Trypanosoma brucei. Among the human CLC family members, the T. brucei proteins display highest similarity to CLC-6 and CLC-7. TbVCL1, but not TbVCL2 and TbVCL3 is able to complement growth of a CLC-deficient Saccharomyces cerevisiae mutant. All TbVCL-HA fusion proteins localize intracellulary in procyclic form trypanosomes. TbVCL1 localizes close to the Golgi apparatus and TbVCL2 and TbVCL3 to the endoplasmic reticulum. Upon expression in Xenopus oocytes, all three proteins induce similar outward rectifying chloride ion currents. Currents are sensitive to low concentrations of DIDS, insensitive to the pH in the range 5.4 to 8.4 and larger in nitrate than in chloride medium.CLC type anion transport proteins are homo-dimeric or hetero-dimeric with an integrated transport function in each subunit. We have identified and partially characterized three members of this family named TbVCL1, TbVCL2 and TbVCL3 in  Trypanosoma brucei ssp. are the causative agents of human African trypanosomiasis (HAT), also called sleeping sickness, and of a related disease in livestock, called nagana. Untreated sleeping sickness is fatal. The parasites are transmitted by the tsetse fly (Glossina spp.). During their life cycle, the parasites must tightly regulate their internal ion composition to survive and proliferate in different environments such as the tsetse fly gut, salivary glands, or the human blood. Until recently, transporters or channels selective for inorganic ions have attracted little attention. Research in T. brucei transport systems has so far mainly concentrated on transporters of organic nutrients such as amino acids, sugars, nucleosides, nucleobases and metabolites [abolites –5.Torpedo marmorata and described as voltage-gated chloride channel [-/H+-exchangers [Anion transport proteins that are members of the CLC gene family are found in all three domains of life. The first CLC member was identified in  channel . Later ichangers . CLCs archangers ,9. Formachangers . Some machangers –12. In tchangers ).T. brucei procyclic forms. After expression Xenopus oocytes the resulting currents were functionally characterized. All three proteins are reversibly inhibited by 4,4-diisothiocyanostilbene-2,2-disulphonate (DIDS) and transport different anions such as chloride, bromide, iodide and nitrate. In T. brucei procyclic forms all three proteins localize intracellularly.In trypanosomes, no chloride transporters have yet been described. Here, we show that three proteins conveying this function are present in Unless otherwise noted, all reagents were obtained from Sigma-Aldrich .Arabidopsis thaliana, Saccharomyces cerevisiae and three members of the tri-trypanosomatid group  were aligned with MUSCLE [Arabidopsis genes from AtClCa to AtClCg are as follows; AT5G40890, AT3G27170, AT5G49890, AT5G26240, AT4G35440, AT1G55620, AT5G33280. Following the alignment, we grew a minimum evolution tree. The complete analysis was carried out in MEGA6 [Protein sequences of CLC proteins in humans, h MUSCLE . Loci foin MEGA6 . Visualiin MEGA6 .Xenopus laevis, we scanned all peptides predicted from gene models version 1.8.3.2 of the assembled Xenopus laevis genome version 9.1, acquired from http://www.xenbase.org/ as follows. As query, we used the Pfam family PF00654 HMM logo, obtained from http://pfam.xfam.org/, and searched with HMMsearch version 3.1b2. The search produced 18 peptides, whereof 15 had E-values between 2.5E-53 and 9.1E-110 for the full sequence. The remaining three hits were of low confidence . In the 15 hits with a low E-value, we found the genes clcn1 through clcn7, clcnkb, and three additional genes. The genes clcn1 through clcn4 were found on both short and long chromosomes; clcn5 through clcn7 and clcnkb were only found on a long chromosome. In addition, we found loc100488828 on a long and loc100487225 on a long and a short chromosome, also bearing the CLC family member motif.To obtain all CLC family proteins in E. coli protein ClC-ec1, and the three T. brucei proteins were aligned using MUSCLE. Alignment and graphical representation was carried out in CLC sequence viewer 7.7.Protein sequences for the described human CLC proteins, the yeast protein GEF1, the Xenopus laevis to obtain the oocytes was done under anesthesia with 0.2% tricaine solution  and analgesic treatment with 25mg/kg Flunixin-Meglumin. Xenopus laevis oocytes were prepared, injected and defollicated as described previously [in vitro with the mMESSAGE mMACHINE kit . Oocytes were injected with 50 nl of solution containing cRNA coding for one of the genes (0.4 μg/μl) or water and then incubated in modified Barth’s solution 2, 0.41 mM CaCl2, 100 units/ml penicillin, 100 μg/ml streptomycin) at 18°C for 3 days before measurements.Animal experiments were carried out in strict accordance to the Swiss ethical guidelines, and have been approved by the Kantonstierarzt of the Canton Bern, Kantonaler Veterinärdienst Bern (BE85/15). Surgery of eviously . PolyadeElectrophysiological experiments were performed using an Oocyte Clamp OC-725  two-electrode voltage clamp amplifier. Currents were digitized at 5 kHz with MacLab/200 .2, 1 mM CaCl2 and 5 mM Na-HEPES (pH 7.4) (chloride medium). The perfusion solution (6 ml/min) was applied through a glass capillary with an inner diameter of 1.35 mm, the mouth of which was placed about 0.5 mm from the surface of the oocyte. The ground electrode contained 3% agar in 3M KCl. Other media used for the functional characterization were the following: gluconate medium where gluconate was used as the major anion containing 90 mM Na-gluconate, 1 mM KCl, 1 mM MgCl2, 1 mM CaCl2 and 5 mM Na-HEPES (pH 7.4). To determine the anion selectivity, 90 mM NaCl in the chloride medium were replaced by either 90 mM NaBr (bromide medium), 90 mM NaI (iodide medium) or 90 mM NaNO3 (nitrate medium).The holding potential was -40 mV. With a frequency of 1 Hz discrete voltage steps of 300 ms duration to potentials ranging from -120 mV to +80 mV in steps of 10 mV were applied. The perfusion medium contained 90 mM NaCl, 1 mM KCl, 1 mM MgClXhoI/NotI and NotI/BamHI, respectively. TbVCL3 was cloned into pDR196 between restriction sites SpeI and EcoRI. S. cerevisiae Δgef1::KanR mutant strain Y16838 , was transformed as described [600) of ~0.6. Cells were centrifuged, adjusted to an O.D.600 of 0.1 in water, washed once and resuspended in SC medium to an O.D.600 of approximately 0.02. A serial dilution (1:2) of bathophenanthrolinedisulfonic acid (BPS)  was prepared in a 96-well plate with round bottom wells, containing 100 μL per well. One hundred microliters of resuspended cells were added to each well in duplicates and incubated at 28°C with constant shaking. As control yeast were grown in the absence of BPS. At defined time points, the cell suspensions were mixed using a multichannel pipette and the O.D.600 was measured using a microplate reader .TbVCL1 and TbVCL2 were cloned into the yeast expression vector pDR195  between S. cerevisiae, cells were pre-incubated at 30°C for 30 min in 200 μL lyticase lysis solution . One hundred and fifty microliters of Promega SV RNA lysis solution  was added to each sample and RNA isolation was continued following the manufacturer’s instructions. RNA samples were treated with DNase I  for 25 min at 37°C, followed by phenol/chloroform extraction and ethanol precipitation. DNase I-treated RNA (0.5 μg) was used for cDNA synthesis using Takara PrimeScript reverse transcriptase . Quantitative RT-PCR was performed using a LightCycler 480 System (Roche). The reaction mixtures consisted of 1x SYBR green premix, Ex taq  and 0.2 μM forward and reverse primers. Two reference genes [5’- CACGGATAGTGGCTTTGGTGAACAATTAC-3’ and ALG9_R, 5’-TATGATTATCTGGCAGCAGGAAAGAACTTGGG-3’; and TAF10_F, 5’- ATATTCCAGGATCAGGTCTTCCGTAGC-3’ and TAF10_R, 5’- GTAGTCTTCTCATTCTGTTGATGTTGTTGTTG-3’. For quantification of TbVCLs-transcripts the same primers were used as for the analysis of parasites (described below).For total RNA extraction from T. brucei 29–13 procyclic forms [7 cells/ml) in SDM-79  supplemented with 10% (v/v) fetal bovine serum  at 27°C. Parasites (4–5 × 107) were harvested by centrifugation at 1250 × g for 10 min, washed once in transfection buffer , resuspended in 450 μl of the same buffer, and mixed with 15 μg of linearized plasmid. Electroporation was performed with a BTX Electroporation 600 System  with one pulse  using a 0.2-cm pulse cuvette . Electroporated cells were immediately inoculated in 10 ml SDM-79 containing 10% heat-inactivated FBS. Clones were obtained by limited dilution in 24-well plates in SDM-79, containing 20% conditioned medium, in the presence of 2 μg/ml puromycin  for selection. Antibiotic-resistant clones were tested for the presence of the introduced constructs by PCR. RNA interference and expression of HA-tagged constructs was induced by addition of 1 μg/ml tetracycline to parasite cultures.ic forms  were culATCGGAATCTAAGCTTGGATCCTGGTCTGTTTGCGCTGATC and reverse primer ATCGGAATCTTCTAGACTCGAGGTGACCAAGCCCACAAACTT. For TbVCL2 a 427bp fragment spanning nucleotides 1436–1863 was amplified using forward primer ATCGGAATCTAAGCTTGGATCCTGACGTGTCGGTCATATCGT and reverse primer ATCGGAATCTTCTAGACTCGAGTTGTGCCACCCACAAAACTA. For TbVCL3 a 354bp fragment spanning nucleotides 403–757 was amplified with forward primer ATCGGAATCTAAGCTTGGATCCTATCTTGAGCGCCGGAAG and reverse primer ATCGGAATCTTCTAGACTCGAGACAGGAACACCGGCAGATAC. The amplified fragments were cloned into vector pALC14  aNA genes , allowinNA genes , a deriv7 parasites previously transfected with the pALC14-TbVCL2-RNAi construct with 10 μg pMS14v5-TbVCL3-RNAi construct as described before [Transfection for double-knockdown of TbVCL2 and TbVCL3 mRNA was carried out by electroporating 4 × 10d before , using pGATGCCTGTTTTCCACCAGT and CGTCTACAACCACCATGTGC were used to amplify a 138 bp long product at position 2512–2650 of the gene. For TbVCL2 forward and reverse primer ACCGTACCGGGTATTCATCA and AGCGCACCACAAATAAAACC were used to amplify a 141 bp long fragment at position 974–1115 of the gene. For TbVCL3 forward and reverse primer CTGCGGATCTTACCCACATT and TCAGGTCAAGCAGTGAGTGG were used to amplify a 104 bp long product at position 1802–1906 of the gene. For Tb927.11.10190 we used the primers described before [-∆∆CT method [Induced and non-induced RNAi cultures were harvested 48 h post induction and total RNA was extracted according to the manufacturers’ protocol using the Promega SV total RNA extraction Kit  or the RNeasy Mini Kit  using the on-column DNase treatment. RNA concentration and purity were measured with a spectrophotometer  and 10 μg total RNA from each time point were subjected to a second DNaseI treatment  when isolated with the Promega SV kit. Afterwards, the RNA was purified by a phenol/chloroform extraction followed by precipitation. RNA concentration was again determined with a spectrophotometer and 2 μg total RNA were used for reverse transcription with random primers using the High Capacity cDNA Reverse Transcription Kit  or 500 ng total RNA were used in combination with the Superscript III Reverse Transcriptase . The resulting cDNA was diluted to the desired concentrations and analyzed by quantitative PCR using the Fast SYBR Green Kit (Applied Biosystems) according to the manufacturer’s instruction. As validated internal control the expression level of Tb927.11.10190 mRNA was measured in parallel . The sped before . Each saT method .NotI, extracted with phenol/chloroform and precipitated prior to transfection. For immunolocalization, procyclic form trypanosomes were cultured in presence of 1 μg/ml tetracycline for 24 h to induce expression of the tagged proteins. Parasites were harvested by centrifugation at 800 x g for 10 min, washed with cold phosphate-buffered saline  and then spread on Superfrost Plus Microscope Slides . Preparation of the slides for microscopy was done as described earlier [To generate cell lines overexpressing HA-tagged versions of TbVCL1, TbVCL2 and TbVCL3 we used the pALC14 vector as described before . For all earlier .2XRos (Invitrogen) and subsequent washing prior to spreading the cells on the microscope slides.The following primary antibodies were used; mouse monoclonal anti-HA , rabbit anti-TbGRASP , rabbit anti-TbCatL and rabbit anti-BiP  at dilutions of 1:250, 1:1000, 1:500 and 1:1000, respectively. The corresponding secondary fluorophore-conjugated goat anti-mouse Alexa Fluor 488 or goat anti-rabbit Alexa Fluor 594 (Invitrogen) antibodies were added at dilutions of 1:1000. Mitochondrial staining was done by incubation of the parasites for 20 min with 0.5 μM MitoTracker Red CM-HFluorescence microscopy was performed on a Leica DMI6000 B microscope (Leica Microsystems). Deconvolution and analysis of the images was done with the Leica Application Suite X software provided by the manufacturer. Separation of Golgi and HA signals in HA-TbVCL1 cells was measured in Icy . To detein silico screen of the predicted T. brucei proteome (v4.0 from ftp.sanger.ac.uk) for ion channels using the hmmer-3.0b3 program [Voltage-gated Chloride channel Like), TbVCL2 and TbVCL3, respectively. Amino acid identity between the members was 24% to 27% as determined with a global alignment [T. cruzi and L. major share 54–61% and 42–49% amino acid identity, respectively. In human proteins highest similarity is found for CLC-6 (AAI17421.1) and CLC-7 (AAF34711.1) with 26–27% for TbVCL1 and TbVCL3 and 21–22% for TbVCL2. All three T. brucei VCLs contain CBS domains  in their C-terminal regions that have been shown to be involved in the functional regulation of certain members of the CLC family [S. cerevisiae, A. thaliana and humans , TbVCL2, does not contain any of the consensus residues.PMA1) promoter in an S. cerevisiae gef1∆ mutant (strain Y16838). Deletion of the GEF1 gene, which codes for a yeast Cl-/H+-exchanger belonging to the CLC family, causes various phenotypes, but sensitivity to iron deprivation can easily be measured [T. brucei VCLs were able to complement the mutant phenotype, we cultivated the cells in the presence of increasing concentrations of the iron chelator bathophenanthrolinedisulfonic acid (BPS). Expression of TbVCL1 led to almost full recovery of growth in the presence of 15 μM BPS compared to the corresponding wild-type strain (BY4742) transformed with the vector pDR (gef1∆ mutant expressing TbVCL2 or TbVCL3 at the same BPS concentration (50) was in the same range as for the wild-type control, while TbVCL2- and TbVCL3-expressing gef1∆ cells as well as the gef1∆ mutant transformed with the vector showed a 3- to 4-fold higher sensitivity to BPS . Increased outward currents were never detected in water-injected control oocytes or in oocytes expressing the T. brucei myo-inositol transporter [T. brucei AAT6 amino acid transporter [T. brucei (see below), the observed currents are due to a spillover of the transport function to the oocyte plasma membrane. It is not clear why this spillover follows an all-or-none logic. Large current signals were detected in 52% of injected oocytes for TbVCL1 (n = 29), 97% for TbVCL2 (n = 29) and 39% for TbVCL3 (n = 26). It should be noted that we can not exclude the possibility that the trypanosome proteins combine with one of the 18 CLC-like proteins found in Xenopus laevis  and outside (95 mM) the oocytes, indicating outward rectification. Experiments with TbVCL1 and TbVCL3 gave comparable results   . TbVCL1 , n = 6)  showed a- and NO3- medium from an oocyte expressing TbVCL2. -, Br-, I- and NO3- as major anion. For all three transport proteins, the anion selectivity was NO3- > I- > Br- > Cl-.In addition, we investigated the anion selectivity of the currents. We found that the current amplitudes at +80 mV elicited by the individual transport proteins were independent of the pH in the range of 5.4–8.4 .T. brucei procyclic forms, we attempted to stably express tetracycline-inducible C- and N-terminal hemagglutinin (HA)-tagged versions of TbVCL1, TbVCL2 and TbVCL3. TbVCL1 only showed fluorescence with the C-terminal tag while TbVCL2 and TbVCL3 showed fluorescence only in the N-terminally tagged version. Expression was confirmed by Western blotting  revealed that 53% of parasites contained one major punctum, with the remainder containing either no (6%), two (29%) or more (12%) puncta of similar intensity. The puncta showed no co-localization with lysosomes (CatL) or the Golgi (TbGRASP), however in most cases they localized close to TbGRASP , we decided to downregulate the expression of these two proteins in the same cell line. After transfecting the TbVCL2 RNAi cell line with a second RNAi construct targeting TbVCL3, we obtained a downregulation of 74% and 62% for TbVCL2 and TbVCL3, respectively, in one biological duplicate, and 73% and 58% in the other one. Despite the reduction in TbVCL2 and TbVCL2 mRNA levels, no difference in growth was observed between induced and non-induced cells over seven days .T. brucei, named TbVCL1, TbVCL2 and TbVCL3. The three proteins share only low amino acid residue identity of < 27% amongst each other. They are also quite distant from the closest human homologs CLC-6 and CLC-7 with an identity of < 27%. CLC-6 and CLC-7 have been shown to catalyze electrogenic Cl-/H+-exchange [T. brucei coding for a putative β-subunit of chloride transport proteins. In the following we compare the TbVCLs with human CLC-6 and CLC-7.We describe the identification and localization of three different anion transport proteins in exchange ,41 and aexchange . We did Xenopus oocytes. As mentioned above, we can not exclude that these proteins form dimers with endogenous CLC like proteins. In all three cases only a fraction of injected oocytes showed extra chloride currents over the plasma membrane. In case of human CLC-6 efficient re-routing to the oocyte plasma membrane required N-terminal attachment of GFP [TbVCL1, TbVCL2 and TbVCL3 induced chloride currents upon expression in t of GFP  and in ct of GFP  were reqt of GFP , while Ct of GFP . We failt of GFP ,41.3- > I- > Br- > Cl-. Anion selectivity of TbVCL1, TbVCL2 and TbVCL3 is different from CLC-6 with NO3- > Cl- > I- [- > Br- > NO3- > I- [The anion selectivity of all three TbVCLs was NOCl- > I-  and CLC-O3- > I- .- conducting proteins, the currents induced by TbVCL1, TbVCL2 and TbVCL3 were reversibly inhibited by DIDS. Other examples of the CLC family that show reversible inhibition by DIDS include CLC-Ka, CLC-2 and CLC-ec1 [50 value for TbVCL2 was about 30 μM. DIDS sensitivity of the Arabidopsis AtClCa transporter has not been reported.Like many Cl CLC-ec1 –44. The S. cerevisiae gef1∆ mutant. It is interesting to note that CLC-6, but not CLC-7 is able to rescue the growth phenotype of this mutant [Arabidopsis CLCs, only AtClCd and AtClCf were able to complement the gef1 mutant phenotype [Arabidopsis localized in the Golgi. In contrast, at least two of the Arabidopsis CLC transporters, AtClCa and AtClCe, that failed to complement the gef1∆ mutant phenotype were localized in chloroplast membranes and at the tonoplast, respectively [While all three TbVCLs induced currents in oocytes, only TbVCL1 was able to complement the phenotype of the s mutant . Of the henotype . Consistectively .T. brucei procyclic forms. TbVCL1 showed a different intracellular localization than TbVCL2 and TbVCL3. The few puncta detected in proximity of the nucleus did not co-localize with markers for lysosomes or Golgi. We speculate that nonetheless, TbVCL1 might be associated with the Golgi apparatus, mainly because of the adjacent localization of the HA-TbVCL1 signal to the Golgi marker TbGRASP. The average number of HA-TbVCL1 puncta per cell  correlated with the number of kinetoplasts , the correlation coefficient, however, was low (r = 0.35). The correlation coefficients of the two indicators, HA-TbVCL1 and Golgi apparatus, showed only weak correlation, too (r = 0.36). Interestingly, TbVCL1 compensated for loss of a S. cerevisiae chloride channel that locates to the late Golgi and the prevacuole in this organism [Localization of the anion transport proteins was investigated in organism . In contorganism . The reaT. brucei. Yeast complementation experiments indicate that at least TbVCL1 works as a genuine chloride transporter.In summary, three putatively independent anion transport proteins of the CLC family have been identified in S1 FigRobustness of the tree depicted in (PDF)Click here for additional data file.S2 FigT. brucei proteins were aligned to known CLC proteins by MUSCLE. Key residues for the permeation pathway are highlighted following their numbering in E. coli; S107, Y445, and E148 –also known as gating glutamate or Eext. In addition, E203 –also known as the proton glutamate or Eint−is depicted, it is an additional key residue for chloride transport in ClC-ec1. Residue background coloring represents amount of identity ranging from dark blue (0%) to bright red (100%), summarized below the alignment.The three (PDF)Click here for additional data file.S3 FigThe mRNA abundance in TbVCL1, TbVCL2 and TbVCL3-expressing yeast, respectively, was quantified by qRT-PCR and normalized to reference genes ALG9 and TAF10, respectively. TbVCL mRNA was not detected in yeast cells transformed with empty vector. The values are mean ± SD of three technical replicates.(PDF)Click here for additional data file.S4 FigThe current-voltage relationships were determined in medium containing chloride as major anion , in medium containing gluconate as major anion  and chloride medium supplemented with 300 μM DIDS (open circles). Substitution of chloride by gluconate leads to a substantial reduction of the outward currents observed during pulses to positive potentials compared to medium containing chloride as major anion. Averaged I/V-relationships obtained from TbVCL1-expressing oocytes  and TbVCL3-expressing oocytes  are shown in panel (A) and (B), respectively.(PDF)Click here for additional data file.S5 Fig50 of 56 ± 18 μM.Concentration-dependent inhibition of the current observed at +80 mV of TbVCL3-expressing oocytes in chloride medium with increasing concentrations of DIDS . The inhibition was fitted with an IC(PDF)Click here for additional data file.S6 FigAll measurements were performed with chloride medium. Measured steady-state currents at a potential of +80 mV were normalized to the value observed at pH 7.4 . We did not find an influence of the pH on the current amplitudes in the measured range.(PDF)Click here for additional data file.S7 FigT. brucei cell lines generated for localization experiments were analyzed by Western blot to confirm overexpression of the tagged TbVCLs. About 5x106 cells of each clone were subjected to 8% SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes, and probed with mouse anti-HA antibody. Expression of the tagged TbVCLs could be confirmed . As loading control the membranes were reprobed with rabbit anti-Bip antibody . Relative position of the protein-ladder is shown on the left side.Induced (+) and non-induced (-) (PDF)Click here for additional data file.S8 FigT. brucei cells are shown as immunofluorescence image and right below the same transmitted light microscopy pictures obtained by differential interference contrast. C-terminally HA-tagged TbVCL1 is shown in green, the Golgi marker TbGRASP in red and the nuclear and kinetoplast DNA stain DAPI in blue. Scale bars indicate 10 μm. TbVCL1 localizes always close to the Golgi apparatus.Ten representative procyclic (PDF)Click here for additional data file.S9 FigCumulative growth of procyclic parasites in absence (open circles) and presence (squares) of 1μg / mL tetracycline was monitored for 7 days. Two biological duplicates are shown (A and B). Induction of RNAi against both TbVCL2 and TbVCL3 showed no growth retardation under standard culture conditions. All data points represent means from two independent experiments.(PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.


Escherichia coli O104 serotypes. All O104 strains examined were intimin negative and belonged to the B1 phylogroup, and some were Shiga toxigenic. We report here the genome sequences of bovine O104:H7 (n = 5), O104:H23 (n = 2), O104:H8 (n = 1), and O104:H12 (n = 1) isolates and human clinical isolates of O104:H7 (n = 5).Cattle harbor and shed in their feces several  Escherichia coli O104:H4, a hybrid pathotype of enteroaggregative and Shiga toxin-producing E. coli (STEC), was responsible for a major foodborne illness outbreak in Germany in 2011  isolated from cattle feces and five O104:H7 human clinical strains. They are members of the B1 phylogroup, which includes a variety of enteropathogenic, enterohemorrhagic, and Shiga toxigenic strains . A complete list of the strains and their genomic characteristics is provided in E. coli O104 strains will further help elucidate the pathogenic potential of E. coli O104 serotypes.Genomic DNA of the target strains was extracted from 1 ml of overnight culture using the Qiagen DNeasy blood and tissue kit . The purity of the DNA was assessed spectrophotometrically using a NanoDrop spectrophotometer . Genomic libraries of all the strains were constructed using Nextera XT DNA library preparation kit, and whole-genome sequencing was performed on an Illumina MiSeq sequencer  using the MiSeq version 2 reagent kit with 2 × 250 cycles. The whole-genome shotgun sequences have been deposited at DDBJ/ENA/GenBank under the accession numbers listed in


Synechocystis sp. PCC 6803 that the Psb29 subunit, originally identified as a minor component of His-tagged PSII preparations, physically interacts with FtsH complexes in vivo and is required for normal accumulation of the FtsH2/FtsH3 hetero-oligomeric complex involved in PSII repair. We show using X-ray crystallography that Psb29 from Thermosynechococcus elongatus has a unique fold consisting of a helical bundle and an extended C-terminal helix and contains a highly conserved region that might be involved in binding to FtsH. A similar interaction is likely to occur in Arabidopsis chloroplasts between the Psb29 homologue, termed THF1, and the FTSH2/FTSH5 complex. The direct involvement of Psb29/THF1 in FtsH accumulation helps explain why THF1 is a target during the hypersensitive response in plants induced by pathogen infection. Downregulating FtsH function and the PSII repair cycle via THF1 would contribute to the production of reactive oxygen species, the loss of chloroplast function and cell death.One strategy for enhancing photosynthesis in crop plants is to improve their ability to repair photosystem II (PSII) in response to irreversible damage by light. Despite the pivotal role of thylakoid-embedded FtsH protease complexes in the selective degradation of PSII subunits during repair, little is known about the factors involved in regulating FtsH expression. Here we show using the cyanobacterium This article is part of the themed issue ‘Enhancing photosynthesis in crop plants: targets for improvement’. One ofixation . Irreverfixation ,5, but afixation ,6. Only fixation , is a poRepair of PSII occurs in all organisms that carry out oxygenic photosynthesis ,9. AlthoSynechocystis sp. PCC 6803 (hereafter Synechocystis 6803), electron microscopy has revealed the isolated FtsH complex to be hexameric and composed of alternating FtsH2 and FtsH3 subunits [var2 and var1 null mutants [The main pathway for degrading damaged D1 during repair involves proteolysis by specific members of the FtsH family of ATP-dependent metalloproteases in both cyanobacteria ,15 and csubunits , which, subunits ,21. Althsubunits ,21, withthf1 (thylakoid formation 1) mutant of Arabidopsis [Synechocystis 6803 [Synechocystis 6803 psb29 null mutant, but specific effects on FtsH were not examined [psb29 null mutant of the cyanobacterium Synechococcus sp. PCC 7942, but changes to the expression of individual FtsH subunits were not investigated [How expression of FtsH complexes is regulated in response to light stress is unclear. Recent studies of the variegated bidopsis  have indbidopsis ,24. The tis 6803  and a roexamined . A reducstigated . In addistigated .Synechocystis 6803, like THF1 in Arabidopsis, is important for normal accumulation of the FtsH heterocomplex involved in PSII repair. Furthermore, affinity purification data suggest that Psb29 physically interacts with FtsH complexes in vivo. To gain further insights into Psb29, we have determined the crystal structure of Psb29 encoded by Thermosynechococcus elongatus, a thermophilic cyanobacterium widely used to study structural aspects of PSII assembly and repair [Here we show that Psb29 in d repair ,29. Psb22.(a)Synechocystis sp. PCC 6803 [Synechocystis 6803 were grown on an orbital shaker in BG11 medium in 250 ml conical flasks at 29°C under moderate light conditions (40 µmol photons m−2 s−1). For purification of protein complexes, the FtsH2-FLAG strain was grown as described above in 500 ml of medium using 2 l conical flasks. For purification of Psb29-FLAG protein complexes, 4 l of Psb29-FLAG strain were grown in a 10 l flask in BG11 medium supplemented with 1 mM glucose, agitated with magnetic stirrer and bubbled with air. In both cases, surface irradiance was increased to 100 µmol photons m−2 s−1 of light to compensate for the longer path length of the flasks. For spot growth tests, 2.5 µl of mixotrophic culture and 102, 103 and 104 serial dilutions were spotted onto BG11 agar plates and grown for 7 days.All mutants were constructed in the glucose-tolerant WT-P strain of PCC 6803  and growPCC 6803 . For mix(b)psb29 gene in Synechocystis 6803 (Cyanobase designation sll1414) was constructed in two steps. First, the flanking sequencing of sll1414, 445 bp upstream and 555 bp downstream, was PCR amplified with primer set sll1414-1F (AGTTTCTCGTTCTGCCGCCTCAGCTCTT) and sll1414-2R (AATGGGGCCTCATAGTGGGGCATGGATTGAAGATATCAGGGCCGATTACAAAGGGGGGGATAGT), and sll1414-3F (ACTATCCCCCCCTTTGTAATCGGCCCTGATATCTTCAATCCATGCCCCACTATGAGGCCCCATT) and sll1414-4R (ATTAACTCCCCATCCACTTCCACTTCGATGAT). The resulting PCR products were then mixed as DNA template for overlap extension PCR with primer set sll1414-1F and sll1414-4R. The fused PCR fragment containing an EcoRV restriction site instead of sll1414 ORF was then cloned into pGEM-T Easy vector. In the second step, a DNA cassette that confers chloramphenicol resistance was inserted into the EcoRV site. Two transformation vectors were selected due to the nature of blunt-end ligation: pSll1414camA has the chloramphenicol marker integrated in the same direction as sll1414, whereas, pSll1414camB has the marker in the opposite direction. Both plasmids were used to transform the glucose-tolerant WT-P strain of Synechocystis 6803, yielding strains ΔPsb29camA and ΔPsb29camB.The transformation vector for disruption of psbA2 locus were generated by cloning PCR fragments into the NdeI and NheI sites of pPD-CFLAG [psb29 (sll1414) was amplified with primer pair CF-Psb29-F (TTTTTTCATATGACTAAAATTCGCACTGTTTCTGACGCCAA) and CF-Psb29-R (TTTTTTGCTAGCGCTTTCGGAACTCTCCGCTGTGGTT) and the coding sequence of ftsH2 (slr0228) was amplified with primer set CF-FtsH2-F (TTTTTTCATATGAAATTTTCCTGG.AGAACTGCCCTACTT) and CF-FtsH2-R (TTTTTTGCTAGCTAGTTGGGGAATTAACTGTTCCTTGACGGGA). The Synechocystis 6803 mutant ΔPsb29camA was used as background strain to generate Psb29-FLAG/ΔPsb29 and insertion mutant Rslr0228::cm [The transformation vectors for expressing C-terminal 3xFLAG-tagged derivatives of Psb29 and FtsH2 at the PD-CFLAG . The cod228::cmR  was tran(c)Synechocystis Psb29 conjugated to keyhole limpet haemocyanin .Preparation of membranes by breaking cells using a Mini-Beadbeater-16 (BioSpec) and anti-FLAG pull downs were performed as described in . The chl(d)The MS analyses of protein bands excised from gels were done on a NanoAcquity UPLC (Waters) on-line coupled to an ESI Q-ToF Premier mass spectrometer (Waters), as described in .(e)ftsH2 and ftsH3 transcript levels by quantitative PCR was performed as described in [ftsH2 and ftsH3 and Transcriptor Reverse Transcriptase (Roche). The rnpB gene encoding the B subunit of ribonuclease P was used as a reference and the analysis was performed in triplicate using three independent cultures.Determination of the ribed in  using sp(f)T. elongatus (Cyanobase designation: Tlr1134) was cloned into the BamHI and XhoI sites of the modified pRSETA expression vector [psb29 using primer set Tlr1134-F (GGATCCGTGCAAAATCCTCGAACTGTCTCTGATACCAAACG) and Tlr1134-R (CTCGAGTCAAGCGGGTGCATCGGAGCTGGCAT). The resulting vector pRSETAPsb29 encodes a recombinant protein consisting of a 6xHis tag at the N-terminus followed by a thrombin cleavage site then Psb29. The E. coli strain KRX was used for recombinant Psb29 expression. Psb29 expression in transformed cells was induced at an OD730 of 0.8 with 1 g l−1 rhamnose and cells were then grown at 18°C overnight. Cells were lysed by sonication in lysis buffer . In some preparations, the lysis buffer was supplemented with a Complete Protease Inhibitor Cocktail Tablet – EDTA . The supernatant was mixed with a Ni-IDA resin . Non-specifically bound proteins were removed by washing three times with wash buffer  and Psb29 was eluted with elution buffer . The protein was concentrated to around 10 mg ml−1 in 20 mM Tris-HCl pH 7.9, 500 mM NaCl and used for crystallization trials. Concentrated samples were placed in sitting drop vapour diffusion crystallization screens using a Mosquito® robot .The coding sequence of Psb29 from n vector  followin322, which diffracted very weakly. If protease inhibitor was omitted, crystals were readily obtained in three crystal forms. Two of these were in P21 (designated A-P21 and B-P21) and the third in I222. The structure was solved by single-wavelength anomalous dispersion (SAD) with the A-P21 crystal form, soaked overnight with 1 mM dipotassium tetraiodomercurate (Jena Bioscience). The P6322 form was soaked overnight in 1 mM 4-(Chloromercuri)benzensulfonic acid sodium salt (Jena Bioscience), but this was not used for phase determination. Crystals were cryoprotected in the mother liquor with 30% glycerol added, and flash-cooled in a loop into liquid nitrogen. Diffraction data were collected at Diamond Light Source and processed using xia2 [1 were found and the structure phased using the autoSHARP [1,I222 and P6322 crystal forms were solved by molecular replacement with Phaser [1 structure as a model. These structures were refined with REFMAC or phenix.refine [For preparations in the presence of protease inhibitor, the only crystals obtained were of needle morphology in P6ing xia2  with XDSing xia2 . See tabutoSHARP  pipelineutoSHARP  and refiutoSHARP . The B-Ph Phaser  using thx.refine . Structux.refine .Table 1(g)Synechocystis 6803 (sll1414 gene product) against UniProt KnowledgeBase Reference proteomes (http://www.uniprot.org). The cut-off threshold was empirically set to 1×10−4 after manually examining the resulting hits. 103 records were from cyanobacteria, 84 from plant, 11 from green algae, 12 from red algae and one from a virus that infects the green alga Chlorella sp. strain NC64A. 211 sequences were then aligned using MAFFT version 7 programme with the ‘G-INS-I’ setting applied [Arabidopsis thaliana THF1, predicted by ChloroP 1.1 Server [211 Psb29 sequences were retrieved by blasting Psb29 from  applied . Gaps wi applied  and then applied  was used applied . The fin applied . Subsets applied . The evo applied . The abo1 Server , were al3.(a)Synechocystis 6803, we performed an immunoblotting analysis of membranes isolated from a psb29 null mutant, ΔPsb29camA, in which the psb29 gene was replaced by a chloramphenicol-resistance cassette . Cultures grown to late-exponential phase under either photoautotrophic or mixotrophic conditions were analysed. Antibodies specific for each of the four FtsH proteins encoded by Synechocystis 6803 revealed that levels of FtsH2 and FtsH3 were decreased substantially in the mutant compared to the WT control, consistent with a specific effect on the accumulation of the FtsH2/FtsH3 hetero-complex, whereas there was less of an impact on FtsH1 and FtsH4 (a). Similar results were also obtained with a psb29 null mutant, ΔPsb29camB, containing the chloramphenicol-resistance cassette inserted in the opposite orientation . Reverse-transcription PCR confirmed that ftsh2 and ftsH3 were still transcribed in ΔPsb29camA so the effect of Psb29 on the expression of FtsH2 and FtsH3 occurred after transcription (b). The 2–5-fold increase in ftsH2 and ftsH3 transcripts in ΔPsb29camA might reflect a compensatory mechanism to increase expression. Importantly, immunoblotting experiments showed that FtsH2 and FtsH3 expression was reduced but not blocked totally in the absence of Psb29 .To test whether Psb29 plays a role in the expression of FtsH in nd FtsH4 a. Similacription b. The 2–(b)Synechocystis 6803 expressing either Psb29 or FtsH2 tagged at the C-terminus by addition of a 3XFLAG tag. Expression of the tagged proteins under the control of the psbA2 promoter in the relevant ftsH2 or psb29 null mutant restored photoautotrophic growth at high irradiances, indicating that the tagged proteins were still functional . Immunoaffinity purification of Psb29-FLAG from detergent-solubilised membranes using anti-FLAG antibodies, followed by 2D gel electrophoresis (clear-native in the first dimension and denaturing in the second) and detection of proteins by protein staining, immunoblotting and mass spectrometry revealed the presence of large complexes containing FtsH2, FtsH3 and FtsH1 (a), which we assign to FtsH2/FtsH3 and FtsH1/FtsH3 heterocomplexes based on previous studies [b). Overall these data support the direct interaction of Psb29 with FtsH2/FtsH3 complexes.To test whether Psb29 interacts with FtsH we generated two strains of nd FtsH1 a, which  studies . Also de studies  and Phb3 studies . Psb29-F(c)T. elongatus as an N-terminal His-tagged protein in E. coli and isolated the protein by Ni-affinity chromatography. Four crystal forms were obtained by hanging drop vapour diffusion; X-ray diffraction data were collected at resolutions from 3.6 Å to 1.4 Å and the structure of Psb29 determined by heavy atom SAD . Each Psb29 subunit consists of 9 alpha helices . It is likely that proteolytic cleavage of the C-terminal helix allows more compact higher resolution crystal lattices to form, as there is insufficient space in these lattices to accommodate the C-terminal helix observed in the P6322 crystal form. The I222 crystal form shows a domain-swapping of the N-terminal helix from the N-terminus to residue Ile22, creating a domain-swapped dimer . Given that the domain-swap is not observed in the other crystal forms, this is probably a crystallization artefact.Psb29 in the other crystal forms was proteolytically cleaved at the C-terminus. In the B-P2(d)Chlorella sp. strain NC64A that possesses a Psb29-encoding gene closely related to green algal Psb29 sequences . In the proteome database interrogated on 11th November 2016, 103 out of 106 cyanobacteria were found to encode Psb29 homologues. The genome sequences of the three remaining cyanobacteria, Limnoraphis robusta CS-951, Leptolyngbya valderiana BDU 20041, and Cyanobium sp. PCC 7001 (Synechococcus sp. PCC 7001) are still incomplete and so still yet might encode Psb29.Bioinformatic analyses revealed that Psb29 and its eukaryotic homologue THF1 are found solely in oxygenic photosynthetic organisms . One exception is a virus infecting the green alga T. elongatus shows a mean sequence similarity of 59.2% with the 102 cyanobacterial Psb29 sequences examined and 53.7% with the 84 plant THF1 sequences. Six residues are totally conserved in cyanobacterial and plant Psb29/THF1 sequences : based on the structure described here, F14, V35, L39, G55 and G138 (T. elongatus numbering) appear important for the packing of alpha helices and R133 at the beginning of helix 7 is within H-bonding distance of E36 in the middle of helix 2 . These sequence identities would suggest a high degree of conservation of tertiary structure between Psb29 and THF1 in this region of the molecule. A ConSurf analysis in which all Psb29/THF1 sequences were fitted into the T. elongatus structure revealed high sequence conservation on one face of the molecule, which would indicate an important role for this region in protein function , in the more divergent region of Psb29. The C-terminal end of the protein is also poorly conserved .The alignment of Psb29/THF1 sequences revealed a variety of small insertions and deletions. In the case of plant THF1, these insertion/deletion events correspond to 4.Synechocystis 6803 are likewise reduced in psb29 null mutants (c). These data suggest a conserved role for Psb29/THF1 in fine-tuning the expression of thylakoid FtsH heterocomplexes.Previous work in Arabidopsis has shown that the absence of THF1 leads to a 40–80% decrease in the amount of the type A and type B FTSH subunits involved in PSII repair as judged by immunoblotting . We showa), it is possible that Psb29 is also involved in the accumulation of FtsH1/FtsH3 heterocomplexes [psb29 null mutant under the conditions examined  proteins involved in plant immunity [We have also presented the first structural information on Psb29. The first 3 and last 16 residues could not be identified in the most complete crystal structure, possibly because of structural flexibility or because of some proteolytic degradation. The fitting of cyanobacterial and plant Psb29/THF1 proteins into the  figures  and 5. Rimmunity . Thus soPSII repair is one of several photoprotective mechanisms used by plants . DespiteWork in cyanobacteria has highlighted D1 synthesis as a weak link in PSII repair due to reactive oxygen species (ROS)-mediated oxidation of elongation factor EF-G required for protein translation . Attemptthf1 null mutant [Chlamydomonas reinhardtii [Although upregulating FtsH activity and the PSII repair cycle would seem beneficial for plant growth, there appear to be situations where plants deliberately downregulate chloroplast FtsH activity, which is known to lead to the enhanced production of ROS even under non-photoinhibitory conditions . The soul mutant  and/or bl mutant  and Chlanhardtii  suggests


Protein–protein interaction studies further demonstrated that yeast yUtp23 and human hUTP23 directly interact with the H/ACA snoRNP protein yNhp2/hNHP2, the RNA helicase yRok1/hROK1(DDX52), the ribosome biogenesis factor yRrp7/hRRP7 and yUtp24/hUTP24. yUtp23/hUTP23 could therefore be central to the coordinated integration and release of ES6 binding factors and likely plays a pivotal role in remodeling this pre-rRNA region in both yeast and humans. Finally, studies using RNAi-rescue systems in human cells revealed that intact PIN domain and Zinc finger motifs in human hUTP23 are essential for 18S rRNA maturation.Two proteins with PIN endonuclease domains, yUtp24(Fcf1)/hUTP24 and yUtp23/hUTP23 are essential for early pre-ribosomal (r)RNA cleavages at sites A0, A1/1 and A2/2a in yeast and humans. The yUtp24/hUTP24 PIN endonuclease is proposed to cleave at sites A1/1 and A2/2a, but the enzyme cleaving at site A0 is not known. Yeast yUtp23 contains a degenerate, non-essential PIN domain and functions together with the snR30 snoRNA, while human hUTP23 is associated with U17, the human snR30 counterpart. Using  Saccharomyces cerevisiae) or 47S (Homo sapiens) precursor (pre-rRNA) by a series of endonucleolytic cleavages followed by exonucleolytic trimming, which results in the concomitant removal of external  and internal  transcribed spacer sequences RNAs are processed from an initial 35S . The. TheSaccin vitro and yeast yUtp24 was shown to crosslink to both the U3 snoRNA and close to the A1 cleavage site in the pre-rRNA in vivo  endonucleases yUtp24/Fcf1 (hUTP24 in humans) and yNob1 (hNOB1 in humans) are critical for 18S rRNA processing. Early pre-rRNA cleavages at three sites, A0, A1/1 and A2/2a, require the presence of yUtp24/hUTP24 and the PIN endonuclease domain of the protein is essential for cleavages at A1/1 and A2/2a . The yea in vivo . yNob1/h in vivo ,8–11. Onin vitro and specifically associates with the snR30 H/ACA snoRNP in vivo , is essential for early cleavages at A0, A1/1 and A2/2a ,13. Howe in vivo ,15. The  in vivo ,17, and  in vivo ,19. Inte in vivo ), yRrp7  in vivo ), yUtp24 in vivo ). Releas in vivo –24.Metazoa but similar to the yeast protein, it is required for pre-rRNA cleavages in the 5΄ ETS and ITS1  (Considerably less is known about UTP23 in nd mice) ,13 and ind mice) . Howeverin vivo and in vitro approaches to determine the role of yUtp23/hUTP23 in both yeast and human ribosome biogenesis. In vivo RNA-protein crosslinking studies (CRAC) generated a transcriptome-wide RNA binding profile for yeast yUtp23, which provides new insights into its relationship with snR30 and the ES6 region of the 18S rRNA. We also performed in vitro assays using recombinant proteins to determine RNA and protein interaction partners of yeast yUtp23 and human hUTP23 in the SSU processome. Finally, we established RNAi-rescue systems in HEK293 cells to study the effect of mutant hUTP23 on pre-rRNA cleavage in the human system.Here, we present a combination of S. cerevisiae strain expressing genomically encoded, C-terminal HTP-tagged (His6-TEV-protA) yUtp23 under the control of its endogenous promoter (The promoter  was cons600 ∼0.5) were UV-irradiated in a 1.2 m metal tube for 100 s at 254 nm to generate RNA–protein crosslinks. The CRAC method (http://www.novocraft.com). Sequencing data from this publication were analyzed as previously reported .Actively growing yeast cultures in SD medium .The open reading frames of the protein genes listed in 8-PP (PreScission protease recognition site)-2xHA (hemagglutinin) tag was amplified by PCR and cloned into the pcDNA5/FRT/TO vector (Invitrogen) for protein expression under the control of a tetracycline-inducible promoter. The coding sequence of hUTP23 was altered to render it resistant to the siRNAs used to deplete the endogenous mRNA (A human hUTP23 cDNA construct (generated by IDT) containing a C-terminal Hisous mRNA . Point mous mRNA  and conf32P]-αUTP, using plasmid constructs or PCR products with T7 promoter sequences as template ) was added. Products were resolved on 4% polyacrylamide/1x TBE (5% glycerol) native gels and visualized using a Typhoon FLA9000 PhosphorImager.Recombinant proteins were expressed and purified as described previously . RNA subtemplate . RNA bin35S] in vitro translates for 1 h at 4°C in Buffer NB . The beads were washed five times with buffer NB to remove unbound material. Retained proteins were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and visualized by Coomassie staining, immunoblotting or by using a PhosphorImager, respectively.GST-bait proteins were immobilized on glutathione sepharose pre-blocked with bovine serum albumin and incubated with His-tagged recombinant proteins or  5΄ end radiolabeled oligonucleotides (TRI reagent (Sigma-Aldrich) was used to extract RNA from HEK293 cell pellets. For northern blot analysis, 2 μg of total RNA was separated on 1.2% glyoxal–agarose gels, transferred to nylon membrane and hybridized with [leotides . ResultsHEK293 cells expressing HA-tagged hUTP23 proteins were grown on coverslips and induced with tetracycline for 72 h before being fixed with phosphate buffered saline (PBS) containing 4% paraformaldehyde. Immunofluorescence analysis was performed as described in . Briefly32P] 5΄ end radiolabeled oligonucleotides  yUtp23 protein under the control of its endogenous promoter. The affinity-tagged yUtp23 protein supported wild-type growth and actively growing yeast cultures were UV-irradiated as described   Figure . Consist) Figure , the yUtDuring preparation of the cDNA libraries used for sequencing, microdeletions and/or mutations are often introduced at the site of crosslinking and can therefore be exploited to determine precise protein contact sites . Major sin vitro transcribed snR30 RNA, with an estimated dissociation constant (KD) value of ∼300 nM  and might therefore require the presence of the pre-rRNA or other factors within the pre-ribosomal particle.We next examined the yUtp23 crosslinking sites on snoRNAs Figure . For thiM Figure . yUtp23  in vivo . Howeverin vivo , PhosphorImaging (panels C and F) or immunoblotting , respectively.To better understand the role of yUtp23/hUTP23 within this ‘binding platform’ for ribosome biogenesis factors in yeast and humans, we established an proteins  and [35SWe firstly found that yeast yUtp23 interacted with itself (panels B and C), suggesting that, similar to other PIN domain proteins , it mighin vitro transcribed snR30  and yRok1 (panel C). In yeast, snR30 is required for the stable association of yRrp7 to pre-ribosomes but not vice versa . We therin vitro . It is tin vitro  as well as the core box H/ACA protein hNHP2, for their ability to interact with recombinant GST-tagged human hUTP23 . Importantly, positive interactions detected in yeast (panels B and C) were also detected in the human system. Human hUTP23 interacted with hUTP24, hRRP7, hNHP2 and hROK1/DDX52. This indicates that key protein–protein interactions at the 18S ES6 rRNA region are evolutionarily conserved.In conclusion, the observed direct interactions between yUtp23/hUTP23 and factors that also bind to the ES6 region or the snR30/U17 snoRNAs, in combination with the positioning of their crosslink sites on the individual RNAs, provide important insights into their spatial and temporal association within the yeast and human SSU processome (see discussion).In budding yeast, yUtp23 contains only two (D31 and D123) of the four possible acidic residues that make up the characteristic PIN domain endonuclease catalytic center Figure , and mutTET promoter, conditions were established to express WT and mutant proteins at equivalent levels  or downstream of the 2a cleavage site (‘hITS1’) Figure . A 5΄-exSurprisingly, expression of either the PIN  why the PIN and Zinc finger mutations impeded hUTP23 function. The significant accumulation of the 30S pre-rRNA caused by expression of the hUTP23 D31N PIN mutant o Figure , lane 6 o Figure  would clin vitro transcribed U17 snoRNA  that bind the 3΄ hairpin in the snoRNA.Using  Figures . Direct 3 Figure . The top3 Figure  and yUtp3 Figure . Interes3 Figure . This re3 Figure . It was  Figures . Our dat in vivo . We beli in vivo , are sufIn vivo protein–RNA crosslinks were also found between yUtp23 and the ES6 region of the 18S rRNA. The specificity of this interaction was validated with an in vitro RNA binding experiment  also impaired in vitro interactions with snR30 : identifier GSE87238.GEO database (Supplementary DataClick here for additional data file.


SMN1 gene coding for the full-length protein  and the SMN2 gene that preferentially generates an exon 7-deleted protein (SMNΔ7) by alternative splicing. To study SMN and SMNΔ7 degradation in the cell, we have used tagged versions at the N- (Flag) or C-terminus (V5) of both proteins. Transfection of those constructs into HeLa cells and treatment with cycloheximide showed that those protein constructs were degraded. Proteasomal degradation usually requires prior lysine ubiquitylation. Surprisingly, lysine-less variants of both proteins tagged either at N- (Flag) or C-terminus (V5) were also degraded. The degradation of the endogenous SMN protein, and the protein constructs mentioned above, was mediated by the proteasome, as it was blocked by lactacystin, a specific and irreversible proteasomal inhibitor. The results obtained allowed us to conclude that SMN and SMNΔ7 proteasomal degradation did not absolutely require internal ubiquitylation nor N-terminal ubiquitylation . While the above conclusions are firmly supported by the experimental data presented, we discuss and justify the need of deep proteomic techniques for the study of SMN complex components (orphan and bound) turn-over to understand the physiological relevant mechanisms of degradation of SMN and SMNΔ7 in the cell.Spinal muscular atrophy is due to mutations affecting the  SMN1 (telomeric) and SMN2 (centromeric) located in chromosome 5q13   6] and Feptidase . Accordieptidase  in a phyeptidase . The preeptidase  and Itcheptidase  or USP9Xeptidase ,33,34,35The general limitation of this data, extensive to other published studies using similar approaches, is that the mutant constructs, even if used untagged and in cells where the endogenous SMN has been interrupted, can only prove if a determined mechanism is necessary or essential for degradation, but not if it is relevant in normal cell physiology. Those constructs are unlikely to match the post-translational modifications and interactions of the natural cell endogenous protein. More than 40 different proteins are known to interact with SMN through different regions participating in snRNP and SnoRNP assembly, pre-mRNA splicing, actin transport and dynamics, etc. . LysinesDNA constructs for expression of SMN (human SMN) and SMNΔ7 (human SMNΔ7) were generated from human SMN1 cDNA cloned into pDNR-LIB vector   by PCR  using the following oligonucleotides: forward BamH1 SMN, 5′-GACGGATCCCATATGGCGATGAGC-3′ and reverse XhoI SMN 5′-GCGCTCGAGTTAATTTAAGGAATGTGAGCACC-3′. The same forward BamHI SMN and the reverse XhoI non-stop hSMN: 5′-GCGCTCGAGGCTAAATTTAAGGAATGTGAGCACC-3′. Amplified DNA was subcloned into pcDNA 3.1/V5-HisTopo TA vector  after digestion with BamHI/XhoI. SMN2∆7 was generated by PCR amplification with the same template and the forward primer BamH1 SMN as above and the reverse primer XhoI SMN2Δ7: 5′-GCGCTCGAGCTATGCCAGCATTTCCATATAATAGCCAGTATGATAGCCACTCA-3′ or the reverse XhoI non-stop: SMN2Δ7 5′-GCGCTCGAGGCCAATGCCAGCATTTCCATATAATAGCCAGTATGATAGCCACTCA-3′ and inserted after digestion with BamH1/XhoI into pcDNA 3.1/V5-HisTopo TA vector digested with the same restriction enzymes. N-terminal Flag-tagged SMN or SMNΔ7 constructs were generated by PCR using the following oligonucleotides: forward BamHI Flag-SMN 5′ GACGGATCCATGGACTATAAGGACGATGATGACAAGATGGCGATGAGC 3′ and the reverse XhoI SMN as above or the same forward BamHI Flag-SMN and the reverse primer XhoI SMN2Δ7 already described. Amplified DNAs were subcloned into pcDNA3.1 vector (Invitrogen) after digestion with BamHI/XhoI. Lysine-less variants (K0) with all Lys residues changed to Arg of SMN or SMNΔ7 were synthesized by GenScript and subcloned into the appropriate vectors as indicated above. The DNA sequence of all constructs was verified by Sanger sequencing in an ABI Prism 3130XL.2. For transfections, cells were plated at 3 × 105 cells/well in 6-well plates and transfected with Lipofectamine 2000 (Invitrogen). Transfected HeLa cells were plated 24 h after transfection on 60 mm dishes and treated with the protein synthesis inhibitor cycloheximide  for the times indicated, in the absence or in the presence of 20 µM Lactacystin  as indicated. Cells viability after the different treatments (up to 24 h) was >95% as judged by Trypan-blue exclusion. After the treatments, cells were washed with cold phosphate-buffered saline (PBS) and directly lysed in SDS-buffer . Cell extracts were sonicated for 10 min on ice, centrifuged at 14,000× g for 30 min and supernatants used to measure total protein concentration with BCA protein assay kit . Total proteins (20 µg) were separated onto 12% SDS-PAGE gels and transferred to nitrocellulose or PVDF membranes for Western immunoblot analysis. Membranes were blocked with TTBS  with 3% BSA overnight. The blots were then probed with anti-SMN1 antibodies at 1: 1000 , anti-Flag (Sigma) at 1: 5000 or anti-V5 antibodies (Invitrogen) at 1:5000 as indicated. Anti α-Tubulin  monoclonal antibody was used as total protein loading control. Blots were developed with species-specific antisera conjugated with horseradish peroxidase  and detected with a chemiluminiscent method . Blots were analyzed by quantitative densitometry using Totallab TL100 software . Results are expressed as mean ± S.E.M. for a minimal number of three independent experiments.HeLa cells were grown in Dulbecco’s modified Eagle’s medium  supplemented with 10% foetal bovine serum  and 100 µg/mL gentamycin, at 37 °C and 5% COfoci in fluorescence images was analyzed with ImageJ 1.37c software [Cells grown on coverslips were washed 3 times with cold PBS  fixed with 4% paraformaldehyde in PBS for 20 min at room temperature and permeabilized and blocked with 0.1% Triton X-100 containing 3% BSA in PBS for 30 min at room temperature. Primary antibodies  were added in blocking solution without Triton X-100, and incubated 3 h at room temperature. After washing 3 times with PBS, coverslips were incubated with Alexa Fluor 488 conjugated anti-mouse antibody (1:1000 dilution) and 4′,6-diamidino-2-phenylindole  for 1 h, washed again three with PBS. The coverslips were mounted with ProLong Gold antifade reagent for confocal microscopy observation in a laser scanning microscope (Leica TCS SP5). Images were captured with the same settings for all experiments presented. Under these settings no autofluorescence was detected. Controls, omission of primary or secondary antibodies, revealed no fluorescence. Images were processed using ImageJ software . Quantifsoftware  values rTaken together, these data show that SMN and SMNΔ7 proteasomal degradation does not absolutely require internal ubiquitylation nor N-terminal ubiquitylation or acetylation . Nevertheless, we rationalize the need of deep proteomic techniques for the analysis of the turn-over of SMN complex components (orphan and bound) to precisely determine the physiological mechanisms of degradation of SMN and SMNΔ7 in the cell.


Casein kinase 2 (CK2) is highly activated in Alzheimer disease (AD) and is associated with neurofibrillary tangles formation. Phosphorylated SET, a potent PP2A inhibitor, mediates tau hyperphosphorylation in AD. However, whether CK2 phosphorylates SET and regulates tau pathological phosphorylation in AD remains unclear. Here, we show that CK2 phosphorylating SET at Ser9 induced tau hyperphosphorylation in AD. We found that either Aβ treatment or tau overexpression stimulated CK2 activation leading to SET Ser9 hyperphosphorylation in neurons and animal models, while inhibition of CK2 by TBB abolished this event. Overexpression of CK2 in mouse hippocampus via virus injection induced cognitive deficit associated with SET Ser9 hyperphosphorylation. Injection of SET Ser9 phosphorylation mimetic mutant induced tau pathology and behavior impairments. Conversely co-injection of non-phosphorylated SET S9A with CK2 abolished the CK2 overexpression-induced AD pathology and cognitive deficit. Together, our data demonstrate that CK2 phosphorylates SET at Ser9 leading to SET cytoplasmic translocation and inhibition of PP2A resulting in tau pathology and cognitive impairments. Casein kinase 2 (CK2) is highly ubiquitous, evolutionarily conserved in eukaryotic cells . The CK22PP2A), also known as SET, is increased  is compromised in the AD brains , while tncreased . SET is ncreased , where increased , modulatncreased . In the ncreased . Our prencreased . CK2 co-ncreased  while SEncreased , indicatSupplementary Table S1.Site-directed mutations were constructed using the QuikChange site-directed mutagenesis kit (Stratagene) according to the manufacturer’s instructions. Site-directed mutagenesis was carried out to mutate Ser9 of SET to either alanine (S9A) or glutamic acid (S9E), which mimicked the non-phosphorylated or phosphorylated condition of SET respectively. All mutants were generated by PCR and cloned in a His-tagged pcDNA3.1 (-) vector respectively in Xho I and Kpn I restriction sites. We transfected cells with plasmid coding for CK2α, which corresponds to catalytic alpha subunit. All plasmids were sequenced and prepared using an endotoxin-free plasmid extraction kit (Tiangen). AAV2-hTau (full-length 441- amino acid human 4 repeat tau), AAV2-pCAG-SET WT, AAV2-pCAG-SET S9A, AAV2-pCAG-SET S9E, and AAV2-pCAG-CK2 were constructed and packaged by Obio Technology  Co., Ltd. Lipofectamine 2000 transfection reagents were from Invitrogen. Bicinchoninic acid (BCA) protein detection kit was from Pierce . Reagents for cell culture were from Gibco BRL . Antibodies employed in this study are listed in 3×Tg AD mice (PS1m146v/APPswe/TauP301L) were purchased from the Jackson Laboratory. APP/PS1 mice were from the Model Animal Research Center of Nanjing University. Male C57BL/6 mice  were supplied by the Experimental Animal Central of Wuhan University. All the animals were housed in an air-conditioned room  with free access to food and water. The behavioral tests were performed on their active hours.PP2A activity in the cell extracts and brain lysates were measured using the phosphatase kit V2460 according to the manufacturer’s procedure (Promega).Fear conditioning was carried out as described previously . Mice weThe standard Morris water maze (MWM) procedure was used with minor modifications as described previously . The watThe mice were anesthetized by chloral hydrate and perfused intracardially with 400 mL normal saline containing 0.5% sodium nitrite, followed by 400 mL 4% formaldehyde solution and the Golgi dye solution containing 5% chloral hydrate, 4% formaldehyde, and 5% potassium dichromate. After being perfused, the brains were dissected into 5-mm × 5-mm sections and transferred to a vial containing Golgi dye solution for 3 days in dark, then immersed in solution containing 1% silver nitrate for another 3 days. The brains were serially sectioned into 40-μm-thick sections using a vibrating microtome . The number of spines was counted manually from the two-dimensional projections. Classification of mushroom spines was performed as described previously .CK2 activity in the cell extracts and brain lysates were measured using the CK2 kinase Assay/Inhibitor Screening kit CY-1170 according to the manufacturer’s procedure (Cyclex).Primary hippocampus neurons were prepared from E17 to E18. Hippocampus were dissected and gently minced in Hank’s buffered saline solution, then suspended in 0.25% (vol/vol) trypsin solution at 37°C for 15 min. Neurons were cultured in neurobasal medium supplemented with 2% (vol/vol) B-27 and 1× GlutaMAX for AAV infection. All cell culture reagents were purchased from Thermo Fisher Scientific.2 at 37°C. HEK293/tau cells were seeded into 6-well plates 1 day before transfection, performed using Lipofectamine 2000 (Invitrogen), according to the manufacturer’s instruction. Forty-eight hours after transfection with SET [wild type (WT)] or mutants respectively, cells were rinsed twice in ice-cold PBS (pH 7.5) and lysed with buffer containing 50 mM Tris-Cl, pH 8.0, 150 mM sodium chloride, 1% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 0.02% sodium azide, 100 mg/mL phenylmethysulfonyl fluoride, and 10 mg/mL protease inhibitors  followed by sonication for 5 s on ice. After centrifugation at 12,000 g for 5 min at 4°C, supernatants were fetched out and added with equal volume of 2 Laemmli sample buffer. Samples were boiled for 10 min before electrophoresis. Protein concentration was estimated by BCA kit .HEK293 cells were stably transfected with the longest human tau (tau441) (HEK293/tau). The cells were cultured in Dulbecco’s modified Eagle’s medium  in the presence of 200 mg/mL G418 containing 10% fetal bovine serum, and in a humidified incubator aerated with 95% air and 5% CO2, 10% (vol/vol) glycerol, 1:100 PMSF, 1:1,000 protease inhibitor mixture containing 4-(2-Aminoethyl)-benzenesulfonyl fluoride hydrochloride, aprotinin, bestatin, leupeptin, E-64, and pepstatin A. The proteins in the extracts were separated by SDS/PAGE and analyzed by Western blotting using antibodies. Samples of HEK293/tau cell extracts were similarly analyzed. Immunoreactive bands were visualized with the Odyssey Infrared Imaging System (LI-COR Biosciences) and quantitatively analyzed by ImageJ software.Mice brain tissues, cortex, and hippocampus, were quickly dissected out and homogenized on ice to generate 12% (w/v) homogenate in buffer containing 50 mM Tris⋅HCl pH 8.0, 150 mM NaCl, 1% (vol/vol) Triton X-100, 1 mM EDTA, 1 mM MgClCultured cells were fixed in 4% (vol/vol) paraformaldehyde for 15 min and permeabilized in phosphate buffer containing 0.5% Triton X-100 (PBST). Non-specific binding was blocked by incubation in PBST buffer containing 0.1% Triton X-100 and 5% (wt/vol) BSA for 1 h. The primary antibodies against pSET (1:100), pSTAT1 (1:500), or CK2 (1:500) were then applied in blocking solution and incubated at 4°C overnight. The secondary antibodies conjugated to Alexa-Fluor 488/548 were added to the coverslip for 1 h at room temperature, and then Hoechst  for 30 min. The coverslips were washed and mounted onto slides. Images were acquired using a laser two-photon confocal microscope .2PO4, 24 mM NaHCO3, 6 mM MgSO4, 0.5 mM CaCl2, and 10 mM D-glucose. Coronal slices (350-μm thick) containing the dorsal hippocampus were cut at 4–5°C in the slicing solution using a Leica VT1000S vibratome and then transferred to an incubation chamber filled with oxygenated slicing solution in a 30°C water bath for 1 h before recording. Slices were laid down in a chamber with an 8 × 8 microelectrode array in the bottom planar  and kept submerged in artificial cerebrospinal fluid  with a platinum ring glued by a nylon silk. Signals were acquired using the MED64 System . The fEPSPs in CA1 neurons were recorded by stimulating the Schaeffer fibers from DG. LTP was induced by applying three trains of high-frequency stimulation .Mice (2 month-old) were used for all our electrophysiology experiments. Mice were deeply anesthetized as mentioned above. When all pedal reflexes were abolished, brains were removed and placed in ice-cold oxygenated slicing solution containing the following: 225 mM sucrose, 3 mM KCl, 1.25 mM NaHBilateral hippocampus DG zone of mice were injected with AAV respectively as described previously . The injt-test was used to determine the different means among the groups. The level of significance was set at P < 0.05.Data were expressed as means ± standard deviation (SD) and analyzed using commercial software . The two-way analysis of variance or one-way analysis of variance, or a Student’s Figure 1A) and 3xTg AD  mouse models from 3 to 6 month age. Immunoblotting analysis and quantification support that SET Ser9 again was highly phosphorylated in 6-month-old mice compared with 3-month-old in both animal models . In WT C57 mice, no difference was observed in CK2 activity and SET phosphorylation from 3 to 6 month age . As a substrate of CK2 . These data implicate that CK2 activation in AD may bring along with SET hyperphosphorylation in an age-dependent manner.CK2 is activated in AD and we have previously showed CK2 phosphorylates SET in HEK293/tau cells , howevere of CK2 , the posFigure 2A). A similar result was observed by infecting primary neuronal culture with an adeno-associated virus type 2 (AAV2) coding for human Tau (AAV2-hTau) . Immunoblotting validated SET Ser9 was highly phosphorylated following treatment with Aβ  and human tau overexpression . Moreover, treatment with TBB, a CK2 inhibitor, significantly decreased the SET Ser9 hyperphosphorylation induced by Aβ or hTau . Positive control STAT1 was also highly phosphorylated in both conditions by CK2 .To test the possibility that CK2 activation causes SET hyperphosphorylation in AD, we treated primary neuronal culture with Aβ and analyzed CK2 kinase activity. We found that Aβ stimulus resulted in a marked increase in CK2 activity . As positive control, STAT1 was highly phosphorylated and strongly localized in the nucleus. Treatment with TBB abolished STAT1 phosphorylation . Hence our data suggest that Aβ or tau can induce CK2 activation and may subsequently lead to SET Ser9 phosphorylation, resulting in its cytoplasmic translocation.SET is a nuclear protein which translocate to the cytoplasm in AD . To demoSupplementary Figure S3), while SET was prominently phosphorylated in CK2 injected mice . The quantification of cells with cytosolic p-SET in cortex and hippocampus further implicated that SET phosphorylation led to its cytoplasmic translocation . Immunoblotting validated that overexpression of CK2 significantly increased SET phosphorylation at Ser9 compared to the control . Next, we performed fear conditioning experiment. We found that overexpression of CK2 strongly incurred the cognitive deficit . Moreover, MWM test also showed that CK2 overexpression triggered the cognitive impairments . However, overexpression of CK2 did not affect the motor activity because the swimming speed remained comparable in control and CK2 overexpressed animals . To demonstrate indeed CK2-triggered cognitive defect is associated with synaptic plasticity deficit, we performed electrophysiology analysis . We found that overexpressed CK2 substantially decreased the fEPSP slope, supporting that CK2 amplification damaged or sabotaged the synaptic plasticity . To further investigate whether CK2 overexpression affects the synapses, we infected primary hippocampal neurons with control AAV2 or AAV2-CK2. In 3 days, we conducted immunofluorescence staining, and found that overexpression of CK2 decreased the total dendritic length and the average number of dendritic branches. Noticeably, the spine density and mushroom type were reduced by CK2 overexpression as well . To support this observation, we also performed immuno-blotting analysis of a panel of synaptic proteins in the hippocampus of CK2 mice and in primary hippocampal neurons. We found that numerous synaptic proteins including synaptophysin, synapsin1, and NR1 were evidently reduced after CK2 infection , and the quantitative data were summarized in Figures K and Supplementary Figure S10B. Thus, our data strongly support that CK2 overexpression and its activation initiates the cognitive defects while impairing synaptic plasticity and synaptogenesis.To demonstrate whether CK2 activation induces any cognitive defects, we injected C57/BL6 mice dentate gyrus with AAV2 coding for CK2 (AAV2-CK2). In 1 month, immunofluorescent analysis showed that SET translocated from nucleus to cytoplasm in hippocampus dentate gyrus zone  or CK2 infected mice . To further investigate whether CK2-induced tau hyperphosphorylation is mediated via SET phosphorylation, HEK293/tau cells were transfected with WT SET and different SET mutants including non-phosphorylation SET S9A and phosphorylation mimetic SET S9E . We found that overexpression of WT SET led to significant reduction in PP2A phosphatase activity, which was further reduced by SET S9E, while non-phosphorylation SET S9A displayed no any inhibitory activity . Next, we monitored tau phosphorylation status with different antibodies in immunoblotting analysis. Tau phosphorylation at Ser199, Ser202/Thr205 (AT8), Thr231, Ser396, and Ser404 sites tightly correlated with PP2A phosphatase activity , and the quantitative data was summarized in Figure 4C.Previous studies have showed that CK2 immunoreactivity is markedly increased in NFTs-bearing hippocampal neurons with strong anti-tau immunolabeling, compared with those without NFTs . In the Supplementary Figures S12D–F). In 1 month, we analyzed PP2A enzymatic activity. As expected, overexpression of SET significantly inhibited PP2A activity. Noticeably, phosphorylation mimetic SET S9E displayed much stronger inhibitory activity, by contrast, SET S9A exhibited no inhibitory activity . Immunoblotting from hippocampal  and cortical  tissues revealed that the phosphorylation of tau also tightly correlated with the phosphatase activity of PP2A. The quantification data were summarized in Figure 4F and Supplementary Figure S13B. These data further demonstrate that SET phosphorylation inhibits PP2A activity and subsequently induces tau hyperphosphorylation.To assess whether SET phosphorylation is responsible for CK2 overexpression-induced cognitive defects, we injected different AAV2  into the hippocampus of C57/BL6 mice . We made similar observations in fear conditioning assay . Electrophysiology also supported that overexpressed WT SET or S9E evidently reduced synaptic plasticity. In contrast, SET S9A displayed no any effect . Golgi staining also strongly supported that overexpressed WT SET or S9E significantly reduced spine density and mushroom type, by contrast, SET S9A had no any effect . The quantification was summarized in Figures 5I,J. Moreover, we also performed immunoblotting analyses with tissues from hippocampus  or cortex  and found that overexpressed WT SET or S9E robustly reduced the synaptic proteins including synaptotagmin, synaptophysin, NR2B, PSD95, and PSD93. Once again, SET S9A had no any effect, indicating that WT SET or its phosphorylation mimetic mutant S9E substantially reduces the synaptic proteins expression, fitting with the Golgi staining observations. The quantified data were summarized in Figure 5L and Supplementary Figure S14B. Overall, our data strongly suggest that SET phosphorylation induces tau hyperphosphorylation via inhibiting PP2A, leading to the loss of synaptic plasticity.Next, we performed the behavioral assay. We employed MWM and fear conditioning assays. MWM demonstrated overexpression of WT SET or S9E significantly induced the cognitive defects. By contrast, SET S9A overexpressed mice remained comparable with WT control . PP2A phosphatase activity assay supported that SET S9A transfected cells displayed much higher phosphatase activity than WT SET . Immunoblotting assay of p-tau tightly correlated with the phosphatase activity . The quantified data were summarized in Figure 6C. Together, these data strongly suggest that phosphorylation of SET at Ser9 is responsible for CK2 activation-induced cognitive defect through blocking PP2A phosphatase activity.To test whether SET phosphorylation by CK2 is really responsible for its overexpression-induced cognitive deficit, we firstly transfected HEK293/tau cells with WT SET or non-phosphorylation SET S9A in the presence of CK2 . In 1 month, we performed phosphatase PP2A activity analysis . As expected, tau phosphorylation tightly correlated with PP2A phosphatase activity in both hippocampus  and cortex . The quantification of the western blot results was summarized in Figure 6F and Supplementary Figure S16B. These data further confirm that phosphorylation of SET at Ser9 is required for CK2-induced tau pathology.To investigate whether SET phosphorylation is indeed responsible for this event, we co-injected AAV2 SET S9A with AAV2 CK2 into the hippocampus of C57/BL6 mice  and fear conditioning assays . Electrophysiology analysis showed that co-injection of non-phosphorylation SET S9A with CK2 significantly increased the fEPSP slope than WT SET co-injection with CK2 , supporting non-phosphorylation of SET at Ser9 attenuates the loss of the synaptic plasticity. Notably, Golgi staining indicated that SET S9A animals revealed substantially more dendritic spines and mushroom type spines compared to WT SET co-injected ones in the presence of CK2 , supporting that blocking phosphorylation of SET at Ser9 inhibits the CK2-induced synaptic plasticity impairment and the resulting cognitive defect. The quantified data were summarized in Figures 7I,J. Immunoblotting also fitted with this observation as the synaptotagmin, synapsin, PSD95, and PSD93 were clearly increased in SET S9A samples compared with WT SET , supporting that the blocking phosphorylation of SET reverses the synaptic defect induced by CK2.Next, we performed MWM and fear conditioning cognitive functional assays. Again, non-phosphorylation SET S9A completely reversed CK2-induced defects in both MWM (In the current study, we provide extensive evidence demonstrating that CK2 activation by Aβ or tau can induce SET Ser9 phosphorylation, leading to its cytoplasmic translocation, where it blocks PP2A activity, resulting in tau hyperphosphorylation in AD mouse model. Overexpressed phosphorylation mimetic SET S9E mimics the pathology and behavior deficit induced by CK2 strongly supporting that SET Ser9 phosphorylation is responsible for the CK2-mediated pathological effects. On the other hand, we provide additional evidence by co-injecting CK2 virus and non-phosphorylated SET S9A. We found that non-phosphorylated SET reversed CK2 overexpression-induced pathology and cognitive defects. Together, our data strongly support the notion that CK2-mediated phosphorylation of SET induces tau payhology in AD.SET is a nuclear protein , which hOur findings show that CK2 phosphorylates Ser9 on SET leading to its cytoplasmic translocation and inhibition of PP2A resulting in tau phosphorylation in AD. This finding provides a novel molecular mechanism for tau pathology in AD development.All animal experiments were approved by the Animal Care and Use Committee of Huazhong University of Science and Technology and performed in compliance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.XW designed the research. QZ, YX, YW, YS, KZ, YM, FH, MW, DK, and QW performed the research. BZ, RL, J-ZW, KY, and XW analyzed the data. KY and XW wrote the paper.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Here we focus on investigating the changes at the level of membrane proteins in these mutants. Our LC/MS-MS analyses of tandem mass tag (TMT) labeled peptides derived from the cyp79B2/B3 and myb28/29 relative to wild type resulted in the identification of 4,673 proteins, from which 2,171 are membrane proteins. Fold changes and statistical analysis showed 64 increased and 74 decreased in cyp79B2/B3, while 28 increased and 17 decreased in myb28/29. As to the shared protein changes between the mutants, one protein was increased and eight were decreased. Bioinformatics analysis of the changed proteins led to the discovery of three cytochromes in glucosinolate molecular network (GMN): cytochrome P450 86A7 (At1g63710), cytochrome P450 71B26 (At3g26290), and probable cytochrome c (At1g22840). CYP86A7 and CYP71B26 may play a role in hydroxyl-indolic Gls production. In addition, flavone 3′-O-methyltransferase 1 represents an interesting finding as it is likely to participate in the methylation process of the hydroxyl-indolic Gls to form methoxy-indolic Gls. The analysis also revealed additional new nodes in the GMN related to stress and defense activity, transport, photosynthesis, and translation processes. Gene expression and protein levels were found to be correlated in the cyp79B2/B3, but not in the myb28/29.Glucosinolates (Gls) constitute a major group of natural metabolites represented by three major classes  of more than 120 chemical structures. In our previous work, soluble proteins and metabolites in Arabidopsis mutants deficient of aliphatic ( After sulfation, the core Gls structure is formed. In aliphatic Gls biosynthesis, the methionine chain-elongation and the core structure biosynthesis are under the control of three transcriptional factors MYB28, MYB29, and MYB76  as natural anticancer compounds are represented by three major classes of chemical structures , and discovered new nodes in the glucosinolate molecular network (GMN) that include stress and defense related proteins like glucan endo-1,3-beta-glucosidase, glutathione S-transferase F7 and glutathione S-transferase F2 and the electron carriers cytochrome B5 isoform C and cytochrome c oxidase subunit 5b-2  labeling LC-MS/MS based quantitative proteomics. Analyses of protein interaction networks using STRING and functional enrichment of the identified proteins using agriGO allowed us to discover new nodes and edges in the GMN. With qRT-PCR, we were able to determine the correlation between gene transcripts and membrane proteins in the two mutants. Together with our published soluble proteomics work  Heynh ecotype Columbia (Col-0) seeds were obtained from the Arabidopsis Biological Resource Center . The seeds of cyp79B2/B3 and myb28/29 were kindly provided by Dr. John Celenza  and Dr. Masami Hirai , respectively. The mutant genotyping and chemotyping were reported in our previous study , 0.5 M sucrose, and 10 mM phenylmethylsulfonyl fluoride (PMSF). The protein extracts were filtered through cheesecloth and centrifuged at 800 g for 10 min at 4°C. The supernatant was transferred to ultracentrifuge tubes and centrifuged again at 100,000 g for 1.5 h at 4°C. The formed microsomal membrane was washed with 100 mM sodium carbonate using a glass dounce homogenizer, followed by centrifugation at 100,000 g for 1.5 h at 4°C. The microsome pellets were rinsed with 500 μl resuspension buffer containing 100 mM HEPES (pH 7), 1% triton X-100 and 0.5 M sucrose, and centrifuged at 800 g for 10 min at 4°C. Protein was precipitated using 5 volumes ice cold 90% acetone overnight at −20°C, followed by washing the pellets once with ice cold 90% acetone and twice with ice cold acetone before solubilizing in 7 M urea, 2 M thiourea, 4% CHAPS, and 0.25% Triton X-100. The protein amount was assayed using an EZQ assay kit .Protein was extracted according to Pang et al.  by grindg centrifugation at 4°C for 15 min. After washing with ice cold 90% acetone, the pellets were solubilized, reduced, alkylated and digested with modified trypsin  at a 1:25 (w/w) ratio for 16 h at 37°C, followed by TMT labeling according to the TMT 6-plex kit manual . The WT replicates were labeled with 126 and 127 tags, cyp79B2/B3 replicates with 128 and 129 tags and myb28/29 replicates with 130 and 131 tags at room temperature for 2 h. After quenching with 8 μl 5% hydroxylamine for 30 min, the labeled samples were combined and lyophilized. Two independent experiments and four biological replicates each sample were performed.A total of 50 μg protein from each replicate was precipitated with ice cold 90% acetone at −20°C overnight, followed by 20,000 m/z with full MS resolution of 70,000 and 200–2,000 m/z with MS2 resolution of 17,500. The first mass was fixed at 115 m/z, and 445.12003 m/z (polysiloxane ion mass) was used for real-time mass calibration.The TMT labeled peptides were desalted on Macrospin C-18 reverse phase mini-column  and fractionated using an Agilent HPLC 1260 strong cation exchange system as previously described  and methionine oxidation were set as dynamic modifications. Precursor mass tolerance was 10 ppm, fragment mass tolerance was 0.01 Da, spectrum grouping maximum retention time difference was 1.1 and false discovery rate was 0.01 at the peptide level. Proteins quantification based on labeled unique peptides intensities and statistical analyses were performed as previously described .The MS data were searched using Proteome Discoverer 1.4  against the The relationship between the significantly changed proteins and Gls metabolic pathways . This list of primers used in qRT-PCR is provided in Supplementary Table Ct method . The differences in Ct values (ΔCt) between the target gene and two internal controls (AT4G34270 and AT5G44200) were calculated to normalize differences in the starting materials. The expression ratios of cyp79B2/B3 and myb28/29 to WT were calculated and compared to the ratios from the protein data using Pearson's r.To determine whether protein expression levels were correlated with transcript levels, we conducted qRT-PCR of 44 genes selected based on the proteomics data  and 71B26 (CYP71B26) in oxidation reduction reaction and oxygen binding . As we detected a side network correlated to indolic GMN , cellular components (CC), and molecular functions (MF) levels. By annotating 147 changed membrane proteins in the cyp79B2/B3 and myb28/28, respectively  to nt Table . Especiacyp79B2/B3 mutant and six with changes in the myb28/29 mutant. In the cyp79B2/B3 membrane proteome, a group of general stimuli response-related proteins exhibited significant changes compared to WT  lectin family protein (At5g18470) involved in carbohydrate binding forms connections with MYB51 and CYP81F2  involved in chloroplast division and protein binding  in flavonoid metabolism  and L15-1 (RPL15A) might be a component in thiohydroximate synthesis through the connections with GSTF9, GSTF10 and/or GSTF11. Both proteins were increased, presumably to compensate for the deficiency of aliphatic Gls in the mutant  acting on adenine phosphorylation (Allen et al., cyp79B2/B3, the defense and stress-related genes calreticulin 3 (At1g08450; Sun et al., cyp79B2/B3 indicates transcriptional regulation of indole glucosinolates. In myb28/29, although there was no overall correlation between transcript and protein levels, isoform 2 of LysM (At1g21880; Willmann et al., myb28/29.In the cyp79B2/B3 as the indolic Gls mutant and myb28/29 as the aliphatic Gls mutant. We identified 4,673 proteins, out of which 2,171 were membrane proteins. From these membrane proteins and after transmembrane domain analysis, 192 exhibited different levels relative to WT, with cytochrome P450 86A7, cytochrome P450 71B26 and probable cytochrome c representing new cytochromes potentially involved in GMN. Based on our analyses, the first two might play a role in hydroxyl-indolic Gls production. In addition, a flavone 3′-O-methyltransferase 1 is hypothesized to participate in the methylation process of the hydroxyl-indolic Gls to form methoxy-indolic Gls. GO functional enrichment revealed important processes related to stress response, transport activities and photosynthesis in the cyp79B2/B3 and those related to protein translation in the myb28/29. A transcription profiling of both mutants showed a strong correlation between transcript and protein levels in cyp79B2/B3, and no significant correlation in myb28/29. Overall, the new nodes and edges discovered in the GMNs are useful resources for future hypothesis-testing experiments and ultimately toward engineering and breeding of Gls profiles with positive impacts on human health and plant defense.Glucosinolate biosynthetic process is controlled by several cytochrome proteins known to be localized to the membrane, but little is known about how Gls metabolism would affect the membrane proteome. In this study, we aim to address this important question utilizing the TMT labeling based quantitative proteomics of two genetic mutants, i.e., IM performed the experiments, data analysis and paper drafting; MY performed qRT-PCR experiment and data analysis; NZ participated in protein extraction and peptides labeling; SG conducted the statistical analysis; CD contributed in LC/MS analysis of peptides; MA and ME provided supervision and advice, and SC designed the experiments, supervised the work and finalized the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer XH and handling Editor declared their shared affiliation, and the handling Editor states that the process nevertheless met the standards of a fair and objective review.