EMBO/SourceData huggingface dataset:
Figure 2A Structure of autoinhibited ScV1. The catalytic A (pink) and non catalytic B (green) subunits are arranged as a heterohexamer around the central rotor subunits D (red) and F (cyan). The three peripheral stalks (EG heterodimers 1-3, blue and orange, respectively) are bound at the periphery of the B subunits. The inhibitory H subunit (yellow) is unique to the eukaryotic V-ATPase. Unless otherwise stated, the coloring scheme used here for the V1 subunits will be used from here on.B Interaction of the peripheral stalks with the catalytic hexamer. Each peripheral stalk crosses a non-catalytic A/B interface on its way from the top of the V1 to the base of the catalytic core. Representative view of the interaction is shown for EG3.C Interaction of peripheral stalks EG1 and 2 with the catalytic hexamer. The peripheral stalks are bound to the top of the B subunits via their C-termini (boxed). The electron density (contoured at 1.2 σ) for the C-terminal domains of EG1 and EG2 is shown in the panels to the left and right.D The structure of autoinhibited ScV1rotated 90° around the vertical from the view shown in panel A to highlight peripheral stalks EG2 and EG3. Note the bent appearance of EG3 compared to EG2 along with the presence of a partially unstructured region known as the "bulge" in subunit G (indicated with an arrow). The box shows electron density (contoured at 1.2 σ) for the EG3 N-termini contacting the C-terminal domain of the A subunit of a closed catalytic site (see asterisk). The horizontal lines on the structure indicate the sections shown in panels E (upper two lines) and F (lower two lines).E Section through the ScV1 electron density map showing interaction of the A3B3 catalytic hexamer with the peripheral stalks (EG1-3). View is rotated 90° from the view in A, looking up the central rotor. The A/B pairs forming the three catalytic sites are designated (AB)1-3 according to which of the three peripheral stalks is bound to each B subunit. For example, (AB)1 has peripheral stalk EG1 bound to its B subunit. The black arrowheads indicate the non-catalytic A/B interfaces.F Section through the ScV1 electron density map at the level of the N-termini of the peripheral stalks. Note the change in position of the N-termini of the peripheral stalks, which have each crossed a non-catalytic interface (black arrowheads in E) and are now in proximity to the C-termini of the catalytic A subunits (ACT) of the adjacent A/B pairs. While EG1 and EG2 are now near ACT from (AB)3 and (AB)1, respectively (see double headed arrows), only the bent EG3 peripheral stalk is close enough to contact the corresponding ACT from (AB)2 (see asterisk).
Figure 3A The A3B3DF complex shown with the subunits unique to the eukaryotic V1 removed, highlighting the core conserved catalytic complex labeled as "Catalytic core". The three peripheral stalks and inhibitory subunit H are found only in the eukaryotic V1, whereas A3B3DF represents the full complement of subunits in the prokaryotic V1 complex. Notably, the prokaryotic V1 is an active ATP hydrolase (Arai et al, 2009). The orientation of the A and B subunits with respect to the membrane is indicated by the arrow on the left.B Conformational differences in the catalytic A subunits. The catalytic A subunits are shown aligned by their N-terminal β barrels, highlighting the change in conformation of C-terminal α helical bundles associated with the closed ((AB)2 and (AB)3; dark and light gray, respectively) and open ((AB)1; magenta) catalytic sites. Movement of this domain in response to nucleotide occupancy is thought to drive rotation of the central rotor during catalysis.C ScV1's catalytic hexamer contains one open and two closed catalytic sites. Side view of the three catalytic hexamer A/B pairs (shown in surface representation). Note that the two closed pairs (AB)2 and 3 are non-equivalently closed.D Alignment of the A3B3DF from ScV1 and nucleotide-free EhV1 by the catalytic hexamers. The A3B3DF from ScV1 is shown in the same color scheme as in Fig 2, EhV1 is in blue. The alignment shows the difference in position of the central rotor subunit, D. Note that the C-terminus of subunit D (DCT) is longer in eukaryotic V-ATPase (see electron density contoured at 1.2 σ in the box to the right).E ScV1 and EhV1are halted in different rotational positions. Alignment of the A3B3DF of ScV1 and nucleotide-free EhV1 (blue, with dashed lines drawn through the A subunits) by the central stalk subunit D, reveals that the two catalytic cores are halted in different rotational positions. The autoinhibited ScV1 requires 40° rotation to overlay its catalytic hexamer with that of the EhV1.F Comparison of the D subunits of ScV1 (red) and nucleotide free EhV1 (blue). HCT from ScV1 is shown in yellow and A subunit from EhV1 is shown in dark blue.
Figure 4A ScV1-H subunit interactions. Region of electron density (contoured at 1.2 σ) outlined by the box in the side view of ScV1 shown to the right. The H subunit N-terminal domain (HNT) is bound to the N-terminal α helices of peripheral stalk EG1 while its globular C-terminal domain (HCT) is bound at the base of the catalytic hexamer.B Interaction between HNT and peripheral stalk EG1. View of HNT as in panel A rotated 110° along the horizontal, showing the α helical HEAT repeats of HNT forming a pocket for binding the N-termini of peripheral stalk EG1. HNT's α helices forming the pocket are labeled as in (Sagermann et al, 2001). Pro197 on HNT (red sphere) has been previously shown to cross-link subunit E (Jefferies Forgac, 2008).C Interaction between HCT and the base of the catalytic hexamer. The H subunit inhibitory contacts at the base of the ScV1 are mediated by a loop in HCT (highlighted in magenta) that is seen to contact two short segments of the C-terminal domain of the subunit G from the open catalytic site (AB)1 (highlighted in purple) and two turns of the N-terminal a helix of the central rotor subunit D (highlighted in blue).D HCT binds to the open (AB)1 pair. View of ScV1 looking along the central rotor subunit D towards the cytoplasm, showing the position of the H subunit binding at the interface of the open A/B pair (AB)1. The catalytic hexamer is shown in surface representation and the central rotor, peripheral stalks and subunit H are shown as ribbons.
Figure 5C Growth phenotype of H subunit mutants. Deletion of V-ATPase subunits from yeast results in a conditional lethal phenotype (Vma-) characterized by an inability to grow at pH 7.5 in the presence of 60 mM CaCl2 (upper panel), but growth is observed at pH 5 (lower panel). Subunit H mutations were introduced on a plasmid into a yeast strain deleted for the H subunit and their phenotypes assessed. Both humanH subunit isoforms (HsH1, HsH2), a chimeric H subunit (HChim) containing ScHNT and HsHCT, mutations in yeast intended to mimic the length of the human H loop sequence and accessibility (Hloop), and a point mutation of the conserved aspartic acid residue in the loop sequence (HD410A) are shown compared to the H subunit deletion (ΔH) and wild type H on a plasmid (H WTpl). All grow on pH 5 medium, but the human isoforms failed to complement the deletion phenotype.D ATPase activity of H subunit mutants. The ScV1 sectors were purified from each strain that complemented the phenotype as well as from ScV1ΔH, for comparison of MgATPase activity. Average specific activities are shown plotted +/- s.e.m from two independent purifications. The ScV1 sector containing chromosomally encoded H subunit shows no detectable MgATPase activity (Parra et al, 2000; Zhang et al, 2003), whereas ScV1containing plasmid-borne wild type H (H WTpl) exhibits low levels of activity likely due to reduced expression from the low copy plasmid. The HChim and HLoop mutants lead to a loss of activity silencing, displaying nearly the same activity as ScV1ΔH.E SDS-PAGE and Western Blot analysis of ScV1 preparations. Left panel, 15% SDS PAGE of ScV1 purified from the subunit H mutant and WT strains. Note that H WTch is purified from a yeast strain deleted for the C subunit (used for crystallization). The remaining strains contain subunit C, which is co-purified with ScV1 in varying levels. Note that while H WTch ScV1, which is expressed from a strain deleted for subunit C, displays no detectable MgATPase activity, the H subunit mutants co-purified with the most C subunit display the least activity, suggesting that absence or presence of subunit C has no effect on MgATPase activity. Right panel, immunoblot probed with an antibody directed against the N-terminal Myc tagged mutant H subunits expressed from a plasmid. Note that the levels of mutant H expression does not appear to vary from strain to strain.
Figure 6A Overlay of ScV1 and the cryo-EM structure of the V1Vo holoenzyme in "state 2" (Zhao et al, 2015). The structures of ScV1 and ScV1Vo were overlaid by aligning the A3B3 catalytic hexamers, thus highlighting structural differences in the central rotor and peripheral stator subunits. Coloring of ScV1Vo (shown as semi-transparent ribbons) is as used throughout for ScV1 for all but the A3B3F (tan), subunit H (dark green), subunit C and Vo domain (gray). The EG1-HNT interaction is boxed. Note the shift in position of HNT and EG1 between ScV1 and ScV1Vo.B Comparison of peripheral stalk EG1 and HNT in ScV1 versus ScV1Vo. The boxed view in A is shown enlarged. The N-terminal α helices of peripheral stalk EG1 and HNT change in conformation, likely induced by the movement of HCT when going from the holo enzyme to the autoinhibited conformation.C View as in A rotated 150º around the vertical. Comparisons of peripheral stalk EG3 and HCT are boxed.D Comparison of peripheral stalk EG3 in ScV1 versus ScV1Vo. In ScV1Vo, EG3 binds to the C subunit head domain (Chead; gray) whereas in ScV1, EG3 is bent towards the C-terminal domain of a catalytic A subunit (enlarged view of box labeled EG3 in panel C).E Comparison of HCT's conformation in ScV1 versus ScV1Vo. Enlarged view of box labeled HCT in panel C highlighting the conformational changes and binding interactions of HCT; for clarity only aNT, HCT and the central rotor (DF) are shown. In ScV1Vo, the most C-terminal α helix (dark green) of HCT (green) binds to aNT (light gray), with the inhibitory loop (green spheres) rotated away and inaccessible for binding the B subunit and the central stalk (transparent red). In ScV1, the HCT (yellow) inhibitory loop (yellow spheres) binds to the central stalk subunit D (red) and the B subunit of the open (AB)1 catalytic site. The difference in conformation between ScV1Vo and ScV1 involves a ~150° rotation of HCT. Note that the HCT loop (spheres) is rotated away from the central stalk in ScV1Vo whereas in ScV1, the C-terminal α helix (orange) involved in binding aNT is rotated away and unavailable for rebinding to the membrane.F HCT conformational changes. Available subunit H structures as obtained for isolated H (Sagermann et al, 2001) (purple), in ScV1Vo (Zhao et al, 2015) (green) and in ScV1 (yellow) are shown aligned by their N-terminal domains (HNT) illustrating the conformational flexibility of HCT. The loop involved in inhibition of ScV1 is shown as red spheres, highlighting the 180º rotation of HCT when going from the conformation of isolated H (HCT 1ho8) to the inhibitory conformation in ScV1(HCT ScV1).
Figure 3The rapZ mutants were expressed from plasmids under control of the PBAD promoter (induction with arabinose, repression by glucose) and tested for the ability to complement a strain lacking the chromosomal rapZ gene in (b) Northern blotting experiment detecting GlmZ (top panel) and 5S rRNA (bottom panel; loading control) to assess the degree of GlmZ cleavage triggered by the RapZ variants. Processed GlmZ is indicated with an asterisk. Strains Z8 (wild-type, lanes 1, 2) and Z28 (∆rapZ, all other lanes were used. Strain Z28 carried plasmid pBAD33 (lanes 3, 4; empty vector control = VC) or derivatives triggering synthesis of the indicated RapZ variantsThe rapZ mutants were expressed from plasmids under control of the PBAD promoter (induction with arabinose, repression by glucose) and tested for the ability to complement a strain lacking the chromosomal rapZ gene in (c) GlmS protein (MW = 66.9 kDa) levels in the same cultures as revealed by SDS-PAGE of total protein extracts followed by Coomassie blue staining. Strains Z8 (wild-type, lanes 1, 2) and Z28 (∆rapZ, all other lanes were used. Strain Z28 carried plasmid pBAD33 (lanes 3, 4; empty vector control = VC) or derivatives triggering synthesis of the indicated RapZ variantsThe rapZ mutants were expressed from plasmids under control of the PBAD promoter (induction with arabinose, repression by glucose) and tested for the ability to complement a strain lacking the chromosomal rapZ gene in (d) β-Galactosidase activities produced by the same cultures and replicates (mean values and standard deviations are shown). Cells carry a glmS'-lacZ fusion on the chromosome, which requires base-pairing with sRNA GlmZ for high expression. Strains Z8 (wild-type columns 1, 2) and Z28 all other columns) were used. Strain Z28 carried plasmid pBAD33 columns 3, 4; empty vector control = VC) or derivatives triggering synthesis of the indicated RapZ variants
Figure 1C) MXE saturation analysis. Whereas increasing amounts of RNA-seq reads should lead to the confirmation of further MXE candidates, more RNA-seq reads might also result in the rejection of previously validated MXEs. The green curves show the number of validated MXEs in relation to the percentage of total RNA-seq reads used for validation. The orange curves indicate the number of initially 'validated MXEs' that were rejected with increasing amounts of reads. Grey dashed lines indicate the point of saturation, which is defined as the point where a two-fold increase in reads leads to rejection of less than 1% of the validated MXEs. Of note, whereas the rejection of validated MXEs saturates with 20% of the data the amount of novel MXE validations is still rapidly increasing.E) Size and distribution of multi-cluster MXEs.
Figure 3A) Heatmap showing all differentially expressed MXE clusters with at least 3 RPKM. Here, we used the Gini coefficient, which is a measure of the inequality among values of a frequency distribution (Ceriani & Verme, 2012) and has successfully been used to determine tissue-enriched gene sets (Zhang et al, 2017), to determine highly tissue-specific MXEs (maximum normalized Gini index of cluster) and MXEs with a broad tissue expression distribution (minimum Gini index). For each MXE cluster the percent-spliced-in (PSI) value of the ubiquitous MXE (minimum Gini index) is subtracted from the PSI value of the specific MXE (maximum Gini index of cluster) (delta PSI value) and scaled between -1 (broad tissue distribution) and 1 (highly tissue specific). Each column represents an MXE pair and each row represents MXE expression in a tissue, cell-type, or at a developmental time point. The bar graph summarizes counts where the specific MXE is 1.5 fold more spliced in than the ubiquitous MXE.
Figure 4C) Heatmap showing the the delta PSI values (PSI value of the non-SNP-containing MXE subtracted from the PSI value of the SNP-containing MXE) of MXE clusters containing pathogenic SNPs scaled between -1 and 1 (blue = high expression non-SNP-containing MXE, red = high expression SNP-containing MXE). Columns represent MXE clusters and rows tissues, cell types, and developmental stages. The column bar graph summarizes counts where the SNP-containing MXE is 1.5 fold more expressed than the non-SNP-containing MXE, whereas the row bar graph shows this for each tissue, cell type, and developmental stage.
Figure 1B atg19Δ cells containing HTB‐Atg19 were grown to mid‐log phase. Atg19 was affinity purified and subjected to mass spectrometric phosphorylation mapping. Phosphorylation sites: enlarged; lysine substitutions: gray; Atg11 binding region: green.C atg19Δ cells containing GFP‐Atg19 wild‐type or mutants as indicated were grown to mid‐log phase. Processing of endogenous Ape1 was analyzed by Western blotting and quantified by calculating the ratio of cleaved versus uncleaved Ape1 normalized to the wild‐type. Atg19 expression was assessed by anti‐GFP Western blotting.D atg19Δ GFP‐Atg11 Atg1‐TAP cells containing myc‐Atg19 as indicated were grown to log phase. Atg11 was immunoprecipitated, and bound Atg19 was analyzed by Western blotting.E GST‐fused propeptide of Ape1, Atg19 as indicated, and a His‐tagged fragment of Atg11 (amino acids 685-1178) were purified from Escherichia coli, mixed, and propeptide‐bound proteins were isolated and analyzed by Coomassie staining. Quantification of three individual experiments is shown. Error bars represent standard deviation.
Figure 2A GFP‐Atg11 Ape1‐mRuby atg19Δ cells containing myc‐Atg19 as indicated and Cup1‐Ape1 were grown to log phase. Overexpression of Ape1 was induced by addition of 250 μM copper sulfate for 3 h, and autophagy was induced by treating cells for 1 h with rapamycin. Scale bar, 5 μm.B Ape1‐mRuby atg19Δ cells containing GFP‐Atg19 wild‐type or GFP‐Atg19‐3A were analyzed in log phase. Scale bar, 5 μm.C GFP‐Atg8 Ape1‐mRuby atg19Δ cells containing myc‐Atg19 as indicated and Cup1‐Ape1 were analyzed as in (A). Scale bar, 5 μm.
Figure 3B atg34Δ yeast cells containing HTB‐Atg34 were grown to mid‐log phase and treated with rapamycin. Atg34 was affinity purified and subjected to mass spectrometric phosphorylation mapping. Phosphorylation sites: enlarged; Atg11 binding region: green; lysine substitution: gray.C Ams1‐GFP atg19Δatg34Δ cells containing protein A (Stag)‐tagged Atg34 as indicated were starved for 4 h. Processing of endogenous Ams1‐GFP was analyzed by Western blotting and quantified by calculating the ratio of free GFP versus uncleaved Ams1‐GFP normalized to the wild‐type. Expression of Atg34 proteins was assessed by anti‐protein A Western blotting.E GST, GST‐Atg19, and GST‐Atg34 were purified from Escherichia coli and in vitro phosphorylated with immunoprecipitated Atg1‐TAP bound to IgG magnetic beads.F In vitro phosphorylation of recombinant Atg19, Atg19‐3A, and Atg19‐3D using wild‐type and kinase‐dead Atg1‐D211A as described in (E).
Figure 4A Wild‐type, hrr25‐ts, and hrr25‐ts cells containing HRR25 on a centromeric plasmid were grown to mid‐log phase at 18°C followed by 2 and 4 h of log‐phase growth at 37°C. Ape1 processing was analyzed by Western blotting.B atg19Δ and atg19Δ hrr25‐ts cells containing GFP‐Atg19 as indicated were analyzed for Ape1 processing in log phase after 2 h at 37°C.C GFP‐Atg11, GFP‐Atg11 hrr25‐ts, and GFP‐Atg11 atg1‐D211A cells were analyzed for GFP‐Atg11 dot formation. Quantification of at least 70 cells in three individual experiments is shown. Error bars represent standard deviation. Scale bar, 3 μm.D GFP‐Atg11, GFP‐Atg11 hrr25‐ts, and GFP‐Atg11 atg19Δ cells containing BFP‐Ape1 and Cup1‐Ape1 were grown to log phase. Overexpression of Ape1 was induced by addition of 250 μM CuSO4 for 3 h. coloc.: Cells with GFP‐Atg11/BFP‐Ape1 co‐localization. n = 50 (wt), 84 (hrr25‐ts), 127 (atg19Δ). Scale bar, 3 μm.E Hrr25 was immunoprecipitated from Hrr25‐TAP cells and as a control from a non‐tagged wild‐type strain. In vitro phosphorylation of recombinant Atg19, Atg19‐3A, Atg34, and GST using the kinase‐bound beads was performed as described in Fig E. Both Atg19 panels are from the same gel with the same exposure.
Figure 1(B) Immunoblot analysis of the immunoprecipitated HA-SOX9 or FLAG- FBW7α and their pull-down products. The corresponding Western blotting from the whole cell extract (WCE) of the transfected HEK293 showed the levels of exogenous HA-SOX9 or FLAG-FBW7α proteins in the total input. Cells were treated with 10 µM MG132 for 4 hours prior to harvesting for the immunoprecipitation. Blots are representative from 3 independent experiments.(C) FBW7α immunoblot eluted from the agarose bead-bound peptide, which had been incubated with the recombinant SCF-FBW7α for 1 hour at 37◦C. The agarose bead-bound peptide contains either the non-phosphorylated SOX9 amino acid sequence spanning from residue number 231 to 245 (SOX9 peptide), or those with the threonine corresponding to residue number 236 and 240 being phosphorylated (pSOX9 peptide). The input (10%) showed the level of the supplemented recombinant SCF-FBW7α in the in vitro binding reaction. Shown blot is representative from 2 independent experiments.(D) Representative Western blotting set from 3 independent repeats of pT236-SOX9 from lysates of HEK293 transfected with either HA-EV or HA-SOX9. Each lysate were immediately divided and left untreated or subjected to lambda phosphatase (λ-PPase) treatment for 1 hour at 37◦C prior to gel electrophoresis. SOX9 blot showed the presence of SOX9 protein in both the untreated and the phosphatase-treated SOX9-transfected cell lysates.(E) Bead-immobilized in vitro translated (IVT) HA-SOX9 wild type (WT) or T236/240 mutant were either untreated or subjected to in vitro GSK3 kinase reaction for 90 minutes at 37◦C prior to elution and gel electrophoresis. The SOX9 blot showed total SOX9 protein eluted from the beads from each reaction. Shown immunoblots are single representative experiment from 3 independent repeats.(F) GSK3-mediated phosphorylation of threonine 236 promoted SOX9 interaction with recombinant SCF-FBW7α in vitro. Bead-immobilized IVT HA-SOX9 were either untreated or phosphorylated with GSK3 as described in (E) prior to further incubation with recombinant SCF-FBW7α for in vitro binding assay. Immunoblot showed FBW7α protein in the in vitro binding reaction (input) as well as those which had been eluted from the untreated and GSK3-treated immobilized SOX9. The set of blots shown is representative from 3 independent experiments.
Figure 3(B) Immunoblotting of HA-SOX9-WT protein following expression of various FLAG-FBW7α constructs including wild type (WT), or those with R465A or R479A mutation in Daoy. The cells were transfected with equal amount of HA-SOX9-WT and FLAG-FBW7α and the whole cell lysates were collected 24 hours following the transfection. GAPDH proteins were shown to indicate total protein loading for each sample. The set of blots shown is representative from 2 independent experiments.(C) Co-immunoprecipitation of HA-SOX9 with either wild type (WT) or R465A- FLAG-FBW7α. Unlike the wild type protein, FLAG-FBW7α containing arginine 465 mutation did not significantly co-immunoprecipitate with HA-SOX9 (IP with anti HA antibody) and vice versa (IP with anti FLAG antibody). The expressions of HA-SOX9 and FLAG- FBW7α- WT or R465A mutant were assessed in the whole cell extract (WCE) of the transfected HEK293 with GAPDH protein used to indicate total protein loading.(D) Comparison of FBW7 mRNA expression across the medulloblastoma subgroups (WNT, SHH, Group 3, and 4) in a cohort containing 423 medulloblastoma cases. Expression of FBW7 in both normal fetal (n= 5) and adult cerebellum (n = 13) were used for comparison against the medulloblastoma.(E) Overall survival analysis of medulloblastoma patient based upon FBW7 expression level. Analysis was performed on 383 out of the 423 cases from which the survival data were available. Cohort was divided into high- (blue line) and low- FBW7 (red line) expressing subgroup using FBW7 median expression as group classifier. Statistical analysis was determined by using a LogRank test.(F) Correlation analysis between SOX9 protein/mRNA and FBW7 mRNA expression level from medulloblastoma tissue microarray consisting of 142 tissue samples. SOX9 protein level was scored following immunohistochemistry staining, while SOX9 and FBW7 mRNAs expression by the RNA Scope as described in Materials Methods. A modest negative correlation was determined by Kendall's correlation test (τ = - 0.223, p0.001) between SOX9 protein/mRNA and FBW7 expression. Affiliation of samples with medulloblastoma subgroups was indicated by different colored circle (blue=WNT, red=SHH, yellow=Group 3, green=Group 4, grey=Undetermined).