EMBO/SourceData huggingface dataset:
Figure 4GalNAc-T1 affects the organization and function of the endomembrane system. HaCaT WT and GALNT1 KO cells were stained for intracellular trafficking markers (scale bar - 10 μm). (A) Clathrin; (B) Rab5; (C) Rab11.GalNAc-T1 affects the organization and function of the endomembrane system. (D) Steady state intracellular pH measurement in in HaCaT WT and GALNT1 KO cells. Data is presented as average ± SD of 2 independent experiments. ANOVA followed by Dunnet's multiple comparison test(E) GalNAc-T2 is important for cell to matrix adhesion. HaCaT WT and GALNT2 KO cells were used in a cell to matrix adhesion assay using plates coated with different ECM components. Data is presented as average + SD of 3 independent experiments and is normalized to WT. ANOVA followed by Dunnet's multiple comparison test was used to compare mean relative adhesion of 2 different clones of GALNT2 KOs to WT. * p ≤ 0.05; ** p ≤ 0.01.(F) Western blot for GalNAc-T2 specific target and hemidesmosome component COL17A1.(G) Quantification of Western blot bands for COL17A1 (full length) normalized to β actin.(I) GalNAc-T2 may affect tight junction formation and integrity. Upper panel, TEM of WT and GALNT2 KO organotypic tissue sections, where tight junctions are marked with asterisks. Lower panel, IF staining of WT and GALNT2 KO for tight junction marker occludin (scale bar - 50 μm). Inserts depict 3x zoomed in partial images (scale bar - 50 μm).(J) Expression of cyclin A (green) in HaCaT WT and GALNT3 KO organotypic tissue, visualized by IF (scale bar - 20 μm).(L-N) Co-culture of HaCaT WT or GALNT3 KO keratinocytes with N/TERT1 keratinocytes induces cellular differentiation. Cells co-cultured on coverslips were stained for cleaved Notch 1 intracellular domain (L), cytokeratin 10 (M), or GalNAc-T3 (N; inserts depict 2x zoomed in partial images (scale bar - 20 μm)). Green - relevant staining, red - N/TERT1 cells (labeled with CMTMTR), blue - DAPI, scale bar - 20 μm. Boundaries between HaCaT and N/TERT-1 cells in the green panel of (L) are marked with a dashed line.
Figure 1A Representative images of Bem1-GFP and the PS marker LactC2-GFP in wild type and cho1∆ cells. Images show average intensity projections of deconvolved z-stacks. Black and white pixels were inverted to improve contrast for display. B Frequency of cells displaying polarized Bem1-GFP signal in wild type and cho1∆ cells. Values are means ± SD, n > 100 cells in each of 3 independent experiments. Paired Student's t-tests were performed. C Imaging of the PI4P probe GFP-2xPHOsh2 in wild type and cho1∆ cells. Images show average intensity projections of deconvolved z-stacks in which black and white pixels were inverted.D Scatter dot plot showing PI4P levels at the plasma membrane (see experimental procedure for details of the quantification) in wild type and cho1∆ cells. Values are means ± SD, n > 100 cells, observed over 3 experiments. Mann-Whitney tests were performed.E Ratio of PI4P average fluorescence intensity in the bud versus mother cell in the cho1∆ mutant. Values are means ± SD, n > 100 cells, observed over 3 experiments. A value > 1 indicates enrichment of the fluorescence in the bud.F Images of Bem1-GFP in cho1∆ 9xMyc-AID-stt4 cells after 30 min treatment with or without 0.5 mM auxin. Images are average intensity projections of deconvolved z-stacks.G Frequency of cells with polarized Bem1-GFP signal or Bem1-GFP in puncta in cho1∆ 9xMyc-AID-stt4 cells treated with or without auxin as shown in F. Values are means ± SD, n > 100 cells in each of 6 independent experiments. Paired Student's t-tests were performed.H Images of Bem1-GFP (cyan) and Cdc24-mCherry (red) signals in cho1∆ 9xMyc-AID-stt4 cells with or without auxin. Images display maximum intensity projections of z-stacks.I Maximum intensity projections of z-stacks displaying Bem1-GFP in the mutants indicated. Bem1-GFP was imaged in mss4-102 cho1∆ cells after shifting to the restrictive temperature for 30 minutes.J Frequency of cells with polarized Bem1-GFP signal after incubation at the time and temperature shown. Values are means ± SD, n > 200 cells in each of 3 independent experiments. Paired Student's t-tests were performed.
Figure 2B Upper panel. SDS-PAGE stained with Coomassie blue in which Bem1 is indicated. Lower panel. The liposomes were composed of 100% phosphatidylcholine (PC), 80% PC and 20% phosphatidylethanolamine (PE), 95% PC and 5% phosphatidic acid (PA), 95% PC and 5% phosphoatidylinositol (PI), 95% PC and 5% PI(4,5)P2 (PI(4,5)P2), 95% PC and 5% PI4P (PI4P), 80% PC and 20% phosphatidylserine (PS) or 75% PC 20% PS 5% PI4P (PS+PI4P). (S) supernatant, (P) pellet.C Liposomes composed of either 100% PC ± 40% ergosterol or 75% PC, 20% PS, 5% Bem1 ± 40% ergosterol replacing PC were incubated with Bem1 and treated as in panel (B).E Percentage of the indicated bem1 constructs associated with liposomes containing 75% PC, 20% PS and 5% PI4P.F Lipid ordering determined by 2H solid-state NMR analysis of liposomes containing POPC-d31/POPS (4:1 molar ratio) in the presence or absence of Bem1 BC motifs. Calculation of oriented-like spectra from Pake patterns (de-Pake-ing) and simulation of 2H solid-state NMR spectra were applied to measure individual quadrupolar splittings for POPC-d31 and determine order parameter accurately.Lipid ordering determined by 2H solid-state NMR analysis of liposomes in the presence or absence of Bem1 BC motifs. Calculation of oriented-like spectra from Pake patterns (de-Pake-ing) and simulation of 2H solid-state NMR spectra were applied to measure individual quadrupolar splittings for POPC-d31 and determine order parameter accurately. containing POPC-d31/PI4P (19:1 molar ratio).Lipid ordering determined by 2H solid-state NMR analysis of liposomes in the presence or absence of Bem1 BC motifs. Calculation of oriented-like spectra from Pake patterns (de-Pake-ing) and simulation of 2H solid-state NMR spectra were applied to measure individual quadrupolar splittings for POPC-d31 and determine order parameter accurately. containing POPC-d31/POPC/PI4P (15:4:1 molar ratio).Lipid ordering determined by 2H solid-state NMR analysis of liposomes in the presence or absence of Bem1 BC motifs. Calculation of oriented-like spectra from Pake patterns (de-Pake-ing) and simulation of 2H solid-state NMR spectra were applied to measure individual quadrupolar splittings for POPC-d31 and determine order parameter accurately. containing POPC-d31/POPS/Yeast lipid extract/ergosterol (1:1:1:2 molar ratio).
Figure 3B Percentage of the different full-length bem1 bc mutants associated with liposomes containing 75% PC 20% PS and 5% PI4P.C Fluorescence intensity change associated with the nucleotide exchange of GDP-Cdc42 for mant-GTP Cdc42. Fluorescence was measured after the addition of GDP-Cdc42 to reactions containing Mant-GTP (100 nM), GMP-PNP (100 µM) and the proteins indicated.D Ten-fold serial dilutions of cells and subsequent colony formation on the indicated plates, where expression of wild type GALp-CDC42 is either induced in the presence of Gal or repressed in Dex. Note how mutation of the wild type Cdc42 (KKSKK) to MMSMM is lethal (see blue box), whereas appending the Bem1 BC-1 motif to this cdc42 mutant restores viability (red box).E Representative images of the mEOS-cdc42 mutants signal (cyan) after inducing the expression of GAL1p-CDC42 in the presence of Gal or repressing it in the presence of Dex. The cells in the blue and red boxes correspond to the cells in the blue and red box in panel (D). Images are average fluorescence intensity projections in which black and white pixels have been inverted.
Figure 4A Ten-fold serial dilutions of the indicated mutant cells and subsequent colony formation at the temperatures indicated. Note how mutations on both the BC motifs and PX in bem1 compromise growth at the restrictive temperature.B Western blots probed with anti-HA antibody to detect the indicated bem1 constructs tagged with 3XHA (top panel). Detection of Ade13 was used as loading control (bottom panel).C Representative images of the indicated bem1 mutants tagged with GFP at the BEM1 locus. The inverted fluorescence images are average intensity projections.D Frequency of cells with polarized Bem1-GFP. E Scatter dot plot showing the fluorescence levels of the indicated bem1 strains at the cell pole. The measurements were made on average intensity projections.
Figure 5B Percentage of full-length Cdc24 and cdc24 ph domain mutant (cdc24 ph) associated with liposomes containing 75% PC 20% PS and 5% PI4P.C Percentage of Cdc24 and cdc24 ph mutant associated with liposomes of the composition shown in (B) in the presence of the indicated bem1 protein. Note how additive mutations in the Bem1 lipid binding sequences reduce the percentage of Cdc24 associated with the liposomes.D Percentage of Cdc24 and cdc24 ph associated with liposomes of the composition shown in (B) as a function of the indicated bem1 protein concentration. The curves denote the regression fit of the data to equation 2 in the materials and methods.E Ten-fold serial dilutions and subsequent colony formation of the indicated mutant cells at the indicated temperatures.F DIC images of the indicated bem1 and cdc24 mutants showing the increased morphological defects ensuing from loss of lipid tethering in the bem1 and cdc24 mutants.G Western blots probed with anti-HA antibody to detect Cdc24 or cdc24 ph tagged with 3XHA in the indicated mutant strains (top panel). Detection of Ade13 was used as a loading control (bottom panel).
Figure 6B Global average MSD curves of mEOS-Cdc42 in the strains indicated at the Pole (P) and Non-Pole (NP) of cells. Trajectories longer than 6 frames were analyzed. Number of trajectories analyzed: BEM1 (n = 11 cells: Non Pole: 1814 tracks; Pole: 706); bem1 bc-14E px (n = 10 cells: Non Pole: 1793 tracks; Pole: 908); bem1 bc-14E (n = 13 cells: Non Pole: 1714 tracks; Pole: 975). Error bars display s.e.m. C D coefficients of mEOS-Cdc42 in the strains indicated (in box-plots displaying the median (line), the 25-75 percentiles (box) and the mean (cross)), which were compared using a non-parametric, two-tailed Mann-Whitney rank sum test. D SR-Tesseler images of mEOS-Cdc42 nanocluster organization in BEM1 (5312 localizations shown in image) and bem1 bc-14E px cells (3739 localizations shown). Insets show mEOS-Cdc42 after 491 nm widelfield laser excitation to identify the cell pole. Scale bars: 2 μm. A zoom of the pole region shows the organization of the detected nanoclusters, circled in light blue, in the strains indicated. Scale bar in the zoom: 60 nm.E Distribution of nanocluster area at the pole (P) and non-pole (NP) regions of BEM1 (diameter NP: 59 nm ± 1 nm (s.e.m); P: 74 nm ± 3.2 nm) (n = 16 cells. NP: 570 clusters; P: 162 clusters) and bem1 bc-14E px cells (diameter NP: 59 nm ± 1.5 nm (s.e.m); P: 57 nm ± 2.4 nm) (n = 15 cells. NP: 421 clusters; P: 141 clusters). Data are presented as scatter dot-plots displaying the median as a line and the 25-75 percentiles. Data were compared using non-parametric, two-tailed Mann-Whitney rank sum test.F Active Cdc42-GTP levels were quantified in the strains indicated using a gic2(1-208)-yeGFP probe. Values are mean ± SD for n = 30 cells observed over 2 experiments. Data were compared using a Mann-Whitney test.
Figure 1(c) Substrate independent in vitro SUMOylation assay with SUMO2WT, SUMO2K11Ac or SUMO2K11Q was performed and analyzed by immunoblotting with the indicated antibodies.(d) Substrate independent in vitro SUMOylation assay using either SUMO2WT or SUMO2K11Ac in the presence of increasing amounts of ZNF451-N. Reaction products were analyzed by immunoblotting with the indicated antibodies. Note that - when compared to (c) assays were done at reduced concentrations of E1 and E2(e) In vitro SUMOylation assay using a fixed amount of SUMO2WT (220 ng) with increasing concentrations of SUMO2K11Ac. Ratios ranged from 3:1 (unmodified/K11Ac) to 1:2 (unmodified/K11Ac)(f) In vitro SUMOylation assay, using SUMO2WT or SUMO2K11Ac, with p53 as a substrate. p53 was generated by in vitro translation/transcription in a rabbit reticulocyte lysate system. SUMO chain formation on p53 was analyzed by immunoblotting with α-p53 antibodies.
Figure 2(a) In vitro deacetylation of recombinant SUMO2WT or SUMO2K11Ac (2.65 μM each) with recombinant SIRT1 in the presence or absence of NAD+.(b) In vitro deacetylation of recombinant SUMO2WT, SUMO2K11Ac, SUMO2K33Ac and SUMO2K35Ac. Immunoblotting was performed with antibodies as indicated.(c) In vitro deacetylation of SUMO2K11Ac with increasing concentrations of SIRT1 or SIRT2 (0.74 µM, 1.5 μM, 3 µM), followed by immunoblotting with α-SUMO2K11Ac.(d) Combination of in vitro deacetylation and substrate independent in vitro SUMOylation with SUMO2WT and SUMO2K11Ac in the presence or absence of SIRT1 (1.5 μM).(e) In vitro deacetylation of SUMO2WT or SUMO2K11Ac with or without addition of SIRT1, followed by substrate dependent in vitro SUMOylation of p53.(f) Enrichment of acetylated proteins after SIRT1 inhibition in HeLa cells. Western blot analysis of SUMO2K11Ac after treatment of cells with the SIRT1 inhibitior NAM (10 mM) for 5h and subsequent enrichment for acetylated proteins via acetyl-lysine immunoprecipitation. As a control cells were either treated with control siRNA or siSUMO2/3. The samples were analyzed by immunoblotting with α-SUMO2/3 and α-SUMO2K11Ac antibodies. The α-SUMO2/3Ac and α-SUMO2K11Ac reactive species migrating at ~200 kDa are marked with an asterisk.
Figure 3(b) Western blot analysis with α-RGS-His and α-HA was performed to monitor poly-SUMOylation of HA-PML. The remaining samples were measured by LC-MS/MS and final data analysis was done with MaxQuant. A K11 SUMOylated SUMO2 peptide was identified and is depicted with an Andromeda score of 49.3. Intensities for this SUMO2 peptide for the control (gray) and arsenic (purple) treated samples are blotted as a bar graph. The diagram shows a representative dataset derived from two replicates. The corresponding peptide is displayed below the graph. The SUMOylation site is marked with SUMO2 (green) and peptide fragment ions are indicated by y- (red) and b- (blue).(c) The degradation of the PML reactive band (~ 120 kDa) was determined by immunoblotting. HeLa cells were either mock transfected or transfected with siRNA directed against SUMO1/2/3. The expression of RGS-His-tagged SUMO2WT or SUMO2K11Q was induced by DOX for 12 h. Prior to lysis, cells were treated with 1 μM As2O3 for 6 h. Western blot analysis for wild-type SUMO2 and SUMO2K11Q were blotted with α-RGS-His. Immunoblotting with α-Tubulin served as a loading control. Immunoblots for knockdown controls (SUMO1 and SUMO2/3) can be found
Figure 4(b) Immunoblotting of samples with α-RGS-His (SUMO variants) and α-PCNA as loading control.(c) Bar graph, comparing the identified SUMO-SUMO2 linkages of control and heat shock samples for SUMO2WT (gray) and SUMO2K11Q (purple). The relative abundance (y-axis) of the indicated SUMO-SUMO2 linkage sites (x-axis) are shown. The measured intensity for K11 in the SUMO2WT under control conditions was set to 100% and the relative abundance for the individual residues was calculated in relation to this value. One of two independent replicates is shown.
Figure 1(A) Sirt5 deficiency leads to higher ROS levels in MEFs. ROS levels were determined in the indicated MEFs as described in 'Materials and Methods'. The results are average ± SD of 3 independent experiments **p<0.01 (two-tailed unpaired t-test).(B) Sirt5 deficiency suppresses GSH production in MEFs. The ratio of [GSH/GSSG] was determined in cell extracts as described in 'Materials and Methods'. The results are average ± SD of 3 independent experiments *p<0.05 (two-tailed unpaired t-test).(C) Sirt5 deficiency inhibits NADPH production in MEFs. The ratio of [NADPH/NADP+] was determined in cell extracts as described in 'Materials and Methods'. The results are average ± SD of 3 independent experiments ***p<0.001 (two-tailed unpaired t-test).(D) Sirt5 deficiency decreases cell growth. MEF cells were seeded in 6-well plates at a density of 50,000 cells/well, and cell growth rate was carefully monitored every 1-2 days by cell counting for 8 days. The results are average ± SD of 3 independent experiments *p<0.05, ***p<0.001 (two-tailed unpaired t-test).(E) Sirt5 deficiency leads to higher sensitivity of MEFs to Paraquat. The levels of cleaved PARP and Caspase-3 were determined by western blot analysis.(F) Sirt5 protects MEFs from ROS-induced cell death. Wild-type and Sirt5 KO MEFs were treated with Paraquat (500 μM) for 24 hrs, and then cell viability was determined by counting the remaining adherent cells. The results are average ± SD of 3 independent experiments **p<0.01 (two-tailed unpaired t-test).(G-I) Sirt5 deficiency inhibits the production of NADPH and GSH and increases lipid peroxide in mouse brains after Paraquat injection. Female Sirt5 KO and WT littermates (n=3~5 per group) were intraperitoneally injected with saline or paraquat (10 mg/kg) once a week for 3 consecutive weeks. The ratios of [NADPH/NADP+] and [GSH/GSSG] and the level of lipid peroxide were determined in tissue extracts. The results are average ± SD. *p<0.05, n.s.: not significant (two-tailed unpaired t-test).(J-K) Sirt5 KO mice are more sensitive to Paraquat-induced nigrostriatal dopaminergic degeneration. Female Sirt5 KO and WT littermates were treated with paraquat as in (G-I). The brain sections were stained to detect tyrosine hydroxylase (TH) positive dopaminergic neurons in the SNc region, as described in 'Materials and Methods'. Representative immunohistochemistry images (original magnification, 100×; scale bar, 100 μm) and the corresponding quantifications are shown in (J) and (K), respectively. The results are average ± SD. *p<0.05, n.s.: not significant (two-tailed unpaired t-test).
Figure 2(A)Knockdown of SIRT5 inhibits NADPH production in HEK293T cells. The ratio of [NADPH/NADP+] was determined in cell extracts as described in 'Materials and Methods'. The results are average ± SD of 3 independent experiments **p<0.01, ***p<0.001 (two-tailed unpaired t-test).(B) Knockdown of SIRT5 suppresses GSH production in HEK293T cells. The ratio of [GSH/GSSG] was determined in cell extracts as described in 'Materials and Methods'. The results are average ± SD of 3 independent experiments **p<0.01 (two-tailed unpaired t-test).(C) Knockdown of SIRT5 leads to higher ROS levels in HEK293T cells. ROS levels were determined in the indicated stable cells as described in 'Materials and Methods'. The results are average ± SD of 3 independent experiments ***p<0.001 (two-tailed unpaired t-test).(D) Knockdown of SIRT5 leads to higher sensitivity to Paraquat. The expression levels of PARP and Caspase-3 were determined by western blot analysis.(E) SIRT5 protects HEK293T cells from ROS-induced cell death. The indicated stable HEK293T cells were treated as mentioned above in (D), and then cell viability was determined by counting the remaining adherent cells. The results are average ± SD of 3 independent experiments **p<0.01 (two-tailed unpaired t-test).(G) The activities of G6PD/6PGD and IDH1/2 are reduced in stable HEK293T cells with SIRT5 knockdown. Activities of the indicated endogenous proteins were determined in cell extracts as described in 'Materials and Methods'. The results are average ± SD of 3 independent experiments *p<0.05, **p<0.01, ***p<0.001, n.s.: not significant (two-tailed unpaired t-test).(H) G6PD and IDH2 are activated by SIRT5 in vitro. Upon pre-incubation with SIRT5 in vitro, the activities of the indicated ectopically expressed proteins were determined as described in 'Materials and Methods'. The results are average ± SD of 3 independent experiments **p<0.01, n.s.: not significant (two-tailed unpaired t-test).
Figure 3(A) G6PD activity is not regulated by lysine succinylation. Purified Flag-tagged G6PD was incubated with or without succinyl-CoA (1 mM) at 30°C for 15 mins, followed by measurement of G6PD enzyme activity as described in 'Materials and Methods'. The results are average ± SD of 3 independent experiments. n.s.: not significant (two-tailed unpaired t-test).(B) IDH2 activity is suppressed by lysine succinylation. Purified Flag-tagged IDH2 was incubated with or without succinyl-CoA (1 mM) at 30°C for 15 mins, followed by measurement of IDH2 enzyme activity as described in 'Materials and Methods'. The results are average ± SD of 3 independent experiments *p<0.05 (two-tailed unpaired t-test).(C) SIRT5 knockdown increases G6PD glutarylation and IDH2 succinylation, respectively, and decreases both enzyme activity. Flag-G6PD or Flag-IDH2 was ectopically expressed in the indicated stable HEK293T cells. G6PD/IDH2 proteins were purified by immunoprecipitated with Flag beads and eluted with Flag peptide, followed by determination of their lysine modifications and enzyme activity. The results are average ± SD of 3 independent experiments **p<0.01, ***p<0.001 (two-tailed unpaired t-test).(D) SIRT5 decreases G6PD glutarylation and IDH2 succinylation, and increases both enzyme activity. Flag-G6PD or Flag-IDH2 was co-overexpressed with HA-SIRT5 in HEK293T cells. G6PD and IDH2 proteins were purified by immunoprecipitation with Flag beads and eluted with Flag peptide, followed by determination of their lysine modifications and enzyme activity. The results are average ± SD of 3 independent experiments *p<0.05, **p<0.01 (two-tailed unpaired t-test).(E) Cartoon representation of porcine mitochondrial IDH2 structure shows isocitrate binding pocket (PDB ID: 1LWD) [33], made by Pymol (www.pymol.org). The protein is colored in light green and isocitrate in rainbow, with Lys 360 and Lys 413 residues in IDH2 being colored in red.(F-G) K413 in IDH2 may be a key succinylation site that is directly targeted by SIRT5. The indicated Flag-tagged IDH2 proteins were overexpressed in HEK293T cells with stable SIRT5 knockdown or SIRT5 co-overexpression. Wild-type and mutant IDH2 proteins were purified by Flag beads and eluted with Flag peptide, followed by measurement of IDH2 enzyme activity. The results are average ± SD of 3 independent experiments *p<0.05, n.s.: not significant (two-tailed unpaired t-test).