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Uba1 functions in Atg7- and Atg3-independent autophagy

Nature Cell Biology volume 15, pages 1067–1078 (2013)Cite this article

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Abstract

Autophagy is a conserved process that delivers components of the cytoplasm to lysosomes for degradation. The E1 and E2 enzymes encoded by Atg7 and Atg3 are thought to be essential for autophagy involving the ubiquitin-like protein Atg8. Here, we describe an Atg7- and Atg3-independent autophagy pathway that facilitates programmed reduction of cell size during intestine cell death. Although multiple components of the core autophagy pathways, including Atg8, are required for autophagy and cells to shrink in the midgut of the intestine, loss of either Atg7 or Atg3 function does not influence these cellular processes. Rather, Uba1, the E1 enzyme used in ubiquitylation, is required for autophagy and reduction of cell size. Our data reveal that distinct autophagy programs are used by different cells within an animal, and disclose an unappreciated role for ubiquitin activation in autophagy.

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Figure 1: Atg18 and Atg2 are required for programmed cell size reduction in the Drosophila midgut.
Figure 2: Programmed size reduction is cell autonomous and requires Atg18 and Atg1.
Figure 3: Autophagy is necessary and sufficient for cell size reduction.
Figure 4: Atg7 is not required for programmed cell size reduction and autophagy.
Figure 5: Autophagy is required for clearance of mitochondria.
Figure 6: Uba1 is required for midgut cell programmed size reduction and autophagy.
Figure 7: Role of Uba1 in midgut autophagy.
Figure 8: Uba1 is required for clearance of mitochondria.

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References

  1. Mizushima, N. & Komatsu, M. Autophagy: renovation of cells and tissues. Cell 147, 728–741 (2011).

    Article  CAS  PubMed  Google Scholar 

  2. Levine, B. & Yuan, J. Autophagy in cell death: an innocent convict? J. Clin. Invest. 115, 2679–2688 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Baehrecke, E. H. Autophagy: dual roles in life and death? Nat. Rev. Mol. Cell Biol. 6, 505–510 (2005).

    Article  CAS  PubMed  Google Scholar 

  4. Berry, D. L. & Baehrecke, E. H. Growth arrest and autophagy are required for salivary gland cell degradation in Drosophila. Cell 131, 1137–1148 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Hou, Y. C., Chittaranjan, S., Barbosa, S. G., McCall, K. & Gorski, S. M. Effector caspase Dcp-1 and IAP protein Bruce regulate starvation-induced autophagy during Drosophila melanogaster oogenesis. J. Cell Biol. 182, 1127–1139 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Nezis, I. P. et al. Autophagic degradation of dBruce controls DNA fragmentation in nurse cells during late Drosophila melanogaster oogenesis. J. Cell Biol. 190, 523–531 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Mizushima, N., Levine, B., Cuervo, A. M. & Klionsky, D. J. Autophagy fights disease through cellular self-digestion. Nature 451, 1069–1075 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Tsukada, M. & Ohsumi, Y. Isolation and characterization of autophagy-defective mutants of Saccharomyces cerevisiae. FEBS Lett. 333, 169–174 (1993).

    Article  CAS  Google Scholar 

  9. Thumm, M. et al. Isolation of autophagocytosis mutants of Saccharomyces cerevisiae. FEBS Lett. 349, 275–280 (1994).

    Article  CAS  PubMed  Google Scholar 

  10. Harding, T. M., Morano, K. A., Scott, S. V. & Klionsky, D. J. Isolation and characterization of yeast mutants in the cytoplasm to vacuole protein targeting pathway. J. Cell Biol. 131, 591–602 (1995).

    Article  CAS  PubMed  Google Scholar 

  11. Harding, T. M., Hefner-Gravink, A., Thumm, M. & Klionsky, D. J. Genetic and phenotypic overlap between autophagy and the cytoplasm to vacuole protein. J. Biol. Chem. 271, 17621–17624 (1996).

    Article  CAS  PubMed  Google Scholar 

  12. Schulman, B. A. & Harper, J. W. Ubiquitin-like protein activation by E1 enzymes: the apex for downstream signalling pathways. Nat. Rev. Mol. Cell Biol. 10, 319–331 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Denton, D. et al. Autophagy, not apoptosis, is essential for midgut cell death in Drosophila. Curr. Biol. 19, 1741–1746 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Lee, C-Y., Cooksey, B. A. K. & Baehrecke, E. H. Steroid regulation of midgut cell death during Drosophila development. Dev. Biol. 250, 101–111 (2002).

    Article  CAS  PubMed  Google Scholar 

  15. Micchelli, C. A., Sudmeier, L., Perrimon, N., Tang, S. & Beehler-Evans, R. Identification of adult midgut precursors in Drosophila. Gene Expr. Patter. 11, 12–21 (2011).

    Article  CAS  Google Scholar 

  16. Scott, R. C., Juhász, G. & Neufeld, T. P. Direct induction of autophagy byAtg1 inhibits cell growth and induces apoptotic cell death. Curr. Biol. 17, 1–11 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Juhász, G., Erdi, B., Sass, M. & Neufeld, T. P. Atg7-dependent autophagy promotes neuronal health, stress tolerance, and longevity but is dispensable for metamorphosis in Drosophila. Genes Dev. 21, 3061–3066 (2007).

    Article  PubMed  PubMed Central  Google Scholar 

  18. Belote, J. M. & Fortier, E. Targeted expression of dominant negative proteasome mutants in Drosophila melanogaster. Genesis 34, 80–82 (2002).

    Article  CAS  PubMed  Google Scholar 

  19. Pandey, U. B. et al. HDAC6 rescues neurodegeneration and provides an essential link between autophagy and the UPS. Nature 447, 859–863 (2007).

    Article  CAS  PubMed  Google Scholar 

  20. Fujita, N. et al. An Atg4B mutant hampers the lipidation of LC3 paralogues and causes defects in autophagosome closure. Mol. Biol. Cell 19, 4651–4659 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Pircs, K. et al. Advantages and limitations of different p62-based assays for estimating autophagic activity in Drosophila. PLoS ONE 7, e44214 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Jiang, C., Baehrecke, E. H. & Thummel, C. S. Steroid regulated programmedcell death during Drosophila metamorphosis. Development 124, 4673–4683 (1997).

    CAS  PubMed  Google Scholar 

  23. Itakura, E. & Mizushima, N. p62 targeting to the autophagosome formation site requires self-oligomerization but not LC3 binding. J. Cell Biol. 192, 17–27 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Fujita, K., Maeda, D., Xiao, Q. & Srinivasula, S. M. Nrf2-mediated induction of p62 controls Toll-like receptor-4-driven aggresome-like induced structure formation and autophagic degradation. Proc. Natl Acad. Sci. USA 108, 1427–1432 (2011).

    Article  CAS  PubMed  Google Scholar 

  25. Lum, J. J. et al. Growth factor regulation of autophagy and cell survival in the absence of apoptosis. Cell 120, 237–248 (2005).

    Article  CAS  PubMed  Google Scholar 

  26. Hosokawa, N., Hara, Y. & Mizushima, N. Generation of cell lines with tetracycline-regulated autophagy and a role for autophagy in controlling cell size. FEBS Lett. 580, 2623–2629 (2006).

    Article  CAS  PubMed  Google Scholar 

  27. Nishida, Y. et al. Discovery of Atg5/Atg7-independent alternative macroautophagy. Nature 461, 654–658 (2009).

    Article  CAS  PubMed  Google Scholar 

  28. Tian, Y. et al. C. elegans screen identifies autophagy genes specific to multicellular organisms. Cell 141, 1042–1055 (2010).

    Article  CAS  PubMed  Google Scholar 

  29. McPhee, C. K., Logan, M. A., Freeman, M. R. & B., E. H. Activation of autophagy during cell death requires the engulfment receptor Draper. Nature 465, 1093–1096 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Johansen, T. & Lamark, T. Selective autophagy mediated by autophagic adapter proteins. Autophagy 7, 279–296 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Lenk, S. E., Dunn, W. A. J., Trausch, J. S., Ciechanover, A. & Schwartz, A. L. Ubiquitin-activating enzyme, E1, is associated with maturation of autophagic vacuoles. J. Cell Biol. 118, 301–308 (1992).

    Article  CAS  PubMed  Google Scholar 

  32. Taillebourg, E. et al. The deubiquitinating enzyme USP36 controls selective autophagy activation by ubiquitinated proteins. Autophagy 8, 767–779 (2012).

    Article  CAS  PubMed  Google Scholar 

  33. Scott, R. C., Schuldiner, O. & Neufeld, T. P. Role and regulation of starvation-induced autophagy in the Drosophila fat body. Dev. Cell 7, 167–178 (2004).

    Article  CAS  PubMed  Google Scholar 

  34. Chang, Y. Y. & Neufeld, T. P. An Atg1/Atg13 complex with multiple roles in TOR-mediated autophagy regulation. Mol. Biol. Cell 20, 2004–2014 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Shravage, B. V., Hill, J. H., Powers, C. M., Wu, L. & Baehrecke, E. H. Atg6 is required for multiple vesicle trafficking pathways and hematopoiesis in Drosophila. Development 140, 1321–1329 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Juhász, G. et al. The class III PI(3)K Vps34 promotes autophagy andendocytosis but not TOR signaling in Drosophila. J. Cell Biol. 181, 2347–2360 (2008).

    Article  Google Scholar 

  37. Lee, T. V. et al. The E1 ubiquitin-activating enzyme Uba1 in Drosophila controls apoptosis autonomously and tissue growth non-autonomously. Development 135, 43–52 (2008).

    Article  CAS  PubMed  Google Scholar 

  38. Denton, D. et al. Relationship between growth arrest and autophagy inmidgut programmed cell death in Drosophila. Cell Death Differ. 19, 1299–1307 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Yamashita, S. The post-embedding method for immunoelectron microscopy of mammalian tissues: a standardized procedure based on heat-induced antigen retrieval. Methods Mol. Biol. 657, 237–248 (2010).

    Article  CAS  PubMed  Google Scholar 

  40. Dutta, S. & Baehrecke, E. H. Warts is required for PI3K-regulated growth arrest, autophagy, and autophagic cell death in Drosophila. Curr. Biol. 18, 1466–1475 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank A. Bergmann (University of Massachusetts Medical School, USA), M. Brodsky (University of Massachusetts Medical School, USA), S. Cherry (University of Pennsylvania, USA), G. Juhasz (Eotvos Lorand University, Hungary), P. Meier (The Institute of Cancer Research, UK), T. P. Neufeld (University of Minnesota, USA), H. D. Ryoo (New York University, USA), H. Stenmark (Oslo University Hospital, Norway), the Bloomington Stock Center, the VDRC and the Developmental Studies Hybridoma Bank for flies and antibodies, G. Juhasz for advice about immuno-electron microscopy, T. Fortier for technical support, and M. Freeman, S. Doxsey and the Baehrecke laboratory for constructive comments. This work was supported by NIH grants GM079431 to E.H.B., GM095567 to J.W.H., and S10RR027897 to the UMass EM Core, and by Millennium Pharamceuticals to J.W.H. E.H.B. is an Ellison Medical Foundation Scholar and a member of the UMass DERC (DK32520).

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Authors and Affiliations

  1. Department of Cancer Biology, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA

    Tsun-Kai Chang, Bhupendra V. Shravage, Christine M. Powers, Rachel T. Simin & Eric H. Baehrecke

  2. Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115, USA

    Sebastian D. Hayes & J. Wade Harper

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Contributions

T-K.C., B.V.S., S.D.H., J.W.H. and E.H.B. designed the experiments. All experiments were performed by T-K.C., except Fig. 4d,e by B.V.S., TEM by T-K.C., B.V.S., C.M.P. and R.T.S. and E1 charging assays by S.D.H. T-K.C., B.V.S. and E.H.B. wrote the manuscript and all authors commented on it.

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Correspondence to Eric H. Baehrecke.

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Integrated supplementary information

Supplementary Figure 1 Knockdown of Atg1 does not alter cell growth, and Atg13, Atg6, Atg5, and Atg16 (CG31033), but not caspases, are required for midgut cell size reduction in Drosophila.

(a) Midguts dissected from early third instar larvae that mis-express Atg1IR (GFP in nucleus and cytoplasm) and are stained to detect Discs large (green) on the cortex of all cells. Representative images are shown. (b) Cell size quantification (μm2) from a, n = 10 animal intestines/genotype with 1-5 cells measured/intestine. (c) Midguts dissected from animals at puparium formation that contain Atg13Δ74 loss-of-function mutant cell clones (lacking GFP) and analyzed by fluorescence and DIC microscopy. Wild-type (+/+) control cells possess stronger GFP and heterozygous Atg13Δ74/wild-type (Δ74/+) cells have weaker GFP. Representative images are shown. (d) Cell size quantification (μm2) from c, nn = 6 animal intestines/genotype with 1-5 cells measured/intestine. (e) Midguts dissected from animals at puparium formation that contain Atg61 loss-of-function mutant cell clones (lacking RFP) and analyzed by fluorescence and DIC microscopy. Wild-type (+/+) control cells possess stronger RFP and heterozygous Atg61/wild-type (1/+) cells have weaker RFP. Representative images are shown. (f) Cell size quantification (μm2) from e, n = 8 animal intestines/genotype with 1-5 cells measured/intestine. (g) Midguts dissected from animals expressing Atg5IR specifically in GFP-marked clones of cells at puparium formation and analyzed by fluorescence and DIC microscopy. Representative images are shown. (h) Cell size quantification (μm2) from g, n = 16 animal intestines/genotype with 1-5 cells measured/intestine. (i) Midguts dissected from animals expressing Atg16IR specifically in DsRed-marked clones of cells at puparium formation and analyzed by fluorescence and DIC microscopy. Representative images are shown. (j) Cell size quantification (μm2) from i, n = 7 animal intestines/genotype with 1–5 cells measured/intestine. (k) Midguts dissected from animals expressing p35 in DsRed-marked clones of cells at puparium formation and analyzed by fluorescence and DIC microscopy. Representative images are shown. (l) Cell size quantification (μm2) from k, n = 13 animal intestines/genotype with 1-5 cells measured/intestine. Quantification is shown as mean ±s.d.. N.S.: no significance. Scale bars represent 20 μm.

Supplementary Figure 2 Atg7 is required for nutrient deprivation-induced autophagy in starved larval fat body and Vps34 is required for GFP–Atg5 puncta formation in the midgut at puparium formation.

(a,b) Fat body dissected from early 3rd instar larvae and subjected to 4 h of starvation. Representative images are shown. (a) Fat body expressing mCherry-Atg8a in all cells, and Atg7IR in GFP-marked clones of cells analyzed by fluorescence microscopy. (b) Atg7d4 loss-of-function mutant cells are shown lacking of myrRFP. Wild-type and heterozygous Atg7d4/wild-type control cells possess myrRFP. All fat body cells express GFP–Atg8a. (c) Midguts dissected from animals expressing Atg7IR specifically in DsRed-marked clones of cells at puparium formation and analyzed by fluorescence and DIC microscopy. Representative images are shown. (d) Cell size quantification (μm2) from c, n = 10 animal intestines/genotype with 1-5 cells measured/intestine. (e) Midguts expressing GFP–Atg5 in enterocytes (larger nuclei) and with Vps34Δm22 loss-of-function clone cells (lacking RFP) at puparium formation. Wild-type control cells possess stronger RFP and heterozygous cells have weaker RFP. Representative images are shown. Quantification is shown as mean ±s.d.. N.S.: no significance. Scale bars represent 20 μm.

Supplementary Figure 3 Screen for ubiquitin-like activating enzyme genes that function in midgut programmed cell reduction in size in Drosophila.

Representative images of midgut cells from animals expressing RNAi in GFP-marked clones of cells against (a) Uba2 (CG7528-1, n = 11, VDRC TID 110173, n = 14, and TRiP HM05055, n = 9), (b) Aos1 (TID 47256, n = 8, and HM05183, n = 9), (c) CG13343 (CG13343-3, n = 12, CG13343-4, n = 14, and TID 105141, n = 8), (d) APP-BP1 (TID 7728, n = 13), (e) CG13090 (TID 110326, n = 13, and TID 43558, n = 10), and (f) CG1749 (TID 110395, n = 14, and HMS01352, n = 12). n represents number of animal intestines analyzed/genotype. Scale bars represent 20 μm.

Supplementary Figure 4 Uba1 influences autophagy and programmed reduction of midgut cell size.

(a) Midguts expressing mCherry-Atg8a in all cells, and expressing Uba1IR specifically in GFP-marked clones of cells at puparium formation. Representative images are shown. (b) Midguts dissected from early third instar larvae that mis-express Uba1IR (GFP in nucleus and cytoplasm) and were stained to detect Discs large (green) on the cortex of all cells. Representative images are shown. (c) Cell size quantification (μm2) from b, n = 10 animal intestines/genotype with 1-5 cells measured/intestine. (d) Midguts from animals expressing CL1-GFP in all cells and temperature sensitive Dts7 specifically in DsRed-marked clones of cells at puparium formation. Representative images are shown. Quantification is shown as mean ±s.d.. N.S.: no significance. Scale bars represent 20 μm.

Supplementary Figure 5 Uba1 is not required for autophagy that is induced by stress in different tissues.

(a) Fat body dissected from early third instar larvae and subjected to 4 h of starvation that contain Uba1H33 loss-of-function MARCM mutant cell clones (GFP-positive) that also have mCherry-Atg8a expressed in all cells and analyzed by fluorescence microscopy. Control wild-type and heterozygous cells have no GFP. Representative images are shown. (b-f) Early third instar midguts expressing mCherry-Atg8a in all cells and expressing either Uba1IR (b,d,f) or Atg1IR (c,e) in GFP-marked clones of cells in control untreated animals (b), 4 h after starvation (c,d), or 7 h after 1.5 % H2O2 treatment (e,f). Representative images are shown. Scale bars represent 20 μm.

Supplementary Figure 6 Uba1, but not loss of Atg3, influences autophagy and programmed reduction of midgut cell size.

(a,b) Loading controls for the E1 charging assays, n = 2. Representative images are shown. (c,d) Representative TEM images of intestine cells 2 h after puparium formation. (c) Atg310/wild-type control (Atg3/+) and (d) Atg310/Df(3L)cat mutant (Atg3/Df) cells both possess autophagic structures. (e) Starved third instar larval fat body expressing mCherry-Atg8a in all cells and Atg3IR in GFP-marked clones of cells and analyzed with fluorescence microscopy. Representative images are shown. (f,g) Midguts dissected from animals at puparium formation that mis-express Uba1IR (GFP in nucleus and cytoplasm) and stained to detect either BrC (f, red) or Hid (g, red). Representative images are shown. Scale bars represent 1 μm (c,d) and 20 μm (e-g).

Supplementary Figure 7 Full scans of blots in Fig. 7a (a), Fig. 7b (b), Supplementary Fig. S6a (c), Supplementary Fig. S6b (d), and Fig. 7c (e,f).

Supplementary Table 1 Fly strains used in this study.
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Chang, TK., Shravage, B., Hayes, S. et al. Uba1 functions in Atg7- and Atg3-independent autophagy. Nat Cell Biol 15, 1067–1078 (2013). https://doi.org/10.1038/ncb2804

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