EMBO/SourceData huggingface dataset:
Figure 7A, Cross-species integration of bulk RNA-Seq datasets. Naked mole-rat BM populations CP1-4 were matched to human and murine counterparts for GSEA with MSigDB hallmark genesets. Primitive stem and progenitor [left], hsa: LT-HSC, LIN-/CD34+/CD38lo/CD90+/CD45RA- (n=7); HSPC, LIN-/CD34+/CD38lo (n=11); MPP, LIN-/CD34+/CD38lo/CD90-/CD45RA- (n=4); mmu: LT-HSC, LIN-/Sca-1+/Kit+/CD150+/CD48- (n=25); MPP, LIN-/Sca-1+/Kit+/CD150-/CD48+ (n=6); hgl: LTC, LIN-/Thy1.1int/CD34+ (n=5). Lymphomyeloid [center], hsa: LMPP, LIN-/CD34+/CD38lo/CD123lo/CD45RA+ (n=9); CLP, LIN-/CD34+/CD38hi/CD10+/CD45RA+ (n=6); MLP, LIN-/CD34+/CD38lo/CD90-/CD45RA+/CD71- (n=4); CMP, LIN-/CD34+/CD38hi/CD123lo/CD45RA- (n=26); GMP, LIN-/CD34+/CD38lo/CD123lo/CD45RA+ (n=18); mmu: LMPP, LIN-/Sca-1+/Kit+/Flt3hi (n=5); CMP, LIN-/Sca-1-/Kit+/CD16/32lo/CD34+ (n=8); GMP, LIN-/Sca-1-/Kit+/CD16/32hi/CD34+ (n=16); hgl: CP1, LIN+/Thy1.1int/CD34+ (n=5). Erythroid [right], hsa: MEP, LIN-/CD34+/CD38hi/CD123-/CD45RA- (n=23); MKP, LIN-/CD41a+/CD42b+ (n=3); ERY, LIN-/CD34lo/-/CD36+/GYPA+/CD71+ (n=12); mmu: MEP, LIN-/Sca-1-/Kit+/CD16/32-/CD34- (n=15); hgl: CP3, LIN-/Thy1.1lo/CD34+ (n=5); CP4, LIN-/Thy1.1-/CD34+ (n=5). Within each group, cell types were pooled for each species. Shown are pathways related to proliferation and metabolism normalized enrichment score; GeneRatio, (signature ∩ term) / (signature ∩ all terms); FDR, false discovery rate.B, Seahorse assay with cell types sorted according to (A); hOPP, human oligopotent progenitors LIN-/CD34+/CD38+; mOPP, LIN-/Sca-1-/Kit+. ECAR, extracellular acidification rate; OCR, oxygen consumption rate. Error bars, s.e.m.; Human cell types, n=4; mouse, n=3, naked mole-rat, n=3.C, Mitotracker Red staining in mouse (n=4), human (n=4) and naked mole-rat (n=5) BM, histogram of merged per-species data. unstained, BM from mouse [solid], human [dotted] or NMR [area under curve (AUC)]; yLSK, LIN-/Sca-1+/Kit+, 3 month old; oLSK, 24 month; yLTC, LIN-/Thy1.1int/CD34+, 3 year old; hHSC, human CD34+/CD38lo;F, Confocal fluorescence imaging of sorted mouse LT-HSCs (LIN-/Sca-1+/Kit+/CD150+/CD48-) and NMR LTCs across age; Y, young (mouse, 4 month-old; NMR, 3 year-old); O, old (mouse, 24 month-old); MA, middle-aged (NMR, 11 year-old). Scale bar 10µm.G, H Polarization quantification for G, mouse LT-HSC (biological n=3); and H, NMR LTC (Y, n=5; MA, n=4). Line at mean; error bars, s.e.m.; p-value determined by Sidak's Two-way ANOVA.
Figure 1A U2OS cells were transfected with Flag-NAT10 or control vectors. Forty-eight hours later, cells were harvested and whole cell extracts were immunoprecipitated with Flag-antibody affinity resin. The NAT10-binding proteins were resolved by SDS-PAGE and detected by silver staining.B-F U2OS, HCT116 p53+/+ or HCT116 p53-/- cell lysates were immunoprecipitated with control IgG, anti-NAT10 (B and F), anti-p53 (C) and anti-Mdm2 (D and E) antibodies. The immunoprecipitates were subsequently immunoblotted with the indicated antibodies.G Purified NAT10 was incubated with GST, GST-p53 or GST-Mdm2 proteins coupled to Glutathione Sepharose 4B. Proteins retained on the Sepharose were then analyzed by western blot using the antibodies as indicated. The amount of GST fusion proteins are shown in the lower panel.H Full-length GST-p53 fusion protein, its deletion mutants or GST protein was used to pull-down experiments with purified NAT10 protein. The levels of the GST fusion proteins are shown in the left panel.I GST pull-down assay was performed using purified GST-NAT10 deletion mutants or GST protein and overexpressed Flag-p53 or Mdm2 protein in HEK293T cells. Schematic diagram represents the constructs of GST-NAT10 deletion mutants (right panel).
Figure 2A Recombinant His-p53 was incubated with or without purified Flag-NAT10 as described in Experimental Procedures. The reaction products were resolved by SDS-PAGE and immunoblotted using anti-acetyl-lysine antibodies. Flag-PCAF was used as a positive control.B In vitro acetylation was performed and the acetylated p53 was detected as in A.C In vitro acetylation assay was performed as described in A. The reaction products were resolved by SDS-PAGE and acetylated p53 was purified from the SDS-PAGE and subjected to mass spectrometry analysis.D His-p53 or His-p53-K120R fusion protein was incubated with purified NAT10 or NAT10 GE mutant as described in Experimental Procedures. Reaction mixtures were subjected to western blot using the site specific monoclonal anti-Ac-p53-K120 antibody.E H1299 cells were transfected with the indicated vectors. Total proteins and the anti-Flag antibody M2-specific immunoprecipitates were analyzed by western blot using the indicated antibodies.
Figure 3A U2OS cells were transfected with the indicated siRNAs. Seventy-two hours later, cell lysates were prepared and proteins from lysates were subjected to western blot using the indicated antibodies.B U2OS cells were transfected as in A. Seventy-two hours later, total mRNAs were extracted and subjected to RT-qPCR for the indicated genes.C U2OS cells were transfected with increasing amount of plasmid DNA expressing NAT10. Proteins from whole cell extracts were subjected to western blot using the indicated antibodies.D U2OS cells were transfected with control or NAT10 siRNAs. Seventy-two hours later, cells were treated with cycloheximide (CHX) and harvested at the indicated time points. Proteins from cell lysates were subjected to immunoblot for the evaluation of NAT10 and p53 (upper panels). Beta-actin was evaluated as a loading control (lower panel). p53 half-life (t1/2) obtained from three independent experiments was presented as mean ± SEM at the bottom.E U2OS cells were treated with MG132 (10μM) for 4 hours after transfected with the indicated siRNAs. Proteins from cell extracts were detected by western blot for the indicated proteins.F HCT116 p53+/+ cells were transfected with the indicated constructs and treated with MG132 for 4 hours before harvest. p53 was immunoprecipitated with anti-p53 polyclonal antibody and immunoblotted with anti-Ub antibody.G p53 and Mdm2 double null MEF cells were transfected with the indicated constructs and treated with MG132 for 4 hours before harvest. Proteins from whole cell extracts were subjected to western blot using the indicated antibodies.H U2OS cells were transfected with vectors as indicated. Forty-eight hours later, cells were harvested and proteins from whole cell extracts were subjected to western blot using the indicated antibodies.
Figure 4A HCT116 cells were transfected with increasing amount of DNA expressing NAT10. Forty-eight hours later, whole cell lysates were prepared and proteins from lysates were analyzed by western blot using the indicated antibodies.B HCT116 cells were transfected with Flag-NAT10 and harvested at indicated time points after treated with CHX. Proteins from cell lysates were immunoblotted with the antibodies as indicated (left panel). Relative Mdm2 protein levels standardized by β-actin are shown in the right. Mdm2 half-life (t1/2) obtained from three independent experiments was presented as mean ± SEM at the bottom.C HCT116 cells transfected with the indicated constructs were harvested after treatment with MG132 for 4 hours. The Mdm2 protein was immunoprecipitated with anti-Mdm2 antibody and immunoblotted with anti-HA antibody.D In vitro ubiquitination was performed with recombinant GST-Mdm2 (left) or GST-Mdm2 C464A mutant (right) and Flag-NAT10 or Flag-NAT10 GE in the presence of E1, E2 and Ub in vitro as described in Experimental Procedures. The reaction products were resolved by SDS-PAGE, followed by western blot for evaluation of the indicated proteins.E Mdm2 is ubiquitinated by NAT10 in a dose-dependent manner. In vitro ubiquitination assay were carried out with Mdm2 and different doses of purified Flag-NAT10 as described in D.G U2OS cells transfected with NAT10 siRNA-3 (targeting 3'-UTR of NAT10 mRNA) or control siRNA were co-expressed different NAT10 deletion mutants. Cell lysates were extracted and proteins from lysates were subjected to western blot using the indicated antibodies. Relative p53 protein levels standardized by β-actin are shown in the lower.H In vitro ubiquitination reaction was conducted using recombinant GST-Mdm2 C464A and full-length Flag-NAT10 or Flag-NAT10-D5. Ubiquitinated Mdm2 was detected as in D.I HCT116 cells were transfected with the indicated constructs. Cells were harvested after CHX treatment for the indicated times and cell lysates were prepared. Mdm2 protein levels were evaluated by western blot. Relative Mdm2 protein levels standardized by β-actin are shown in the right.
Figure 5A U2OS cells were treated with 10nM actinomycin D and cell extracts were prepared at indicated time points. Proteins from extracts were analyzed by western blot using the indicated antibodies.B U2OS and H1299 cells were treated with actinomycin D for 12 hours. Cells were harvested and proteins from cell lysates were subjected to western blot probed with the indicated antibodies.C U2OS cells transfected with the indicated siRNAs were treated with actinomycin D and harvested at the indicated time points. Proteins from cell lysates were subjected to western blot.D HCT116 cells were treated with 10nM of actinomycin D or 4 μM of doxorubicin (Dox) for 12 hours. Cells were harvested and total proteins were immunoprecipitated with anti-NAT10 antibody. Immunoprecipitates were analyzed by western blot for the indicated proteins.E HCT116 cells were treated with actinomycin D for the indicated times. Cells were fixed and immuno-stained as indicated. Immunofluorescence images were taken by confocal microscopy. Scale bar represents 10 μM.F H1299 cells were transfected with the indicated vectors. After treated with actinomycin D, cells were harvested and the whole cell extracts were subjected to western blot using the indicated antibodies.G H1299 cells were transfected with GFP-NAT10 vectors. After treated with actinomycin D for 12 hours, cells were fixed and nuclei were stained with DAPI. Bar represents 10μM.H HCT116 cells transfected with the indicated constructs were treated with actinomycin D or doxorubicin. Western blot was performed with total cell extracts and Mdm2-specific immunoprecipitates for the indicated proteins.I HCT116 cells were transfected with the indicated constructs. Cells were harvested after treatment with actinomycin D. p53 protein was immunoprecipitated with an anti-p53 polyclonal antibody and immunoblotted with p53 monoclonal antibody (DO-1).
Figure 6A p21 promoter luciferase reporter plasmid was cotransfected with the indicated siRNAs into U2OS cells. Luciferase activity was determined as described in Experimental Procedure.B U2OS cells were transfected with siRNAs as indicated. Seventy-two hours later, cells were harvested after treated with actinomycin D for 12 hours. Relative mRNA levels for the indicated genes were analyzed by RT-qPCR.C-E U2OS cells (C and D) and H1299 cells (D) transfected with the indicated siRNAs were treated with actinomycin D and harvested at the indicated time points. Cell cycle profiles were determined by FACS. Results from three independent experiments are shown as mean ± SEM. (two-tailed Student's t test)F U2OS cells were transfected with the indicated siRNAs. Cells were irradiated with 50 J/m2 UV. Forty-eight hours later, apoptotic cells were determined. Quantification of apoptotic cells is presented. (two-tailed Student's t test)G U2OS cells transfected with the indicated siRNAs were plated. Cell numbers were determined as described in Experimental Procedures at the indicated time points.H Colony formation assay were performed with U2OS cells transfected with the indicated siRNAs. The right panel shows the quantification of formed colonies. **p<0.01, ***p<0.001 (two-tailed Student's t test)
Figure 7A Twenty pairs of fresh frozen CRC tissues and corresponding normal tissues were lysed and total RNAs were extracted and subjected to RT-qPCR. (N, Normal tissue; T, Tumor tissue) **p<0.01 (non-parametric Mann-Whitney t-test)B Immunohistochemical staining of NAT10 in CRC tissues and normal colon tissues. NAT10-specific immuno-complex was detected with DAB as shown as brown color. Quantification of NAT10-positive or NAT10-negative CRC cases is shown in the lower table.C Cell extracts were prepared from different human colon epithelial cell lines as indicated at the top. Proteins from the extracts were subjected to western blot for the evaluation of NAT10 and p53.D CRISPR-Cas9 system mediated NAT10 gene editing. The genome targeting for knockdown NAT10 was performed as described in Experimental Procedures. The HCT116 NAT10 knockdown cell lines were harvested and western blot was performed on the cell lysates for the indicated proteins.E HCT116 Ctrl or c2-4 cells with NAT10 disruption were treated with actinomycin D and harvested at the indicated time points. Cell cycle profiles were determined by FACS. Results from three independent experiments are shown as mean ± SEM. (two-tailed Student's t test)F HCT116 Ctrl cells or c2-4 cells with NAT10 disruption were treated with actinomycin D Cells were harvested at the indicated time points and lysed. The total proteins were analyzed by western blot using the indicated antibodies.G HCT116 Ctrl or c2-4 cells were irradiated with 80 J/m2 UV. Forty-eight hours later, apoptotic cells were determined. Results from three independent experiments are shown as mean ± SEM. (two-tailed Student's t test)H Colony formation assay were performed with HCT116 cells (Ctrl, c1-1 and c2-4). The right panel shows the quantification of formed colonies. (two-tailed Student's t test)I HCT116 c1-1 and c2-4 cell lines were plated and cell growth curves were determined as described in Fig 6F. Error bars represent the SEM of the triplicate experiments.
Figure 1Representative western-blots of the cortical (Ctx) or hippocampal (Hip) extracts from mice at the indicated time after seizure activity in pilocarpine (A,B) models. (C,F) Statistics of Gli1 or Shh expression levels shown in A,B or D,E respectively. n=8-14 mice in C and n=8-23 mice in F.Representative western-blots of the cortical (Ctx) or hippocampal (Hip) extracts from mice at the indicated time after seizure activity in kindling (D,E) models. For D and E, samples obtained from mice evoked with a single kindling stimulation to induce seizure activity as evidenced in EEG. (C,F) Statistics of Gli1 or Shh expression levels shown in A,B or D,E respectively. n=8-14 mice in C and n=8-23 mice in F.(G) Shh levels assayed by ELISA from mouse cortex and hippocampus at the indicated time after the initiation of status epilepticus (SE) induced by pilocarpine (n=7-10).(H,I) Shh levels assayed by ELISA in the medium of slices (H, n=9) or hippocampal neurons (I, n=6) incubated with picrotoxin (Pic) or Mg2+-free (0Mg) for the indicated times.(J) Representative western-blots and statistics of Gli1 expression levels from hippocampal neurons incubated with 0Mg for the indicated times (n=13-19). a-tubulin (a-Tub), a loading control. Data were mean + SEM. *p<0.05; **p<0.01; ***p<0.001 vs. Control (Ctrl) with student's t test.
Figure 2(A-C) Whole-cell recordings of spontaneous epileptiform activity from cultured hippocampal neurons. A, representative traces showing the neuronal activity under the indicated treatments. Ctrl: extracellular solution. Cyclo: cyclopamine. B, the expanded view of a single burst (arrow) from A; C, statistics of the percentage of neurons showing epileptiform activity (left, n=6-7) and the burst frequency (right, n=18-19) shown in A. Cells were incubated in the presence of Cyclo in extracellular solution for 30 min, then transferred to 0Mg plus Cyclo (0Mg+Cyclo) and incubated for another 30 min.(D,E) The effects of Sant-1, robotnikinin (Robot) or 5E1 on the percentage of neurons showing epileptiform activity (D, n=6-10) and the burst frequency (E, n=15-22). (F-H) Extracellular recordings of spontaneous epileptiform activity from CA1 stratum pyramidal of hippocampal slices. F, representative traces under the indicated treatments. Ctrl: artificial cerebrospinal fluid (aCSF).(F-H) Extracellular recordings of spontaneous epileptiform activity from CA1 stratum pyramidal of hippocampal slices. F, representative traces under the indicated treatments. Ctrl: artificial cerebrospinal fluid (aCSF). G, the expanded view of a single burst (arrow) in F. Slices were incubated in the presence of vehicle or Cyclo in aCSF for 30 min, then transferred to picrotoxin in aCSF (Pic) or with Cyclo (Pic+Cyclo) and recorded for 30 min. H, statistics of the spontaneous burst frequency of F from 10 slices (7 rats).(I,J) Left are representative traces showing the D-CPP-sensitive currents recorded from cultured hippocampal neurons (I) or slices (J) incubated in 0Mg or Pic, respectively, with or without Cyclo. Black lines: application of D-CPP. Right are statistics of D-CPP-sensitive current density from 18-26 cells (I) or 16-18 slices (15 rats) (J). Unless stated, 10 µM cyclopamine was used. Data were mean + SEM. *p<0.05,**<0.01,***p<0.001 vs. Ctrl; #p<0.05,##<0.01,###p<0.001 vs. 0Mg or Pic; &amp;amp;p<0.05 vs. 0Mg + IgG. C-E used one-way ANOVA. H-J used student's t test.
Figure 3(A) Glutamate levels assayed by HPLC in the medium of neurons incubated with indicated agents. n=4-6.(B) The paired-pulse ratio of field excitatory postsynaptic potentials recorded in CA1 of hippocampal slices treated with vehicle (Ctrl) or Shh (13-14 slices, 6 rats). Insets: representative traces recorded in response to paired pulse stimuli with different intervals.Statistics of 3H-glutamate uptake by neurons treated with indicated agents (C, n=10)Statistics of 3H-glutamate uptake by neurons transfected with two lentivirus-based RNAi against EAAC1 (R2 and R3) or nonsense RNAi (Non) in response to vehicle (Ctrl) or Shh (D, n=3-6). No difference between R2 or R3 in Ctrl and those in Shh. Inset of (D): representative western-blots for EAAC1.Aspartate (Asp)-evoked currents at -70 mV from hippocampal neurons transfected with RNAi or Non (E, n=17-23) or treated with indicated agents (F, n=16-18).(G) Upper, representative western-blots of EAAC1 and Smo from HEK293 cells transfected with empty vectors (Ctrl), constitutively active form of Smoothened (SmoA1), EAAC1, or EAAC1 plus SmoA1. Lower, 3H-glutamate uptake by cells transfected with the indicated vectors. n=9.(H,I) Shh effects on 3H-glutamate uptake (H, n=6) or Asp-evoked currents (I, n=17-23) of neurons with or without pertussis toxin (PTX) pretreatment. For E,F,I, the uppers: representative traces. Shh: 500 ng/mL. Data were mean ± SEM. *p<0.05; **p<0.01; ***p<0.001 vs. Ctrl or Non (Ctrl) with student's t test.
Figure 4Effects of Cyclo (A-C, n=14) on the progression of kindling including seizure class (A,D,G,J), evoked electrographic seizure duration (ESD) (B,E,H,K) and the number of stimulations required to reach equivalent seizure intensity (C,F,I,L). In A-F, Ctrl are vehicle-treated mice.Effects of 5E1 (D-F, n=16-20), on the progression of kindling including seizure class (A,D,G,J), evoked electrographic seizure duration (ESD) (B,E,H,K) and the number of stimulations required to reach equivalent seizure intensity (C,F,I,L). In A-F, Ctrl are vehicle-treated mice.Effects of ablation of Smo in CaMKIIα-positive neurons (G-I), on the progression of kindling including seizure class (A,D,G,J), evoked electrographic seizure duration (ESD) (B,E,H,K) and the number of stimulations required to reach equivalent seizure intensity (C,F,I,L). In G-I, Ctrl: Smofl/fl induced by tamoxifen; CaMK: Smofl/flCaMKIIα-CreERT2 induced by tamoxifen, n=14-20.Effects of ablation of Smo in Aldh1l1-positive astrocytes (J-L) on the progression of kindling including seizure class (A,D,G,J), evoked electrographic seizure duration (ESD) (B,E,H,K) and the number of stimulations required to reach equivalent seizure intensity (C,F,I,L). In J-L, Ctrl: Smo+/fl; Aldh1l1: Smo+/flAldh1l1-Cre, n=19-20.(M) Frequency of spontaneous seizures (class 4-5) in mice administrated with Cyclo or vehicle (Ctrl) at 4th, 6th and 8th week after pilocarpine SE induction. n=22-23.(N) Percentage of mice with class 5 seizures within 8 weeks after pilocarpine SE induction. n=22-23. Cyclo: 10 mg/kg. 5E1: 900 ng/mouse. Data were mean ± SEM from at least three independent experiments. *p<0.05; **p<0.01; ***p<0.001 vs. Ctrl with student's t test.
Figure 2Morphological changes of vacuolar membranes and peroxisomal GFP-SKL during microautophagic degradation of peroxisomes after glucose adaptation. Double fluorescence of methanol-grown cells of P. pastoris cells at various times after the switch to glucose medium. (a) 0 h, (b) 30 min, (c) 2 h.
Figure 3Fluorescence and electron microscope images showing various stages during microautophagic degradation of peroxisomes. The three panels in each figure are as follows (a-e, left) FM4-64; (center) GFP-SKL; (right) superimposed FM4-64 and GFP-SKL signals. (a, f) stage 0; (b, g) early stage 1; (c, h) late stage 1; (d, i) stage 2; (e) stage 3. (d, arrowhead) During the capture of these fluorescence images, this peroxisome-containing vesicle had just pinched off from the outer vacuolar membrane. Therefore, the FM4-64-stained vesicle membrane and GFP-SKL could not be superimposed in this picture.
Figure 4Kinetics of morphological changes in vacuoles and peroxisomalGFP-SKL during microautophagic degradation of P. pastoris in glucose adaptation. At least 350 vacuoles or 350 clusters of peroxisomes were counted for each time point. Cells were counted in three randomly selected fields, and standard deviations are shown as vertical bars.
Figure 5Macroautophagic degradation of peroxisomes after transfer of methanol-grown P. pastoris cells to ethanol medium. (a-e) Electron micrographs of intermediates seen during macroautophagy. (a) After 1 h of ethanol adaptation, peroxisomes (P) were often surrounded by autophagosomal membranes, higher magnifications of which are in d and e. (b) After 1 h of ethanol adaptation, the autophagosomal membrane surrounding the peroxisomes appears to have fused with the vacuolar membrane. (c) At 2 h a peroxisome in the form of an autophagic body is seen within the vacuole. Magnified pictures of samples obtained at the (d) 20 min time point showing the nuclear endoplasmic reticulum membrane in close proximity to the membranes surrounding the peroxisomes, and at the (e) 1 h time point demonstrating peroxisomes surrounded by multiple autophagosomal membranes. (f) Kinetics of morphological changes in vacuoles and peroxisomal GFP-SKL. Other conditions are described in Materials and Methods, and are the same as in Fig. 4. (g) Fluorescence images taken after 2 h of ethanol adaptation of wild-type P. pastoris cells.
Figure 6Effect of inhibitors on transport of peroxisomal GFP-SKL to, and degradation in, vacuoles during (a) glucose adaptation and (b) ethanol adaptation. Wortmannin and PMSF were dissolved in DMSO, and added to the glucose medium. (Filled circle) Control (2% DMSO); (open circle) 1 mM wortmannin (with 2% DMSO); (filled square) 3 mM PMSF (with 2% DMSO); (filled triangle) 10 mg/ml cycloheximide.
Figure 7Microautophagic degradation of peroxisomes is not observed in a vacuolar protease-negative strain (STK7) of P. pastoris. Kinetics of morphological changes in vacuoles and peroxisomal GFP-SKL during (a) glucose adaptation and (b) ethanol adaptation. (c) Fluorescence images taken after 2 h of glucose adaptation. Arrows show GFP-SKL in vacuolar matrix. Some cells showed diffusion of GFP-SKL into the vacuolar matrix.
Figure 8(a) Visualization of alcohol oxidase activity in colonies after (a) glucose adaptation for 8 h and ethanol adaptation for 24 h in wild-type (strain PPY12) and pag mutant cells (strains STK1-STK6, Materials and Methods) of P. pastoris. (b) Western-blot analysis of samples taken from cultures shifted from methanol to glucose for the indicated time. Antibodies used were AOX, alcohol oxidase; F1, mitochondrial F1β subunit; G6PDH, glucose-6-phosphate dehydrogenase. Growth curve of the wild-type strain PPY12 and the pag4 mutant (strain STK4) after shift from methanol to glucose.