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O-GlcNAc-modification of SNAP-29 regulates autophagosome maturation

Nature Cell Biology volume 16, pages 1215–1226 (2014)Cite this article

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Abstract

The mechanism by which nutrient status regulates the fusion of autophagosomes with endosomes/lysosomes is poorly understood. Here, we report that O-linked β-N-acetylglucosamine (O-GlcNAc) transferase (OGT) mediates O-GlcNAcylation of the SNARE protein SNAP-29 and regulates autophagy in a nutrient-dependent manner. In mammalian cells, OGT knockdown, or mutating the O-GlcNAc sites in SNAP-29, promotes the formation of a SNAP-29-containing SNARE complex, increases fusion between autophagosomes and endosomes/lysosomes, and promotes autophagic flux. In Caenorhabditis elegans, depletion of ogt-1 has a similar effect on autophagy; moreover, expression of an O-GlcNAc-defective SNAP-29 mutant facilitates autophagic degradation of protein aggregates. O-GlcNAcylated SNAP-29 levels are reduced during starvation in mammalian cells and in C. elegans. Our study reveals a mechanism by which O-GlcNAc-modification integrates nutrient status with autophagosome maturation.

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Figure 1: Loss of function of ogt-1(bp815) promotes autophagy activity in C. elegans.
Figure 2: Knockdown of OGT in mammalian cells promotes autophagosome maturation.
Figure 3: OGT knockdown promotes assembly of the Stx17–SNAP-29–VAMP8 SNARE complex.
Figure 4: SNAP-29 is O-GlcNAc-modified.
Figure 5: SNAP-29 is O-GlcNAc-modified at Ser 2, Ser 61, Thr 130 and Ser 153, and O-GlcNAcylation-defective SNAP-29 promotes autophagy activity.
Figure 6: C. elegans SNAP-29 is O-GlcNAcylated and participates in autophagy regulation.
Figure 7: Levels of SNAP-29 O-GlcNAcylation are reduced under starvation conditions.
Figure 8: Model for the role of O-GlcNAc-modification in regulating autophagosome maturation.

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Acknowledgements

We thank I. Hanson for editing services and F. Shao (NIBS) for OGA proteins. Some strains were obtained from CGC, which is funded by the National Institutes of Health (P40 OD010440). This work was supported by the National Basic Research Program of China (2013CB910100, 2011CB910100) and also a grant from the National Natural Science Foundation of China (31225018) to H.Z. The research of H.Z. was supported in part by an International Early Career Scientist grant from the Howard Hughes Medical Institute.

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  1. Bin Guo and Qianqian Liang: These authors contributed equally to this work.

Authors and Affiliations

  1. State Key Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China

    Bin Guo, Qianqian Liang, Fan Wu, Peipei Zhang & Hong Zhang

  2. National Institute of Biological Sciences, Beijing 102206, China

    Lin Li, Yongfen Ma, Zhiyuan Zhang & She Chen

  3. Institute of Neurology, Key Laboratory of Age-Associated Cardiac-Cerebral Vascular Disease of Guangdong Province, Affiliated Hospital of Guangdong Medical College, Zhanjiang 524001, China

    Zhe Hu, Bin Zhao & Du Feng

  4. Department of Anatomy, Cell and Developmental Biology, Eötvös Loránd University, Budapest 1117, Hungary

    Attila L. Kovács

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Contributions

B.G., D.F., Z.Z., S.C. and H.Z. designed the experiments. B.G., Q.L., L.L., Z.H., P.Z., F.W., Y.M. and B.Z. performed the experiments. A.L.K. and D.F. analysed the electron micrographs. B.G., S.C. and H.Z. wrote the manuscript.

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Correspondence to Hong Zhang.

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Integrated supplementary information

Supplementary Figure 1 bp815 promotes autophagy activity in C. elegans embryos and larvae.

(a) Weak expression and diffuse cytoplasmic localization of SQST-1 in wild-type embryos. (b) SQST-1 forms numerous aggregates in epg-5(bp450) mutant embryos. (c) Far fewer SQST-1 aggregates are formed in epg-5(bp450); bp815 double mutants. (d) Number of SQST-1 aggregates per focal plane in epg-5 and epg-5; bp815 mutant embryos. n = 5 focal planes from 5 embryos. ∗∗p < 0.01. (e) sqst-1 mRNA levels are unchanged in epg-5 and epg-5; bp815 animals (p = 0.60) by real-time PCR. Data are shown as mean ± s.d. from n = 3 independent experiments. (f–h) PGL granules are absent in somatic cells in wild-type embryos (f). Accumulation of PGL granules in somatic cells in epg-5 mutants (g) is suppressed by bp815 (h). The two PGL-1-positive cells are germline precursors. (i) Number of PGL-1 aggregates in somatic cells per focal plane in epg-5 and epg-5; bp815 mutants. n = 5 focal planes from 5 embryos. ∗∗p < 0.01. (j–k) Wild-type embryos contain no SEPA-1 aggregates at the 4-fold stage (j), while numerous SEPA-1 aggregates accumulate in epg-5(bp450) mutants (k). (l) Far fewer SEPA-1 aggregates are present in epg-5(bp450); bp815 double mutants. (m) Number of SEPA-1 aggregates per focal plane in epg-5 and epg-5; bp815 mutants. n = 5 focal planes from 5 embryos. ∗∗p < 0.01. Data in (d,i,m) are shown as mean ± s.d. Statistical significance was determined by a two-tailed, unpaired Student’s t-test. (n–o) bp815 suppresses the accumulation of SQST-1 aggregates in epg-5(tm3425) mutants. (p) Western analysis showing epg-5(tm3425); bp815 mutants contain less SQST-1::GFP than epg-5(tm3425) mutants. (q–t) bp815 suppresses the accumulation of SQST-1::GFP aggregates in hypodermal cells in epg-5(tm3425) mutant larvae. (q) and (s): DIC images of animals in (r) and (t). (u–v) bp815 suppresses accumulation of SQST-1 aggregates in bec-1 hypomorphic mutants. (w–x) Accumulation of SQST-1 aggregates in atg-9(bp564) mutants is unaffected by bp815. (y–z) Accumulation of SQST-1 aggregates in atg-3 hypomorphic mutants (y) is suppressed by bp815. (a2–d2) bp815 has no effect on accumulation of SQST-1 aggregates in atg-18(gk378) null mutants (a2–b2) and epg-9(bp320) null mutants (c2–d2). Scale bars: 5 μm (a–c,f–h,j–l,n,o,u–x); 20 μm (q–t,y–d2).

Supplementary Figure 2 bp815 promotes autophagy activity independent of Tor signaling inactivation, transcriptional regulation of autophagy genes or ER stress.

(a–b) rpt-3(RNAi) does not cause accumulation of SQTS-1::GFP in embryonic cells. L4 larvae were used for rpt-3 RNAi feeding and the F1 progeny were arrested at the ∼100 to 200 cell stage. (c–d) rpt-3(RNAi) does not cause accumulation of ectopic SEPA-1 granules at the ∼100 to 200 cell embryonic stage. (e–f) rpt-3(RNAi) has no effect on SQST-1::GFP distribution in larvae. L1 larvae were used for RNAi feeding and arrested animals were examined. (g–h) Simultaneous depletion of rpt-3 does not cause the re-appearance of SQST-1::GFP aggregates in epg-5; bp815 mutants. (i–j) No SQST-1::GFP aggregates are formed in rpl-43; rpt-3 bp815 mutants. (k–l) Simultaneous depletion of xbp-1 does not cause re-appearance of SQST-1::GFP aggregates in rpl-43; bp815 mutants. (m–o) Accumulation of SQST-1 aggregates in epg-5 mutant embryos is not affected by loss of let-363 (encoding the C. elegans Tor homolog) or rheb-1. (p) Relative mRNA levels of autophagy genes in wild type and bp815 mutants. Data are shown as mean ± s.d. from n = 3 independent experiments. There was no significant difference between wild type and bp815 mutants. Statistical significance was determined by a two-tailed, unpaired Student’s t-test. (q–r) A transgene expressing wild-type ogt-1 causes accumulation of a large number of SQST-1 aggregates in epg-5; ogt-1(bp815) larvae. (s–t) A transgene expressing an ogt-1::gfp reporter causes accumulation of many SQST-1 aggregates in epg-5(bp450); ogt-1 double mutant embryos. (u–x) Expression of ogt-1::gfp in intestine (arrow in v), head neurons (arrowhead in x) and hypodermal cells (arrow in x). (y–b2) Accumulation of SQST-1::GFP aggregates in hypodermal cells in epg-5(bp450) mutants is suppressed by ogt-1(tm1046) and ogt-1(ok430). (c2) Western analysis showing that ogt-1(bp815, tm1046 or ok430) greatly reduces SQST-1::GFP levels in epg-5(bp450) mutants. (d2) ogt-1(bp815, tm1046 or ok430) greatly reduces SQST-1::GFP levels in rpl-43 mutants in a western blot assay. (e2–j2) Accumulation of SQST-1::GFP aggregates in rpl-43 mutants is suppressed by ogt-1(ok430) (e2–f2), ogt-1(tm1046) (g2–h2) and ogt-1(RNAi) (i2–j2). (k2–l2) Loss of function of f07a11.2 and f22b3.4 suppresses the accumulation of SQST-1 aggregates in epg-5 mutant embryos. (m2–n2) Accumulation of SQST-1::GFP aggregates in rpl-43 mutants is suppressed by RNAi inactivation of f07a11.2 and f22b3.4. (a), (c), (e), (g), (i), (k), (q), (u), (w), (y), (a2), (e2), (g2), (i2), (k2) and (m2): DIC images of the animals in (b), (d), (f), (h), (j), (l), (r), (v), (x), (z), (b2), (f2), (h2), (j2), (l2) and (n2), respectively. (s): DAPI image of the animal in (t). Scale bars: 5 μm (a–d,m–o,s,t,w,x,k2,l2); 20 μm (e–l,q,r,u,v,y–b2,e2–j2,m2,n2).

Supplementary Figure 3 Loss of OGT, loss of GFAT and Alloxan treatment lead to elevated autophagic flux.

(a–b) OGT siRNA knocks down OGT protein in a western blot (a) and OGT mRNA (b). Data are shown as mean ± s.d. from n = 3 independent experiments. ∗∗∗p < 0.001. (c) Western blot showing LC3 levels in Hela cells treated with or without 5 mM Alloxan for 16 hours under normal conditions or with BafA1. (d) Quantification of LC3 puncta in Hela cells treated with or without 5 mM Alloxan for 16 hours. n = fifty cells pooled from 3 independent experiments were counted in each group. ∗∗p < 0.01. (e) Compared to control siRNA-treated cells, LC3 puncta, detected by anti-LC3, accumulate in Hela cells treated with 5 mM Alloxan for 16 hours under normal conditions or with BafA1. (f) Quantification of LC3 puncta in Hela cells transfected with NC or GFAT1 siRNA. n = fifty cells pooled from 3 independent experiments were counted in each group. ∗∗p < 0.01. (g) Western blot showing phospho-AMPK, phospho-S6K, phospho-4EBP1, OGT and LC3 levels after 2 hours starvation in Hela cells stably expressing control or OGT shRNA. (h) 2 × FYVE-GFP reporter expression in Hela cells transfected with NC or OGT siRNA. Cells were examined after 1 hour starvation. (i) Quantification of 2 × FYVE-GFP puncta in NC or OGT siRNA-transfected Hela cells. n = fifty cells pooled from 3 independent experiments were counted in each group. (j) OGT KD does not significantly increase the mRNA level of several essential autophagy genes. Data are shown as mean ± s.d. from n = 3 independent experiments. Statistical significance was determined by a two-tailed, unpaired Student’s t-test. (k) OGT KD does not increase levels of autophagy proteins. (l–m) Expression of GFP-DFCP1 (l) and Atg16L-GFP (m) in Hela cells transfected with NC or OGT siRNA. Cells were cultured normally or starved for 1 hour before examination. (n–o) Quantification of GFP-DFCP1 (n) and Atg16L-GFP (o) puncta in Hela cells transfected with NC or OGT siRNA that were cultured under normal conditions or starvation conditions. n = fifty cells pooled from 3 independent experiments were counted in each group. Data in (d,f,i,n,o) are shown as mean ± s.d. from 3 independent experiments. (p–r) Kinetic analysis of Atg16L-GFP in control and OGT knockdown cells. Arrowheads indicate puncta that were followed in real-time imaging. (p) shows the duration of Atg16L-GFP puncta. n = 10 puncta pooled from 3 independent experiments. Data are shown as mean ± s.d. ∗p < 0.05. Statistical significance was determined by a two-tailed, unpaired Student’s t-test. Scale bars: 5 μm (q,r); 10 μm (e,h,l,m).

Supplementary Figure 4 OGT KD promotes the colocalization of LC3 puncta with endocytic vesicles and suppresses the autophagic defect in EPG5 KD cells.

(a) Number of RFP-positive and GFP-negative autolysosomes (ALs) in Hela cells treated with or without Alloxan (5 mM) for 16 hours. Cells were examined after 2 hours starvation. n = fifty cells pooled from 3 independent experiments were counted in each group. ∗∗p < 0.01. (b) Number of Rab7-, LBPA- and LAMP1-positive vesicles in control and OGT KD cells. n = fifty cells pooled from 3 independent experiments were counted in each group. (c–d) OGT knockdown increases colocalization of LC3 puncta, detected by anti-LC3, with LBPA-labeled endocytic structures after 2 hours starvation. (d) shows quantification of LC3 puncta positive for LBPA-labeled vesicles. n = fifty cells pooled from 3 independent experiments were counted in each group. ∗∗p < 0.01. (e–f) OGT knockdown increases colocalization of LC3 puncta with LAMP1-Cherry-labeled endocytic structures after 2 hours starvation. (e) shows quantification of LC3 puncta positive for LAMP1-Cherry-labeled vesicles. n = fifty cells pooled from 3 independent experiments were counted in each group. ∗∗p < 0.01. (g–h) Quantification of LC3 puncta positive for Rab7, LBPA or LAMP1 in Hela cells treated with or without Alloxan (g) or with GFAT1 siRNA (h). n = fifty cells pooled from 3 independent experiments were counted in each group. ∗∗p < 0.01, ∗∗∗p < 0.001. (i) DQ-BSA staining in Hela cells stably expressing control or OGT shRNA after 2 hours starvation. DQ-BSA becomes fluorescent upon cleavage in the lysosome, thus indicating lysosomal activity. (j) Percentage of cells positive for DQ-BSA Red fluorescence. n = one hundred cells pooled from 3 independent experiments were counted in each group. ∗∗p < 0.01. (k–l) EM analysis of OGT KD cells under nutrient repletion conditions. Autolysosomes (labeled 1) or hybrid autolysosomes with MVBs or EEs (early endosomes) or LEs (late endosomes) (labeled 3) accumulate. MVBs are labeled 5. (m–n) After 2 hours starvation, OGT KD cells accumulate autolysosomes (1). Hybrid autolysosomes with MVBs or EEs or LEs (3) and MVBs (5) are also indicated. (n) is a magnified image of (m). (o–p) Real-time PCR analysis of siRNA efficiency of OGT siRNA, EPG5 siRNA or OGT siRNA plus EPG5 siRNA. mRNA levels were normalized to that in control cells, which was set to 1. Data are shown as mean ± s.d. from n = 3 independent experiments. ∗∗∗p < 0.001. (q) GFAT1 siRNA treatment suppresses LC3 and p62 accumulation in EPG5 KD cells. Hela cells were transfected with NC siRNA, GFAT1 siRNA, EPG5 siRNA or EPG5 siRNA plus GFAT1 siRNA. Cells were starved for 2 hours before analysis. (r) Alloxan treatment suppresses LC3 and p62 accumulation in EPG5 KD cells. Hela cells were transfected with NC or EPG5 siRNA for 72 hours, then treated with or without Alloxan. After 2 hours starvation, cells were analyzed by immunoblotting using the indicated antibodies. (s) The autophagy defect in Epg5 KD cells is not evidently suppressed by mTOR inactivation. The level of p62 normalized to actin in NC siRNA-treated cells was set to 1. (t) EPG5 siRNA decreases DQ-BSA staining. This is suppressed by simultaneously depleting OGT. (u) Quantification of DQ-BSA Red fluorescence in cells with different knockdown treatments. n = one hundred cells pooled from 3 independent experiments were counted in each group. ∗∗p < 0.01. Data in (a,b,d,e,g,h,j,u) are shown as mean ± s.d. from 3 independent experiments. Statistical significance was determined by a two-tailed, unpaired Student’s t-test. (v–w) EPG5 KD leads to accumulation of autophagic elements. Autophagosomes (2) and autolysosomes (6) accumulate. The autolysosomes are lighter than normal, suggesting that degradation is impaired. MVBs are also indicated (5). (x–y) Simultaneous depletion of OGT suppresses the autophagic flux defect caused by EPG5 siRNA. Mature autolysosomes, which are small and dark (1) accumulate as in OGT KD cells. A hybrid autolysosome, which has fused with an MVB, an early endosome (or EE) or a late endosome (LE), is labeled 3. (y) shows a magnified view of (x). Scale bars: 5 μm (c); 10 μm (f,i,t); 500 nm (k–n,v—y).

Supplementary Figure 5 Loss of OGT activity suppresses the autophagic defect in COPβ and VCP KD cells.

(a–b) OGT KD suppresses the autophagy defect in COPβ or VCP siRNA cells. (c–f) OGT KD suppresses LC3 and p62 accumulation in COPβ or VCP siRNA cells. Hela cells stably expressing control or OGT shRNA were transfected with NC, COPβ or VCP siRNA. After 72 hours, cells were treated with or without 200 nM Rapamycin for 4 hours, then stained with anti-LC3 (a–b) or immunoblotted using the indicated antibodies (c–f). Quantification of LC3 puncta is shown in (c) and (e). n = fifty cells pooled from 3 independent experiments were counted in each group. ∗p < 0.05, ∗∗p < 0.01. (g) VMP1 siRNA treatment causes accumulation of p62 aggregates, detected by anti-p62, which is not suppressed by OGT KD. OGT KD cells contain fewer p62 aggregates than controls. (h) OGT KD does not suppress the autophagic defect in VMP1 siRNA-treated cells. Hela cells stably expressing control or OGT shRNA were transfected with NC or VMP1 siRNA. Cells were starved for 2 hours and stained with anti-p62 (g) or immunoblotted with the indicated antibodies (h). (i) Western blot showing siRNA knockdown of SNAP-29, Stx17 and VAMP8. (j–k) Quantification of LC3 puncta positive for Rab7-GFP (j) or LAMP1-Cherry (k) in Hela cells stably expressing control or OGT shRNA that were also transfected with NC, Stx17, SNAP-29 or VAMP8 siRNA. n = fifty cells pooled from 3 independent experiments were counted in each group. ∗∗p < 0.01. (l–m) Colocalization of LC3 puncta, detected by anti-LC3, with Rab7-GFP (l) and LAMP1-Cherry (m) in OGT KD cells is suppressed by siRNA knockdown of Stx17, SNAP-29 or VAMP8. Cells were starved for 2 hours before anti-LC3 staining. (n–o) Accumulation of p62 aggregates, detected by anti-p62, in EPG5 KD cells 2 hours after starvation (n), is suppressed by simultaneous OGT knockdown (o). This suppression depends on SNAP-29 and VAMP8 activity (o). (p–r) LC3 puncta are separable from Stx17-GFP-labeled structures in EPG5 siRNA-treated cells 2 hours after starvation. Simultaneously depleting OGT promotes colocalization of LC3 puncta with Stx17-GFP in EPG5 siRNA-treated cells. (r) shows quantification of LC3 puncta positive for Stx17-GFP. n = fifty cells pooled from 3 independent experiments were counted in each group. ∗∗p < 0.01. Data in (c,e,j,k,r) are shown as mean ± s.d. from 3 independent experiments. Statistical significance was determined by a two-tailed, unpaired Student’s t-test. Scale bars: 5 μm (l,m,p,q); 10 μm (a,b,g,n,o).

Supplementary Figure 6 OGT KD increases the colocalization of LC3 puncta with Stx17-GFP, SNAP-29-GFP and VAMP8-GFP.

(a–c) After 2 hours starvation, more LC3 puncta colocalize with Stx17-GFP (a), SNAP-29-GFP (b) and VAMP8-GFP (c)-labeled structures in OGT KD cells than controls. Boxed regions are magnified at the right of each image set. (d–f) Number of LC3 puncta positive for Stx17-GFP (d), SNAP-29-GFP (e), VAMP8-GFP (f) in control and OGT KD cells. n = fifty cells pooled from 3 independent experiments were counted in each group. ∗∗p < 0.01, ∗∗∗p < 0.001. (g) Compared to control shRNA cells, OGT KD promotes colocalization of Stx17-CFP-positive LC3 puncta with VAMP8-GFP after 2 hours starvation. (h) Number of Stx17-CFP-positive LC3 puncta colocalized with VAMP8-GFP puncta. n = fifty cells pooled from 3 independent experiments were counted in each group. ∗∗p < 0.01. (i–j) After 4 hours starvation, OGT KD cells contain fewer LC3 puncta positive for Stx17-GFP than controls. (j) shows quantification of Stx17-GFP-positive LC3 puncta. n = fifty cells pooled from 3 independent experiments were counted in each group. ∗∗p < 0.01. Data in (d,e,f,h,j) are shown as mean ± s.d. from 3 independent experiments. (k–m) Time lapse imaging shows that the duration of Stx17+LC3+ vesicles is shorter in OGT KD cells than controls. The puncta were followed after 4 hours starvation. (k) shows quantification of the duration of Stx17+LC3+ puncta in control and OGT KD cells. n = 10 puncta pooled from 3 independent experiments. Data are shown as mean ± s.d. Statistical significance was determined by a two-tailed, unpaired Student’s t-test. Scale bars: 5 μm (a,c,l,m); 10 μm (b,g,i).

Supplementary Figure 7 SNAP-29 is O-GlcNAc-modified.

(a) Levels of endogenous SNAP-29 and VAMP8 co-immunoprecipitated by Flag-Stx17 are increased in OGT KD cells. Cells stably expressing control or OGT shRNA were transfected with Flag-Stx17. Cells were starved for 2 hours before analysis. Flag-Stx17 and actin are loading controls. (b) Relative levels of SNAP-29 and VAMP8 that are co-immunoprecipitated by Flag-Stx17 in control and OGT shRNA cells. Levels of SNAP-29 and VAMP8 are normalized to the corresponding input. The level of SNAP-29 and VAMP8 co-immunoprecipitated by Flag-Stx17 in control cells was set to 1. Data are shown as mean ± s.d. from n = 3 independent experiments. ∗∗p < 0.01. (c) Levels of VAMP4-GFP co-immunoprecipitated by Flag-Stx6 are unchanged in OGT KD cells. Cells stably expressing control or OGT shRNA were transfected with VAMP4-GFP and Flag-Stx6. Cells were starved for 2 hours before analysis. Flag-Stx6 and actin are loading controls. (d) SNAP-29 is O-GlcNAc-modified, detected by anti-O-GlcNAc antibody (RL2), in an O-GlcNAcylation assay. The O-GlcNAcylation substrate Nup62 is a positive control. Coom. Blue, Coomassie staining. (e) The interaction of SNAP-29 with OGT in a co-immunoprecipitation assay is unaffected by mutating the SNAP-29 O-GlcNAcylation sites. Extracts of cells co-expressing HA-OGT with wild-type or mutant Flag-SNAP-29 were precipitated by anti-HA and the co-immunoprecipitants were analyzed by immunoblotting using the indicated antibodies. (f) Tandem mass spectrum of the doubly-charged peptide LKEAIST(GlcNAcS)KEQEAK reveals that Ser153 is O-GlcNAcylated. Recombinant SNAP-29 was purified from E. coli expressing OGT. The b- and y-type product ions are marked on the spectrum, and also illustrated along the peptide sequence shown on top of the spectrum. (g) O-GlcNAcylation is detected on the peptide SKPVETPPEQNGTLTSQPNNR. The exact site could not be identified due to complete neutral loss of the GlcNAc modification group. (h–i) Compared to wild-type SNAP-29-GFP, SNAP-29(QM)-GFP forms more punctate structures and more LC3 and VAMP8-RFP puncta. SNAP-29(QM)-GFP puncta colocalize with VAMP8-positive LC3. (i) shows the number of VAMP8 puncta colocalized with SNAP-29-positive LC3 puncta per cell. n = fifty cells pooled from 3 independent experiments were counted in each group. ∗∗p < 0.01. (j–l) SNAP-29(QM)-GFP forms punctate structures and expression of SNAP-29(QM) increases the number of LC3 puncta under normal conditions or with BafA1 treatment. (j) shows quantification of LC3 puncta under normal conditions or with BafA1. n = fifty cells pooled from 3 independent experiments were counted in each group. Data in (i,j) are shown as mean ± s.d.∗∗p < 0.01, ∗∗∗p < 0.001. Statistical significance was determined by a two-tailed, unpaired Student’s t-test. Scale bar: 5 μm (h); 10 μm (k,l).

Supplementary Figure 8 O-GlcNAcylation-defective mutant SNAP-29 promotes autophagy activity.

(a) Expression of O-GlcNAcylation-defective mutant SNAP-29 (SNAP-29(QM)) suppresses the autophagy defect in EPG5 KD cells. Accumulation of LC3-II and p62 in EPG5 siRNA-treated cells is suppressed by mutant SNAP-29, and to a lesser extent by wild-type SNAP-29. (b) O-GlcNAc-modification reduces the SNAP-29/Stx17 interaction in in vitro pulldown assays. Levels of O-GlcNAc-modified His-SNAP-29 (purified from bacteria co-expressing SNAP-29 and OGT) that were pulled down by GST-Stx17 are much lower than non-modified His-SNAP-29 (purified from bacteria without co-expression of OGT). Co-expressing OGT with the O-GlcNAcylation-defective mutant SNAP-29 has no effect on the SNAP-29/Stx17 interaction. (c) Relative levels of SNAP-29, O-GlcNAcylated SNAP-29 and mutant SNAP-29 pulled down by Stx17. Levels of pulled-down protein are normalized to the corresponding input. The level of non-O-GlcNAcylated SNAP-29 pulled down by Stx17 was set to 1. Data are collected from n = 3 independent experiments and are shown as mean ± s.d.∗p < 0.05. (d) Flag-Stx6 co-immunoprecipitates more HA-SNAP-29 in OGT KD cells than controls. Cells were starved for 2 hours before analysis. Flag-Stx6 and actin are loading controls. (e) Mutant SNAP-29 co-immunoprecipitates more Stx6 than wild-type SNAP-29. Cells were starved for 2 hours before analysis. HA-SNAP-29 and actin are loading controls. (f) Stx6-GFP puncta are separable from LC3 puncta, detected by anti-LC3, in control and OGT KD cells. (g) Western blot showing that Stx6 siRNA knocks down Stx6. (h) OGT-1::GFP and Cherry::SNAP-29 show cytoplasmic localization in intestine. SNAP-29 also forms some punctate structures. (i) Quantification of hTfR::GFP puncta per unit area (250 μm2) in intestine in different genotypes. n = 10 unit areas from 5 L4 larvae. Data are shown as mean ± s.d.∗∗p < 0.01. (j) In wild type intestine, hTfR::GFP puncta mainly localize to the plasma membrane with some in the cytoplasm. (k) snap-29(RNAi) increases cytoplasmic hTfR::GFP puncta. (l–m) ogt-1(bp815) and a transgene expressing O-GlcNAcylation-defective SNAP-29, snap-29(QM), promote hTfR::GFP recycling in intestine. The big and weak fluorescent particles are gut autofluorescence. Scale bars: 5 μm (f); 20 μm (h,j–m). (n) O-GlcNAc-modified SNAP-29 levels are reduced 2 hours after EBSS starvation. (o) UDP-GlcNAc levels are decreased 2 hours after EBSS starvation. Data are shown as mean ± s.d. from n = 3 independent experiments. ∗p < 0.05. Statistical significance was determined by a two-tailed, unpaired Student’s t-test. (p–q) O-GlcNAc-modified SNAP-29 levels are not evidently changed after rapamycin treatment (p) or in EPG5 KD cells (q). Rapamycin-treated, NC siRNA-transfected or EPG5 siRNA-transfected Flag-SNAP-29-expressing Hela cells were lysed, subjected to Co-IP assay with anti-Flag antibody, then immunoblotted with the indicated antibodies.

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Guo, B., Liang, Q., Li, L. et al. O-GlcNAc-modification of SNAP-29 regulates autophagosome maturation. Nat Cell Biol 16, 1215–1226 (2014). https://doi.org/10.1038/ncb3066

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