (Δ = 0.178). In particular, the positions of the O atoms that coordinate to the Tl2 atoms feature a strong deviation (dmax = 0.6356 Å for the O4 atom) showing a distinct distortion of the [SbO6] octahedra around the respective channels. Thus, it appears that the Tl2 channels are responsible for the distinct enlargement of the unit cell of Cs3Sb5O14.The degree of similarity likewise shows a close relationship of the 3 and 0.373 g Sb2O3 (which makes for an approximate molar ratio of 1:1 for Tl:Sb) was heated in a corundum crucible at 1273 K for 12 h in air. From the reaction, a dark-orange powder was obtained. The single crystals formed as rectangular-prismatic plates. Crystals were isolated under a polarizing microscope and cut to an appropriate size for single crystal diffraction of a highly absorbing crystal.A mixture of 0.682 g TlNO3Sb5O14 ] from 0.028 to 0.023}, which might be in part due to the increased number of anisotropic displacement parameters.Crystal data, data collection and structure refinement are summarized in Table 310.1107/S2056989022002869/pk2663sup1.cifCrystal structure: contains datablock(s) I, global. DOI: 10.1107/S2056989022002869/pk2663Isup2.hklStructure factors: contains datablock(s) I. DOI: 2158509CCDC reference:  crystallographicinformation; 3D view; checkCIF reportAdditional supporting information:


Dia2 in S. cerevisiae is involved in the DNA replication and DNA damage stress response, but its effect on cell growth is still unclear. Here, we demonstrate that the absence of Dia2 prolongs the cell cycle by extending both S- and G2/M-phases while, at the same time, activating the S-phase checkpoint. In these conditions, Ctf4—an essential DNA replication protein and substrate of Dia2—prolongs its binding to the chromatin during the extended S- and G2/M-phases. Notably, the prolonged cell cycle when Dia2 is absent is accompanied by a marked increase in cell size. We found that while both DNA replication inhibition and an absence of Dia2 exerts effects on cell cycle duration and cell size, Dia2 deficiency leads to a much more profound increase in cell size and a substantially lesser effect on cell cycle duration compared to DNA replication inhibition. Our results suggest that the increased cell size in dia2∆ involves a complex mechanism in which the prolonged cell cycle is one of the driving forces.Cells have evolved elaborate mechanisms to regulate DNA replication machinery and cell cycles in response to DNA damage and replication stress in order to prevent genomic instability and cancer. The E3 ubiquitin ligase SCF Saccharomyces cerevisiae are Ctf4  with the replisome ,31Ctf4-Gact Dia2 e–g 30]..Ctf4-GFPCtf4-GFP and Ctf4-GFP;dia2Δ strains throughout the entire cell cycle via live-cell microscopy. In order to measure an averaged profile of Ctf4-GFP without data interpolation, we selected cells with cell cycle durations equal to the average for each strain  and chromatin pellet (Pel) fractions were isolated from wild-type and Ctf4-GFP d,e. The Ctf4-GFP d,e. HoweU arrest d,e. In crecovery f,g. Thisrecovery d,f. Thesrecovery d,f. Notast cells e,g. Coll present .Ctf4-GFP , whereas cells with missing Dia2 had an average area of 28.95 ± 5.08 µm2 (n = 90). It is known that mutations that prolong the cell cycle concomitantly increase the cell size in yeast [dia2Δ cells leads to an increase in cell size. Despite the large size of the cells, we do not observe a significant difference in the ratio of the long-to-short (x-to-y) axes . However, 3% of the dia2Δ cells exhibited an elongated phenotype (a ratio of the long-to-short axes is above 1.6) that we do not observe in the wild-type cells  axes b. The avpe cells c. It is s growth ,27,36,37ucleated . This isn = 50 to 385 ± 155 min, n = 50) (2 to 23.4 ± 7.78 µm2 (n = 90) (dia2Δ cells significantly increased the cell cycle duration from 145 ± 30 min to 415 ± 147 min (2 in non-treated cells (n = 90) to 34.24 ± 8.14µm2 in HU treated dia2Δ cells (n = 90). Our data indicates that the additional increase of cell size when the cell cycle is prolonged by HU treatment confirms that extending the cell cycle has a role in cell size alteration  to 1.52 ± 0.50 (n = 90) in dia2Δ cells when treated with HU  c. In add7.78 µm2 d. Furthe(n = 90) e. As sho 147 min . This alteration d. Intere with HU f. Additia2Δcells f, indicadia2Δ involves a complex mechanism for which the extended cell cycle is only one of the triggers.Collectively, we show that the deletion of DIA2 leads to cells with increased cell size and extended S- and G2/M-phases with elevated levels of Ctf4. In addition, a comparison of the effects of DNA replication inhibition and of the absence of Dia2 on cell cycle duration and cell size suggests that the increased cell size in S. cerevisiae cells. It is known that in budding yeast, Dia2 is responsible for maintaining genomic stability and plays a key role in DNA replication. Its levels are low during the G1-phase and increase during the S-phase, corresponding to its function [dia2Δ cells exhibit an extended cell cycle duration due to prolonged S- and G2/M-phases, which also suggests a role of Dia2 in DNA replication and S-phase checkpoint control  and Mrc1-GFP (MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 MRC1-GFP-His3MX6)  [Ctf4-GFP;dia2Δ (MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 CTF4-GFP-His3MX6 dia2Δ::KanMX) and Mrc1-GFP;dia2Δ (MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 MRC1-GFP-HIS3MX6 dia2Δ::KanMX) strains were created. All strains were cultivated in YPD medium (1% (w/v) yeast extract (Difco), 2% (w/v) Bacto peptone (Difco), 2% (w/v) dextrose). As a control, a wild-type (WT) S288C strain without a GFP tag was used.control)  are, witcontrol)  to creatcontrol) . The pai6 cells/mL. Five μL of each dilution were then spotted onto YPD, or YPD supplemented with 50 mM, 100 mM, or 200 mM HU. Plates were incubated at 25 °C for three days.To perform the 10-fold serial dilution assays, yeast samples were prepared from exponentially growing cultures at a concentration of 3.4 × 10w/v) dextrose) with 20 μg/mL extra adenine (Ade) until OD600 = 0.2. A total of 1 mL yeast suspension was centrifuged at 4000 rpm for 1 min, and the pellet was resuspended in 50 μL of fresh CSM with Ade. Prior to microscopy, a concavity microscope glass slide was prepared. The slide contained a cavity in the center, which was filled with CSM and agarose (1.2%), and 2.5 μL of the cell suspension was placed on it. The edge of the hole was greased with Vaseline to create better adhesion between the coverslip and the slide, as well as to reduce deformation of the media for microscopy inside the cavity. The slide was then covered with a cover glass, and slight pressure was applied. All procedures were carried out at 27 °C and according to the protocol described by Silva and co-workers [In order to reduce autofluorescence, prior to live-cell imaging, yeast cells were pre-incubated in CSM media  [Ctf4-GFP and 145 min for Ctf4-GFP;dia2Δ, and the standard error of the mean is shown as error bars. Normalization was carried out, taking the minimum value as 0 and the maximum as 1. Live-cell imaging from a minimum of 3 experiments per strain was also used to determine the duration and the phases of the cell cycle based on characteristic cell and nuclear morphology [p < 0.001; 0.001 < p < 0.01; NS p > 0.01) and the standard deviation was presented as error bars.Time-lapse experiments were performed using a Yokogawa CSU-X1 spinning disc confocal microscope (Andor Revolution XD system with a Nikon TiE microscope stand and an incubator for temperature and humidity control) equipped with an iXon897 EMCCD camera and a CFI Apo TIRF 100X Oil 1.49 NA objective. All experiments were carried out under the following parameters: 11 Z-planes, with a Z-step of 0.5 μm; laser intensity: 8.1% on 488 nm; camera exposure of 200 ms. Frames were taken every 5 min for the whole duration of the experiments, and the maximum intensity projection function was applied to all the acquired images. All data were analyzed using ImageJ and CellTool/Version 1.6.0.2 (ly 2021)  softwarerphology ,30. The 3 was added to 1 × 109 harvested cells treated with 100 mM HU and were incubated for 30 °C. Then, the cells were treated with 3 mL of prespheroplasting buffer (100 mM PIPES (pH 9.4), 10 mM DTT) for 10 min, followed by incubation in 1 mL spheroplasting buffer (50 mM KH2PO4/K2HPO4 (pH 7.5), 0.6 M Sorbitol, 10 mM DTT) for 5 min. Following LongLife™ Zymolyase®  digestion, the spheroplast pellets were washed with 1 mL of ice-cold wash buffer , 2.5 mM MgCl2, and 0.4 M Sorbitol). Next, the cells were pelleted at 4000 rpm for 1 min at 4 °C. The pellets were measured and resuspended in an equal volume of extraction buffer , 2.5 mM MgCl2, 50 mM NaF, 5 mM Na4P2O7, 0.1 mM NaVO3) with 1.5% Triton X-100, 1 mM PMSF, and protease inhibitors cocktail  followed by incubation for 10 min at 4 °C. To remove the aggregated and unlysed cells from the suspension, the lysates were passed through a thin syringe needle and spun at 300 g. Then, the lysates were then under-layered with 30% sucrose and spun at 12,000 rpm for 10 min at 4 °C. The pellet (chromatin-bound) fractions were washed with 25% volume of the EB containing 1.5% Triton X-100 (EBX) and spun again at 10,000 rpm for 5 min at 4 °C. The crude chromatin pellets were dissolved in EBX. The supernatants (Sup) served as a soluble, chromatin-unbound fraction. Finally, the volumes of Sup and Pel were equalized with EBX, and 2× Laemmli buffer was added to each fraction. Samples were boiled for 3 min and spun at 10,000 rpm for 1 min before loading on 6–15% gradient SDS PAGE gels.In order to prepare chromatin-bound and soluble fractions, we used a previously described protocol  with som8 yeast cells were prepared for each time point by TCA precipitation, as described by Foiani and co-workers [3VO4 and 1 mM NaF.For the recovery experiments, cells were first treated with HU for 2 h, and HU was then washed. Total protein extracts from 6 × 10-workers . All solThe samples were loaded on 6–15% gradient SDS-PAGE gels, ran at 140 V, and then transferred onto a Protran blotting nitrocellulose membrane (Cat # 10600003). For detection of the Ctf4 protein, a goat polyclonal Anti-EGFP antibody was used (kindly provided by Max Planck Institute for Molecular Cell Biology and Genetics ). The re2). Finally, the cells were incubated with propidium iodide (50 μg/mL), diluted in 50 mM Tris-HCl (pH 7.8), and analyzed using Becton Dickinson FACScan.Flow cytometry was used to determine the amount of DNA in each cell by measuring its fluorescence signal (λ488), emitted from the intercalated propidium iodide. The yeast cells were pelleted and then fixed using 70% ethanol, followed by resuspension in 0.28 М Tris-HCl (pH 7.5) and sonication for 10 s using a power of 30 V (to disrupt cell adhesion). A total of 2 mg/mL RNAse А was added to each sample, and cells were incubated for 12 h at 37 °C. Following incubation, cells were centrifuged, and the pellet was resuspended in 180 mM Tris-HCl pH 7.5 (190 mM NaCl and 70 mМ MgCl


Actinobacillus for its potential role in nutrient acquisition. The electron density map of 1.76 Å resolution obtained from the crystal structure of the periplasmic binding protein was best fit with a molecular model containing a pyridoxal-5′-phosphate  ligand within the protein's binding site. The identity of the P5P bound to this periplasmic binding protein was verified by isothermal titration calorimetry, microscale thermophoresis, and mass spectrometry, leading us to name the protein P5PA and the operon P5PAB. To illustrate the functional utility of this uptake system, we introduced the P5PAB operon from Actinobacillus pleuropneumoniae into an Escherichia coli K-12 strain that was devoid of a key enzyme required for P5P synthesis. The growth of this strain at low levels of P5P supports the functional role of this operon in P5P uptake. This is the first report of a dedicated P5P bacterial uptake system, but through bioinformatics, we discovered homologs mainly within pathogenic representatives of the Pasteurellaceae family, suggesting that this operon exists more widely outside the Actinobacillus genus.Bacteria require high-efficiency uptake systems to survive and proliferate in nutrient-limiting environments, such as those found in host organisms. ABC transporters in the bacterial plasma membrane provide a mechanism for transport of many substrates. In this study, we examine an operon containing a periplasmic binding protein in  Binding protein–dependent transporter (BPDT) systems are composed of three components: a periplasmic binding protein and a permease–ATPase complex embedded in the inner membrane. The periplasmic binding protein sequesters a specific substrate and recruits it to the permease–ATPase plasma membrane complex, which uses ATP hydrolysis to induce substrate influx from the periplasm into the cytosol .12. Vitamin B12 belongs to the B-type vitamin group that is highly water soluble and contributes significantly to enzymatic catalysis as cofactors. However, among the B vitamins, transporters specific for vitamin B6 remained under investigation , pyridoxamine (PM), pyridoxine, and their corresponding phosphorylated products. However, the biologically active form of vitamin B6 is pyridoxal-5′-phosphate   was identified in A. pleuropneumoniae and, based on sequence homology to FbpA, the periplasmic binding protein AfuA was proposed to be a transporter for ferric iron . We confirm the identity of the ligand in the binding pocket using mass spectrometry and further show, using isothermal titration calorimetry (ITC) and microscale thermophoresis (MST), that P5P binds this periplasmic binding protein with high affinity. Finally, we demonstrate that expressing the operon containing APJL_RS02700 in a strain of Escherichia coli deficient in vitamin B6 synthesis rescues its growth in media containing low levels of P5P. We propose the name P5PA, for P5P binding protein A for this newly characterized periplasmic binding protein. To our knowledge, P5PA is the only bacterial protein shown to promote uptake of the active form of vitamin B6.In this study, we determine the structure of the periplasmic binding protein encoded by APJL_RS02700 in complex with a ligand and identify that ligand to be PLP/P5P (vitamin BA. pleuropneumoniae (APJL_RS02700) with high sequence identity (54%) to AfuA. To determine the function of this homologous protein, we cloned, expressed, and purified it recombinantly in E. coli.Whilst characterizing the function of AfuA , we discA) revealed clear positive density indicating a possible copurifying ligand . An in silico screen for possible ligands revealed that neither G6P nor fructose-6-phosphate, the two primary ligands that bind AfuA, fit the positive density in the binding pocket. Further in silico screening of potential ligands containing six-member rings and phosphate groups indicated that the active form of vitamin B6, called PLP/P5P, best fit in the unmodeled electron density . Thus, we named APJL_RS02700, P5PA for P5P binding protein A.The purified protein was crystalized and subjected to X-ray crystallographic characterization. Using molecular replacement (MR) with AfuA as a model, the structure of “APJL_RS02700” was solved and refined to a resolution of 1.75 Å see . The resFig. 1A and 2A). In addition, the dipole created the N terminus of three alpha helices beginning at residues Thr37, Gly148, and Ser183, respectively, and create a positive environment for the negatively charged phosphate on P5P (B and C). Moreover, the hydrophobic interactions with Gly36, Leu105, and Gln267 contributed to retaining the P5P molecule within this binding pocket (A).The model of P5PA with P5P in the binding pocket reveals a network of hydrogen bonds and hydrophobic clefts that primarily hold the pyridine ring and phosphate group of P5P in place. While Tyr103 interacts with the pyridine ring nitrogen and His203 coordinates the pyridine hydroxyl group, the phosphate group of P5P is stabilized by collective interactions with the side chains of Thr37, Ser147, Thr149, and Ser183 and the backbones of Thr37, Thr149, and Gly148 A and 2A.e on P5P , B and Cg pocket A.Figure 6, which has a pyridine ring as well as a phosphate moiety. Comparing the two binding clefts, it becomes clear that the phosphate moieties of G6P and P5P are both coordinated via hydrogen bonds formed with side chains and backbones of glycine, serine, and threonine (C and D and A and B). In the binding pocket of AfuA, Ser37, Gly38, Thr60, Ser148, Gly149, Thr150, and Gly185 surround the phosphate group of G6P. Similarly, the phosphate moiety of P5P is enclosed by Thr37, Ser147, Gly148, Thr149, and Ser183 in the binding site of P5PA. The ring moieties of G6P and vitamin B6 are also stabilized by the similar intermolecular forces (E and F and A and B). In the binding site of AfuA, the sugar ring is coordinated by forming hydrogen bonds with Tyr103, His205, and Gln269. Likewise, in the binding cleft of P5PA, the pyridine ring of P5P interacts with Tyr103 and His203. However, more hydrophobic interactions are present in the binding cleft of AfuA to seize G6P, including Ser9, Thr11, Gly36, Gly59, Ser184, Ile186, Phe203, Asp 206, and Glu229, whereas only Gly36, Leu105, and Gln267 participate in the hydrophobic network surrounding P5P in the binding pocket of P5PA. It was previously reported that H205A, D206A, or E229A mutants of AfuA have undetectable binding affinity for G6P, implying the loss of capacity to sequester G6P . However, they are involved in transporting different small nutrient molecules. AfuA plays an important role in transporting G6P, which has a sugar ring and a phosphate moiety, whereas P5PA is responsible for taking up vitamin B6 is a ligand of P5PA and determine if P5PA has any other potential copurifying substrates, we extracted any copurifying ligands that were endogenously bound to P5PA and performed LC–MS/MS (6 (P5P) is 247 Da. As a proton was added to ionize P5P, the m/z representing P5P became 248. We used P5P powder dissolved in ultrapure water as a positive control. The peak representing 248 m/z was detected in both the positive control P5P solution and the ligands extracted from P5PA, although the peak in the P5P solution had higher intensity (A and C). In order to identify the molecule represented by the detected 248 m/z peak, the products at 248 m/z were fragmented by nitrogen gas. The expected transition of 248 → 150 m/z can be used to characterize P5P molecules (m/z fragmentation pattern was observed in both the extract (D) and P5P solution (B), implying that P5P was present in the extract of ligand-bound P5PA. Overall, the major peaks present in the P5P solution and the extract had the same m/z but different intensities, suggesting that no other P5P derivatives were identified by LC–MS/MS.To confirm that vitamin BLC–MS/MS . The molntensity , A and Colecules . The 248 extract D and P5Psolution B, implyiKd) of 36 ± 7 nM, which suggests that there is a strong interaction between P5PA and P5P (A). This tight binding activity was further supported by MST results with Kd = 28 nM  exhibited an impaired binding capacity to P5P , the pyr6 that contains multiple vitamers with similar chemical structures, we queried whether P5PA had other potential ligands and was able to bind other vitamin B6 vitamers. We added P5PA to another two vitamin B6 vitamers: PL and PM. The MST-determined Kd for PL and PM was 13 and 8 mM, respectively . This reflects no significant binding activity between G6P and the periplasmic binding protein P5PA supporting our structural analysis that AfuA and P5PA are components of distinct specific nutrient uptake systems.As P5PA is a homolog of AfuA, we wondered whether P5PA is able to interact with hexose phosphate molecules. We measured the interaction between G6P and P5PA using ITC. The released heat was approximately 3 kJ/mol for each G6P to P5PA titration, which was insufficient to calculate a dissociation constant B. This rin vitro, we wondered whether the gene cluster containing P5PA would be able to functionally uptake P5P and thus complement a vitamin B6 deficiency in E. coli. In WT E. coli (strain K-12), the active form of vitamin B6 (P5P) can be synthesized from d-erythrose 4-phospate, pyruvate, and glyceraldehyde 3-phosphate by enzymes encoded by pdx family genes ; however, expression of pSC101, pSC101-P5PAB, and pSC101-AfuABC did not have any effect on the growth of the ΔpdxB mutant (E). When P5P was supplied at 5 μM, the growth defects of the four transformants and the ΔpdxB mutant were partially restored, whereas the pSC101-P5PAB + AfuC transformant grew the fastest of all the transformants to stationary phase (D). Furthermore, the expression of the pyridine ring–related P5PA–binding mutants (Y103A and H203A) did show a growth defect compared with WT P5PA when placed within the pSC101-P5PAB + AfuC growth assay (E. coli verifies that P5PA together with P5PB performs as a high-affinity vitamin B6 (P5P) uptake system.In order to rescue growth of the Δtamin B6 . When P5B mutant E. When Pry phase D. Furtheth assay . Given o6 transporter is conserved in microorganisms other than A. pleuropneumoniae, we searched for homologs using the P5PA amino acid sequence as a template. A sequence was denoted homologous when it was found to share a minimum of 65% amino acid identity with P5PA. To further validate that our predicted P5PA hits were not the G6P-binding AfuA, we collected the sequences identified by BLAST outside the Pasteurellaceae sharing >40% sequence identity over >80% of any P5PA seed sequence. There were 471 sequences meeting this threshold but none that met our P5PA threshold of >65% identity. These 471 sequences were then searched for the P5PA sequence motifs identified by MEME. None fit the motif pattern consistent with P5PA, 468 were consistent with the AfuA motif pattern , and periodontal diseases (Aggregatibacter actinomycetemcomitans) , rabbitbacillus , Mannheinnheimia  and Pastteurella , 26), miribacter ), and biteurella , 26 and acterium ). Moreovomitans) , 30. TakA. pleuropneumoniae, the functional role of P5PA until now remained unknown. It was hypothesized that P5PA could bind to sugar phosphates just as AfuA because of the significant sequence similarity between the two proteins. Another hypothesis was that P5PA could have been the periplasmic binding protein that binds ferric iron as this has not been identified in A. pleuropneumoniae to date and represents an important target as it would traffic the essential nutrient required by bacteria for host colonization. In this study, we structurally characterized P5PA and found it to be a type II periplasmic binding protein capable of binding the active form of vitamin B6 (P5P). We confirmed by ITC, MST, and LC–MS/MS that the interaction between P5PA and P5P is highly specific. Moreover, by expressing P5PAB together with the ATPase AfuC from the AfuABC transporter, we were able to rescue the growth of E. coli K-12 ΔpdxB mutants.Identified as a homolog of the sugar-phosphate transporter AfuA in 6 transport systems in prokaryotes have been poorly characterized. Prior to this study, a very limited number of proteins were disclosed for vitamin B6 binding. PdxU1 and PdxU2 were predicted to bind a vitamer of vitamin B6. This vitamin is called pyridoxine, and this prediction was not supported by any experimental data –dependent pathway where d-erythrose 4-phosphate undergoes a series of biochemical reactions, the product of which then interacts with DXP to form the P5P precursor, pyridoxine-5′-phosphate. This reaction is catalyzed by two key enzymes, PdxA and PdxJ (6) via a salvage pathway. On the other hand, the DXP-independent pathway is found in the majority of bacteria, and also in archaea, fungi, and plants. In this pathway, vitamin B6 is derived from glutamine, ribose 5-phosphate, and glyceraldehyde 3-phosphate with the help of two critical enzymes, PdxS  and PdxT  (Bacteria have two independent and PdxJ . Pyridoxas Pdx2) .A. pleuropneumoniae by Xie et al. , exogenous P5P can diffuse into bacterial cells via passive transport. In fact, previous studies have illustrated that other dephosphorylated vitamers of vitamin B6 enter E. coli K-12 and other bacteria by facilitated diffusion  is near the reported physiological levels of P5P in swine blood supporting the beneficial role of this transporter in high-affinity uptake of the active form of vitamin B6. Another possible reason for expressing a vitamin B6-specific transporter is that nutrient transport is favored over nutrient biosynthesis by microorganisms, since the former consumes less energy  can be taken up from the extracellular environment in E. coli by the ABC importer BtuCDF and synthesized by the enzymes encoded in the cod operon  or on LB agar plates at 37 °C. When antibiotics were needed for plasmid selection, they were added at the following concentrations: 50 μg/ml kanamycin and 50 μg/ml ampicillin.All bacterial strains used in this study are listed in A. pleuropneumoniae strain H49 and integrated into a pET26b expression vector by restriction-free cloning  for large-scale protein expression. IPTG was added to induce protein overexpression with a concentration of 0.5 mM when an absorbance reached 0.6 at 600 nm. After 4 h of incubation at 20 °C, cells were harvested by centrifugation at 7500 rpm at 4 °C and resuspended in lysis buffer  containing 1 mM PMSF, 1 mM benzamidine, 1 mg/ml lysozyme, and 0.05 mg/ml DNases. The cell suspension was sonicated for 15 min with 15 s ON/30 s OFF intervals to lyze the cells. This was followed by centrifugation at 17,000 rpm for 30 min to pellet cell debris (The plasmid pET26b-HisP5PA was transformed into l debris . The sup4R73) as the template (54% sequence identity shared with P5PA). MR and automated refinement were performed using PHENIX. Manual refinement was performed using Coot  was concentrated to 12 mg/ml and used in screen-based sitting-drop crystallization trials. The Gryphon robot (Art Robbins Instrument) was used to set up the MSCG 1 to 4, JCSG1, and index commercial screens with drop sizes of 0.4 μl. P5PA was initially crystallized in two hit conditions: 0.1 M potassium thiocyanate, 30% PEG MME 2000; 0.1 M Ches: NaOH, pH 9.5, and 30% PEG 3000. The crystal hits were manually optimized in a 1:1 volume ratio of protein:precipitant by varying the pH and the concentrations of PEG and glycerol. Candidate crystals were looped from the condition (0.05 M potassium thiocyanate and 27.5% PEG MME 2000) into a cryoprotectant solution consisting of the mother liquor with 20% glycerol and flash frozen in liquid nitrogen. The crystals were sent to the Advanced Photon Source Synchrotron Facility (Argonne) for data collection. MR was performed by running Phaser in PHENIX  using Afing Coot . Moleculing Coot  were genThe purification method described in the P5PA protein purification section was modified as follows in order to purify apo-P5PA for ITC. Before elution, P5PAwas denatured in the gravity column. To denature P5PA, 6 M guanidine hydrochloride (GdHCl) was applied to the column, after which a refolding step was performed by stepwise addition of 4.5 M GdHCl, 3 M GdHCl, and finally 1.5 M GdHCl. The protein was then washed with lysis buffer and eluted with elution buffer . The sub200  at the Structure and Biophysical Core Facility within the Peter Gilgan Centre for Research & Learning . Runs consisted of titrating ligand loaded into the injection syringe against purified protein, which was loaded into the sample cell. About 45 μM of purified P5PA was stored in ITC buffer  at 4 °C. The same buffer was used to make solutions of 300 μM G6P, 300 μM P5P, and 45 μM bovine serum albumin. P5PA, bovine serum albumin, and ITC buffer alone were titrated with P5P or G6P. The parameter setting was 2 μl/injection for a total of 20 injections. Data applied to calculate binding constants were referenced against runs performed with ligand alone to control for the heat of ligand solvation.ITC was performed using an auto-iTCP5PA was fluorescently labeled using RED-NHS Second Generation Protein Labeling Kit (NanoTemper). Briefly, 90 μl of purified apo-P5PA at 10 μM was mixed with 10 μl of the provided labeling dye at 300 μM. The mixture was incubated in dark for 30 min and loaded to a special gravity column that is pre-equilibrated in PBS with 0.05% Tween-20 (PBST). After washing the column with 550 μl of PBST, another 450 μl of PBST was added to elute the fluorescently labeled apo-P5PA. The yield of the labeled P5PA was determined by 280-nm readings made on a Nanodrop.Kd. The corresponding final concentrations of the labeled P5PA and the tested compounds were listed in A target compound was successively diluted in PBST by a dilution factor of two to make a total of 16 different concentrations. Then, 10 μl of the labeled P5PA was added to an equal volume of the diluted compounds and loaded to standard capillaries (NanoTemper). The Binding Affinity Mode of Monolith NT.115 (NanoTemper) was used to detect fluorescence changes and deduct 4Cl to remove excess NaCl and Tris from the solution (Endogenous ligands of P5PA were sought by liquid–liquid extraction. About 1 ml of 80% cold acetone (−20 °C) and 20% cold 2 M HCl was added to P5PA in ITC buffer. The mixture was then vortexed for 5 min, incubated at 4 °C overnight, and centrifuged at 15,000 rpm for 10 min. The supernatant was harvested and dried by SpeedVac SC100 (Savant). The dried pellet was dissolved in milliQ water, and the solution was then used for liquid–liquid extraction with acetonitrile and saturated NHsolution , 44. ThepdxB mutant of E. coli K-12 strain was obtained from the Keio collection B + AfuC, pSC101-P5PA(T149A)B + AfuC, and pSC101-P5PA(H203A)B + AfuC were grown using the same conditions with minor modifications. A 96-well microplate was used and incubated in a Cytation five multimode reader (Biotek USA) with shaking. Values of absorbance at 600 nm were measured every 15 min for a time course of 20 h.ΔAs P5PA and AfuA share significant sequence similarity (54% amino acid identity), a reciprocal BLAST approach was used specifically to identify P5PA homologs and discriminate from AfuA. We first queried a P5PA protein sequence (accession: WP_012262855) against the nr database using BLASTp (v2.10.0+) . The topA similar process was used to identify eight AfuA seed sequences, which were queried against the refseq protein database using BLASTp, and returned 234 hits sharing >65% identity over >80% of the sequence. The 88 putative P5PA hits were then queried against the set consisting of 17 P5PA seed sequences, eight AfuA seed sequences, and 234 AfuA sequences using BLASTp. P5PA hits were only considered valid if all three of the most similar sequences were from the P5PA seed sequences and not from the set of AfuA sequences. All 88 hits passed this threshold, and so when combined with the seed sequences, we were able to identify a total of 105 P5PA sequences. In order to further differentiate the sequences with higher confidence, we used the “differential enrichment” mode of MEME  to ident6WCE. All other data are presented within the article or supporting information.The PDB coordinates for the P5PA structure have been deposited with PDB accession code: This article contains The authors declare that they have no conflicts of interest with the contents of this article.


CRISPR/Cas9-mediated knockout of ATP13A1 in both HAP1.MR1 and THP-1 cell lines revealed a profound reduction in MR1 protein levels and a concomitant functional defect specific to MR1-mediated antigen presentation. Collectively, these data are consistent with the ER-resident ATP13A1 being a key posttranscriptional determinant of MR1 surface expression.The monomorphic antigen-presenting molecule major histocompatibility complex-I-related protein 1 (MR1) presents small-molecule metabolites to mucosal-associated invariant T (MAIT) cells. The MR1-MAIT cell axis has been implicated in a variety of infectious and noncommunicable diseases, and recent studies have begun to develop an understanding of the molecular mechanisms underlying this specialized antigen presentation pathway. However, proteins regulating MR1 folding, loading, stability, and surface expression remain to be identified. Here, we performed a gene trap screen to discover novel modulators of MR1 surface expression through insertional mutagenesis of an MR1-overexpressing clone derived from the near-haploid human cell line HAP1 (HAP1.MR1). The most significant positive regulators identified included β Escherichia coli (E.coli), Salmonella typhimurium, and Mycobacterium tuberculosis (The monomorphic major histocompatibility complex (MHC) class I-related protein 1 (MR1) presents small-molecule metabolites to mucosal-associated invariant T (MAIT) cells , 3. Whilrculosis , 14, 15.rculosis , 17, 18,rculosis , 20, 21.rculosis . Their hrculosis , 23. Furrculosis , 27, 28.2-microglobulin (β2m)-complex translocates to the cell surface  < 0.01), including the MR1 transgene itself and β2m  . Since H+ cells . The res+ cells  (Ac6FP) + cells . The HAP+ cells  (see Expytometry  and sequytometry . In totasertions . The sensertions . Out of  and β2m . Intrigu and β2m ) but als TMEM131 ), endoly LAMTOR2 , 51 and  LAMTOR2 , 53), N-., NAA30 ), and ubg., CUL3 , 56)  to ., VPS29  and VPS5., VPS29 ), cytosk, NHLRC2 , 64), gl., GANAB  and SPPL., GANAB ), mRNA pg., PARN  and ZCCHg., PARN ), and iog., PARN )  was B2M, the gene encoding β2m (2m is a known requirement for MR1 surface translocation (−65), ATP13A1 , GANAB , VPS53 , and IRF2  displayed an MR1 phenotype with at least one of two single guide RNAs (sgRNAs) targeting the gene in an initial validation in bulk HAP1.MR1 cells . Strikingly, total cellular MR1 levels were reduced in all but one HAP1.MR1 ATP13A1 KO clones regardless of the sgRNA used  was transiently transfected with plasmids encoding the Cas9 protein, the fluorescent protein mRuby, and one of three different sgRNAs targeting different regions of the ATP13A1 gene . Single ern blot A. Strikiin yeast . Alteratin yeast . This maC and D). In a T cell stimulation assay, the three HAP1.MR1 ATP13A1 KO clones elicited reduced MAIT cell activation compared with the parental clone when pulsed with the MR1 ligand 5-(2-oxopropylideneamino)-6-D-ribitylaminouracil  + methylglyoxal (MG)) (E). Importantly, ATP13A1 overexpression increased the antigen presentation capacity of all three HAP1.MR1 ATP13A1 KO clones, and this trend was statistically significant at some antigen concentrations ) E. Importtrations E and S7.+ upon sorting but still expressed the protein (“ATP13A1-sufficient clones”). Details on editing of the clones used for the individual experiments can be found in Fig. 2F). This difference was statistically significant in the presence of Ac6FP and as with the total protein content, the reduced MR1 surface expression could be reversed by overexpression of ATP13A1 . Importantly, surface expression of classical MHC class I molecules was not similarly impacted by loss of ATP13A1, suggesting that the phenotype is specific to MR1 . Indeed, MHC class I surface levels were elevated in the ATP13A1 KO clones, possibly because of increased availability of β2m or components of the PLC, which contribute to the stabilization of MR1 in the ER  and one clone per sgRNA that was mRubyn the ER .2m was not differentially expressed in the KO compared with the sufficient clones at the transcript (H) or protein level (C and F), which demonstrates that β2m is not a limiting factor for MR1 expression in these cells. However, MR1 transcript levels were statistically significantly reduced in the ATP13A1 KO clones (I). Thus, we cannot exclude an effect of ATP13A1 on MR1 mRNA stability in the HAP1 system.Additionally, βanscript H or protin level , indicatin level , C and FO clones I. Thus, To confirm that the MR1 phenotype was not specific to the HAP1.MR1 parent clone used in the screen, we used sgRNA2 to knock out ATP13A1 in two other HAP1.MR1 parent clones . The same pattern was evident when analyzing the splice variants of XBP1, a transcription factor that controls the expression of UPR proteins in response to ER stress and is itself activated by alternative splicing of its mRNA (E). The data also indicate that the HAP1.MR1 ATP13A1 KO clones do not suffer from elevated ER stress at baseline, suggesting that the lower MR1 protein levels are not due to a general defect in protein folding.Having established that ATP13A1 KO clones display an MR1 phenotype attributable to ATP13A1 deficiency, we next investigated the mechanism by which ATP13A1 expression modulates MR1 protein levels. Since ATP13A1 has been implicated in the transport of cations , 74, we its mRNA  Fig. 3EEA–D. TheF). Interestingly, the much smaller pool of MR1 molecules in the HAP1.MR1 ATP13A1 KO clones was equally sensitive to the endoglycosidase, indicating that these molecules are glycosylated normally within the ER. This makes it seem unlikely that a general dysfunction of the ER glycosylation machinery is the cause for the reduced MR1 protein levels at baseline. Importantly, protein levels of classical MHC class I molecules were unaffected in the HAP1.MR1 ATP13A1 KO clones  and confirmed disruption of ATP13A1 at the genomic level by Next-Generation Sequencing. This allowed us to investigate whether ATP13A1 deficiency impacted endogenous MR1. While MR1 mRNA levels were unaffected , MR1 surface expression was reduced in five THP-1 ATP13A1 KO clones compared with ATP13A1-sufficient controls . This trend did not reach statistical significance in one repeat due to one sample having high isotype staining . Indeed, the fold-change of MR1 geometric mean fluorescence intensity (GeoMean) induced by Ac6FP was smaller in these clones , suggesting that the observed differences in MR1 surface expression are not simply due to the smaller pool of MR1 molecules available. While surface expression of classical MHC class I molecules was more heterogeneous than MR1 in ATP13A1 KO clones, there was no statistically significant difference compared with ATP13A1-sufficent clones . As expected, MHC class I surface levels were unaffected by incubation with Ac6FP .HAP1 cell cultures are prone to becoming polyploid over time, due to a competitive growth disadvantage of haploid cells . ConsistFig. 4staining . Importae.g., Fig. S13B).When ATP13A1 expression was reconstituted in the THP-1 ATP13A1 KO clone 2, MR1 surface expression was restored . Total MA). This trend was observed across multiple experiments using different MAIT cell donors and 5-A-RU concentrations, although it did not always reach statistical significance (A). Since the antigen presentation pathways for exogenous ligand and intracellular bacteria differ mechanistically . Importantly, this functional defect appeared to be specific to MR1-mediated antigen presentation since the response of a peptide-specific T cell line to peptide-pulsed ATP13A1 KO clones was comparable to that elicited by ATP13A1 sufficient clones . Similarly, presentation of the CD1d ligand α-Galactosylceramide  to sorted human invariant natural killer T (iNKT) cells was not significantly affected by loss of ATP13A1 . Of note, ATP13A1 was not a hit in a recent whole-genome siRNA screen for modulators of antigen presentation by CD1d  to sorted human MAIT cells less efficiently than their ATP13A1 sufficient counterparts A. This tificance A. Since stically , 39, 42, by CD1d .Figure 5E and S14E). Overexpression of ATP13A1 in the THP-1 parent clone, however, did not increase antigen presentation. Thus, it appears plausible that the transporter operates within a tightly controlled regulatory network optimized to prevent aberrant MAIT cell activation.Lastly, when ATP13A1 expression was restored in ATP13A1 KO clone 2, its capacity to present 5-OP-RU was partially rescued , and we found no evidence for a differential global induction of the unfolded protein response in the HAP1.MR1 ATP13A1 KO clones (A), this inhibition did not lead to an increase in full-length MR1 protein levels (B). This suggests that MR1 is not differentially degraded by ERAD upon ATP13A1 KO under homeostatic conditions. Similar results were obtained in the HAP1.MR1 ATP13A1 KO clones (A–E), do not suffer from elevated ER stress at baseline. Like their HAP1.MR1 counterparts, THP-1 cells also responded to thapsigargin with upregulation of HERPUD1, showing that their ER stress response is intact but not triggered by loss of ATP13A1 alone (A).Since MR1 transcript levels did not significantly differ between ATP13A1 KO and sufficient THP-1 clones B, and weO clones , we hypoO clones , 93. In O clones . p97/VCPO clones , 95. EveO clones A, this in levels B. This sO clones . The lowO clones , A–E, doA1 alone A.Figure B, C and E). MR1 transcript levels were less than twofold different (D).To further characterize the effect of ATP13A1 on the kinetics of MR1 expression, we performed pulse-chase experiments after metabolic labeling of nascent proteins with radioactive amino acids. In order to be able to immunoprecipitate nascent MR1 molecules regardless of their folding state, we overexpressed human influenza haemagglutinin (HA)-tagged MR1 in one THP-1 ATP13A1 KO clone and one THP-1 ATP13A1-sufficient clone as well as the THP-1 parent clone . Confirmifferent D.C). However, a trend toward lower MR1 total protein levels was evident even at the time of labeling, although this did not reach statistical significance (E). These differences were small but consistent between experimental repeats and suggest that ATP13A1 modulates either the rate of MR1 protein synthesis or the stability of the nascent MR1 polypeptide immediately after its cotranslational translocation into the ER. Importantly, the abundance and kinetics of folded MHC class I molecules were not affected in ATP13A1 KO cells (D). The increase in MHC class I signal over the course of the experiment can be explained by ongoing postpulse folding, since the antibody used is specific for the folded conformation , MR1 surface levels were reduced in these cells compared with the THP-1 parent and the ATP13A1-sufficient clone 15 across a range of transduction levels (C and E). Importantly, MHC class I surface expression was not affected (D and F). In conclusion, in the absence of ATP13A1, we observed reduced expression of endogenous MR1 as well as of three exogenously expressed constructs, corroborating the conclusion that MR1 protein levels are stabilized by this P5-type ATPase.To exclude the possibility that the reduced MR1-HA expression in the ATP13A1 KO background was caused by differences in transduction efficiency, we transduced the clones with an MR1-Emerald construct featuring an internal ribosomal entry site (IRES) . In thesn levels , C and Eaffected , D and FMAIT cells are one of the most recent additions to the family of innate-like, unconventional T cells . Unlike et al.  were putative negative regulators of MR1 surface expression in our screen. This differs from a recent CRISPR screen performed by McWilliam, Mak et al. , who fouet al. , McWilliet al. , 36. Altlow population in this study, we focused on the P5-ATPase ATP13A1. The data presented here support the conclusion that this transmembrane helix dislocase modulates the size of the intracellular pool of MR1 and consequently, MR1 surface expression and antigen presentation. ATP13A1 is the mammalian orthologue of the yeast ATPase Spf1p  and is unique among the five known mammalian P5-ATPases  are components of a complex that regulates the interconversion of phosphatidylinositol-derived signaling lipids  supplemented with 10% fetal calf serum , 2 mM Glutamine (Sigma-Aldrich), 1 mM Sodium Pyruvate (Gibco), 1× Non-essential amino acids (Gibco), 100 U/ml penicillin +100 μg/ml streptomycin (Sigma-Aldrich), 10 mM HEPES (Gibco), and 50 μM β-mercaptoethanol (Gibco). HEK293T cells were cultured in DMEM (Sigma-Aldrich) supplemented in the same way. Sorted human T cells were cultured in IMDM (Gibco) supplemented with 5% pooled human serum (NHS Blood and Transplant Unit), approximately 5000 U/ml IL-2 . MAIT cells, MelanA-reactive CD8+ T cells, and iNKT cells were sorted from human peripheral blood mononucleocytes (PBMCs) isolated from leukocyte cones obtained from the NHS Blood and Transplant Unit as described below. This work was covered by HTA license number 12433. Cells were kept at 37 °C with 5% COescribed ), and su+ cells by MACS sorting (Miltenyi) according the manufacturer’s protocol. A representative enriched sample was >95% positive for CD2 as determined by flow cytometry . Cells were stored at 4 °C o/n before surface staining for CD3 , CD161 , and the Vα7.2 TCR  to enrich for Vα7.2+ CD161high MAIT cells on a SONY MA900, a FACSAria Fusion, or an FACSAria III sorter (both BD Biosciences) in the WIMM Flow Cytometry Facility. Sort purity was routinely >90%. Invariant natural killer T (iNKT) cells were sorted from PBMC with the antibody clone 6B11 (BioLegend) and maintained as described  as specified in the figure legends, harvested, and transferred to round-bottom 96-well plates for staining with Aqua live/dead stain (Life Technology or Biolegend), followed by Fc receptor blocking , and surface staining on ice. If applicable, cells were split into two wells and primary antibody mix containing either the target antibody or the isotype control antibody was added. When applicable cells were fixed in 2% PFA (Electron Microscopy Sciences) or ICS fixation buffer (eBioscience) before acquisition. Buffer containing 2 mM EDTA was used throughout the procedure for HAP1 cell lines. On occasion, Propidium Iodide (Sigma-Aldrich) was added to samples just before acquisition instead of staining with Aqua live/dead stain. For ploidy staining, cells were fixed in 70% ethanol and stained with 10 μg/ml Propidium Iodide (PI) in the presence of 0.5 mg/ml RNase A before analysis on a flow cytometer in the 561 to 610/20 or the 561 to 620/15 channel on linear scale. Single-color controls were either cells or OneComp eBeads Compensation Beads (Life Technologies). Cells were analyzed on an Attune NxT analyzer equipped with four lasers (Life Technologies), a CyAn ADP analyzer (Beckman Coulter), or an LSR Fortessa X50 fitted with five lasers (BD Biosciences) in the WIMM Flow Cytometry Facility, and data were analyzed and displayed using FlowJo v9 or v10 (FlowJo). Y-axes of histograms show modal counts and x-axes are in logarithmic or biexponential scale except for FSC and SSC, which are on a linear scale. Antibodies used were clone 26.5 for MR1 (BioLegend), clone MOPC-173 as IgG2A control (BioLegend), and clones W6/32 (BioLegend) and G46-2.6 (BD Biosciences) for MHC class I. Fluorophores are indicated on the plot axes.For analysis of HAP1 CRISPR lines generated for bulk validation, cells were dissociated with 0.05% Trypsin-EDTA (Thermo Fisher Scientific), counted using a TC20 automated cell counter (Bio-Rad), and cell number across samples was equalized. Cells were incubated with primary antibody dilution for 1 h at 4 °C. After two washes, cells were incubated with Alexa488-conjugated secondary antibody for 45 min at 4 °C. Cells were fixed in ICS Fixation Buffer for 30 min at RT, washed twice, and incubated in 1× Permeabilization Buffer (BD Bioscience) for 10 min at RT. Cell pellet was then washed and incubated with 0.8 mg/ml RNase A (Applichem) for 1.5 h before two additional washing steps and incubation with 10 μg/ml PI. According to the manufacturer instruction, RNAse A and PI incubation steps were performed in 1× Permeabilization Buffer.E.coli, DH5α bacteria were grown in Luria Broth overnight, washed in PBS, and diluted to an OD600 of 0.5, corresponding to approximately 4 × 108/ml as calculated based on the online Agilent Biocalculator . Stimulation with live bacteria was carried out in medium without antibiotics, and gentamycin (Lonza) was added to a final concentration of 50 μg/ml after 3 h. Peptide  pulsing was performed in serum-free medium. α-Galactosylceramide  was purchased from Enzo Life Sciences. 5-A-RU was synthesized as described previously (2SO4 (Fluka) when the standard curve had fully developed. Color intensity was measured at 490 nm in an iMark plate reader (Bio-Rad) and quantified using a four-parameter standard curve. Supernatants were diluted as appropriate and added in a final volume of 25 μl/well. Standard curves of recombinant IFNγ (PeproTech) were in 50 μl/well.For functional assays, allophycocyanins were incubated with the indicated stimuli for 5 h at 37 °C. Technical duplicates were split either before or after pulsing with the antigen such that the final number of allophycocyanins was 50,000 per well. For stimulation with eviously  and comb2m. Suitable IRDye 680RD and 800CW secondary antibodies were purchased from LI-COR. For quantification of fluorescence-based Western blots, rectangles were drawn around the bands, and signal intensity was determined as median with the border set to right/left and width 3 in the LI-COR Image Studio software. Although we have no definite proof that the data are in the linear range, we assume that this is the case, based on the images.HAP1 cells were harvested for Western blots with Trypsin/EDTA or PBS + 1 mM EDTA to preserve the surface proteome. Sometimes cells were lysed by adding lysis buffer directly to the dish. To make protein lysates, cells were washed in PBS, pelleted, and lysed at 4 °C in either RIPA buffer (Sigma-Aldrich) or self-made lysis buffers  or 20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA pH 8.0, 1% Triton X-100, 1 mM EGTA, 2.5 mM sodium pyrophosphate). All lysis buffers were supplemented with protease inhibitors (Roche). Cell lysates were cleared by centrifugation, and protein-containing supernatants were collected into fresh tubes and quantified with the Pierce BCA protein assay kit (Thermo Scientific) using a twofold dilution curve of BSA (Thermo Scientific) as a standard. HAP1 WT or D9 parent control lysates were not always prepared on the same day as the clone lysates and at times reused in different experiments as indicated in the figure legends. Samples were prepared in 1× Loading Buffer and 1× Reducing Agent (both Invitrogen) to contain equal amounts of protein, boiled, and loaded onto a gel according to their size. Generally, proteins with a molecular weight (MW) below 100 kDa were separated on 4 to 12% Bis/Tris gels (Life Technologies) in MES SDS Buffer (Invitrogen), and proteins with a molecular weight above 100 kDa were separated on 3 to 8% Tris/Acetate gels (Life Technologies) in MOPS Buffer (Invitrogen). Appropriate MW markers were included. Gels were transferred onto a PVDF or nitrocellulose membrane (both Bio-Rad) using the TransBlot Turbo Transfer system (Bio-Rad), and blocked in PBS + 0.1% Tween-20 + 5% bovine serum albumin (BSA) (Sigma-Aldrich or Fisher Scientific). If needed, membranes were cut to size for incubation with different primary antibodies. Membranes were washed in PBS + 0.1 or 0.5% Tween-20 (Acros Organics), incubated with LI-COR secondary antibodies with agitation, washed, and dried between tissues before imaging with the Odyssey Near-Infrared imaging system. For Endoglycosidase H  digestions, equal amounts of protein from RIPA cell lysates were denatured in supplied Denaturation Buffer before splitting each sample in two. One half was incubated with the enzyme in the appropriate Glycobuffer and the other half with Glycobuffer only. Loading buffer and reducing agent were added to each reaction, and samples were analyzed by SDS-PAGE as above. Primary antibodies were clone 6C5  or D16H11  for GAPDH, C4 (Santa Cruz) for ACTB, polyclonal  for MR1, polyclonal  for ATP13A1, clone HC10  for MHC class I, clone EPR9649  for HERPUD1, clone D4B8  for ATF4, and clone EP2978Y  for βFor analysis of HAP1 cells for bulk validation, cells were lysed and collected in RIPA buffer (Sigma-Aldrich) supplemented with protease and phosphatase inhibitors . Lysates were mixed 1:1 with 2× reducing sample buffer (320 mM Trizma base (pH adjusted to 6.8), 40% glycerol, 16 μg/ml bromophenol blue, 8% SDS) and boiled for 10 min. Proteins were separated on 4 to 12% Bis-Tris Gels (Invitrogen) and transferred to PVDF membrane (Biorad). A solution of 0.2% I-Block (Applied Biosystems) in PBS + 0.1% Tween-20 (Sigma-Aldrich) (PBST) was used for blocking and antibody incubation, washing was performed in PBST. Species-specific HRP-conjugated antibody (BioRad) signal was detected using Western Lightning Plus-ECL (PerkinElmer) and imaged on a Universal Hood III machine (Bio-Rad).T value than the samples. qRT-PCRs were run as technical duplicates or triplicates on a QuantStudio7 qRT-PCR machine (Life Technologies), and expression was normalized to a housekeeping gene (HKG). Taqman probes used can be found in RNA was extracted using either the QIAGEN RNeasy kit or the Ambion RNAqueous kit according to the manufacturer’s instructions including the respective DNase digestion protocol. RNA was quantified by Nanodrop (Thermo Scientific), and equal amounts of RNA were reverse transcribed using Ambion or Takara Bio kits. Only cDNA transcribed with the same kit was directly compared in qRT-PCR reactions. HAP1 WT or D9 parent control RNA was not always extracted on the same day as the clone RNA. Pooled “no RT” and “no RNA” controls were routinely included and occasionally gave a signal but always at a much higher CHAP1.MR1-derived clones were treated with 0.1 μM thapsigargin (Sigma-Aldrich) or solvent control DMSO for 3 h. Protein lysates were obtained and Western blots performed as above. In the same experiments, RNA was extracted and reverse transcribed as above. XBP1 cDNA was PCR amplified with previously published primers  Table S and analHAP1.MR1- or THP1-derived clones were incubated in medium containing 10 μM MG-132 (Sigma-Aldrich), 10 μM NMS-873 (Sigma-Aldrich), or the equivalent volume of DMSO for 4 h. Expression of HERPUD1, ATF4, MR1, and GAPDH was analyzed by Western blot as described above.6 THP-1 cells were starved in the absence of methionine and cysteine for 1 h. Following starvation, cells were pulse labeled with 50 μCi of 35S-methionine and -cysteine for 1 min  and immediately transferred into 10× volume of chase medium containing excess amounts of unlabeled cysteine (500 μg/ml), methionine (100 μg/ml), and 100 μM cycloheximide to prevent incorporation of radioactive amino acids. Cells were harvested at 0, 2, and 4 min and lysed in native lysis buffer  Triton X-100) for 1 h. Hemagglutinin-tagged MR1 was immunoprecipitated using monoclonal anti-HA antiserum (clone 12CA5). HLA class I proteins were immunoprecipitated from the supernatant of the first immunoprecipitation with anti-HA, using W6/32 antibody. Quantification was performed in ImageJ by drawing rectangles around the bands, multiplying the mean gray value by the area, and subtracting the value of an equivalent area in the same lane to account for differences in loading across lanes.For metabolic labeling, 1.5 × 107 cells of HAP1.MR1 clone D9 were seeded in a T175 flask on Day 0 and transduced with concentrated gene-trap retrovirus in the presence of 8 μg/ml of protamine sulfate  for three consecutive rounds of transduction on Days 1 to 3 before expansion and freezing. Expanded transduced HAP1.MR1 D9 cells were thawed and approximately 3 × 109 cells were pulsed with 5 μg/ml Ac6FP for 5 h before cell surface staining with anti-MR1 primary antibody (clone 26.5) and goat anti-mouse secondary antibody coupled to Alexa488 (Life Technologies). Stained cells were fixed, permeabilized, and stained with Propidium Iodide before sorting on a LE-SH800 sorter (Sony) fitted with a 100 μm chip over 4 days gating on haploid cells based on PI staining. MR1hi and MR1low populations comprising approximately 1% of total, respectively, were sorted -proximal gene regions were amplified from 4 × 1 μg genomic DNA in four separate reactions per MR1 population using a double-biotinylated primer. Biotinylated single-stranded DNA (ssDNA) was isolated, and Illumina sequencing adapters were added. Libraries were PCR-purified and sequenced on a HiSeq2500 machine (Illumina) generating 50 base pair (bp) single end reads with the 5′ sequencing primer. All primer sequences can be found in et al. , calculated for each gene as shown below. If a gene was sequenced in only one of the populations, one insertion was added in order to enable calculation of the mutational index.Gene trap insertion sites were recovered using linear amplification-mediated polymerase chain reaction (LAM-PCR) similarly to the protocol described by Blomen et al.  and Broc  would underestimate enrichment in the MR1low fraction. Thus, the MI for MR1 was recalculated considering only insertions in exons .Since functional MR1 protein was only produced from the exogenous MR1 cassette, calculating the MI based on insertions in the entire gene body  were generated according to published protocols (http://tko.ccbr.utoronto.ca) (http://genomecrispr.dkfz.de). HAP1 cells were transfected with 2 μg lentiCRISPR v2 bearing the sgRNA of interest in the presence of transfection reagent Turbofectin 8.0 (OriGene). Transfected cells were selected with 2 μg/ml puromycin (InvivoGen) until death of nontransfected control.For bulk validation in HAP1.MR1 parent clone D9, knockout cell lines were generated using the Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system. LentiCRISPR v2 plasmids encoding for rotocols , 127. Tarotocols , TKO v3 onto.ca) , and Genhttps://genome.ucsc.edu/cgi-bin/hgBlat) and cloned into the pX458_Ruby backbone (Addgene #110164). HAP1.MR1 parent clones or a THP-1 WT clone obtained by limiting dilution was transfected with CRISPR plasmids and sorted based on mRuby expression after 2 to 3 days. Single cells were sorted into 96-well plates, and clones were picked for expansion approximately 2 weeks after sorting. For sequencing of CRISPR clones, DNA was extracted using the QIAGEN Blood and Tissue Kit in combination with Ambion Proteinase K. For near-haploid HAP1 clones, genomic DNA around the sgRNA target site was amplified, amplicons were sent for Sanger sequencing at the HIU Sequencing Facility, and sequences were analyzed with SnapGene Viewer (GSL Biotech) and SerialCloner . For polyploid THP-1 ATP13A1 KO clones, regions around the sgRNA target sited were amplified from genomic DNA using primers that generated overhangs compatible with Illumina adapters  target sequence within ATP13A1 each were designed based on the Sabatini/Lander CRISPR pooled library  Table S. sgRNA sadapters . PCR proadapters  web implinkscape.org).Data were analyzed in Excel for Mac 2011 (Microsoft) and plotted using Prism (GraphPad Software) Version 7 and 8. Statistical analysis was performed using Prism (GraphPad Software) Version 7 and 8. Figures were assembled in Inkscape .Data of repeat experiments are included in the Supporting Information whenever practical. Data not included are available upon request (et al. (et al. (et al. (This article contains et al. , Hart et (et al. , and Hol (et al. .M. S. holds consultancies with Nucleome Therapeutics and Enarabio. S. M. N. is a founder, shareholder, board member, and employee of Scenic Biotech BV, a company that employs haploid genetics for target discovery and development. All other authors declare that they have no conflicts of interest with the contents of this article.