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A small GTPase, human Rab32, is required for the formation of autophagic vacuoles under basal conditions

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Abstract

Here we show that a small GTPase, Rab32, is a novel protein required for the formation of autophagic vacuoles. We found that the wild-type or GTP-bound form of human Rab32 expressed in HeLa and COS cells is predominantly localized to the endoplasmic reticulum (ER), and overexpression induces the formation of autophagic vacuoles containing an autophagosome marker protein LC3, the ER-resident protein calnexin and endosomal/lysosomal membrane protein LAMP-2, even under nutrient-rich conditions. The recruitment of Rab32 to the ER membrane was necessary for autophagic vacuole formation, suggesting involvement of the ER as a source of autophagosome membranes. In contrast, the expression of the inactive form of, or siRNA-specific for, Rab32 caused the formation of p62/SQSTM1 and ubiquitinated protein-accumulating aggresome-like structures and significantly prevented constitutive autophagy. We postulate that Rab32 facilitates the formation of autophagic vacuoles whose membranes are derived from the ER and regulates the clearance of aggregated proteins by autophagy.

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References

  1. Klionsky DJ, Emr SD (2000) Autophagy as a regulated pathway of cellular degradation. Science 290:1717–1721

    Article  PubMed  CAS  Google Scholar 

  2. Mizushima N, Ohsumi Y, Yoshimori T (2002) Autophagosome formation in mammalian cells. Cell Struct Funct 27:421–429

    Article  PubMed  Google Scholar 

  3. Hara T, Nakamura K, Matsui M, Yamamoto A, Nakahara Y, Suzuki-Migishima R, Yokoyama M, Mishima K, Saito I, Okano H, Mizushima N (2006) Suppression of basal autophagy in neural cells causes neurodegenerative disease in mice. Nature 441:885–889

    Article  PubMed  CAS  Google Scholar 

  4. Komatsu M, Waguri S, Ueno T, Iwata J, Murata S, Tanida I, Ezaki J, Mizushima N, Ohsumi Y, Uchiyama Y, Kominami E, Tanaka K, Chiba T (2005) Impairment of starvation-induced and constitutive autophagy in Atg7-deficient mice. J Cell Biol 169:425–434

    Article  PubMed  CAS  Google Scholar 

  5. Komatsu M, Waguri S, Chiba T, Murata S, Iwata J, Tanida I, Ueno T, Koike M, Uchiyama Y, Kominami E, Tanaka K (2006) Loss of autophagy in the central nervous system causes neurodegeneration in mice. Nature 441:880–884

    Article  PubMed  CAS  Google Scholar 

  6. Gozuacik D, Kimchi A (2004) Autophagy as a cell death and tumor suppressor mechanism. Oncogene 23:2891–2906

    Article  PubMed  CAS  Google Scholar 

  7. Cao Y, Klionsky DJ (2007) Physiological functions of Atg6/Beclin 1: a unique autophagy-related protein. Cell Res 17:839–849

    Article  PubMed  CAS  Google Scholar 

  8. Suhy DA, Giddings TH, Kirkegaard K (2000) Remodeling the endoplasmic reticulum by poliovirus infection and by individual viral proteins: an autophagy-like origin for virus-induced vesicles. J Virol 74:8953–8965

    Article  PubMed  CAS  Google Scholar 

  9. Dunn WA (1990) Studies on the mechanisms of autophagy: formation of the autophagic vacuole. J Cell Biol 110:1923–1933

    Article  PubMed  Google Scholar 

  10. Mizushima N, Yamamoto A, Hatano M, Kobayashi Y, Kabeya Y, Suzuki K, Tokuhisa T, Ohsumi Y, Yoshimori T (2001) Dissection of autophagosome formation using Apg5-deficient mouse embryonic stem cells. J Cell Biol 15:657–667

    Article  Google Scholar 

  11. Klionsky DJ, Cregg JM, Dunn WA, Emr SD, Sakai Y, Sandoval IV, Sibirny A, Subramani S, Thumm M, Veenhuis M, Ohsumi Y (2003) A unified nomenclature for yeast autophagy-related genes. Dev Cell 5:539–545

    Article  PubMed  CAS  Google Scholar 

  12. Zerial M, McBride H (2001) Rab proteins as membrane organizers. Nat Rev Mol Cell Biol 2:107–117

    Article  PubMed  CAS  Google Scholar 

  13. Gutierrez MG, Munafó DB, Berón W, Colombo MI (2004) Rab7 is required for the normal progression of autophagic pathway in mammalian cells. J Cell Sci 117:2687–2697

    Article  PubMed  CAS  Google Scholar 

  14. Jäger S, Bucci C, Tanida I, Ueno T, Kominami E, Saftig P, Eskelinen E-L (2004) Role of Rab7 in maturation of late autophagic vacuoles. J Cell Sci 117:4837–4848

    Article  PubMed  CAS  Google Scholar 

  15. Munafó DB, Colombo MI (2002) Induction of autophagy causes dramatic changes in the subcellular distribution of GFP-Rab24. Traffic 3:472–482

    Article  PubMed  Google Scholar 

  16. Bucci C, Thomsen P, Nicoziani P, McCarthy J, van Deurs B (2000) Rab7: a key to lysosome biogenesis. Mol Biol Cell 11:467–480

    PubMed  CAS  Google Scholar 

  17. Jäger D, Stockert E, Jäger E, Güre AO, Scanlan MJ, Knuth A, Old LJ, Chen Y-T (2000) Serological cloning of a melanocyte rab guanosine 5′-triphosphate-binding protein and a chromosome condensation protein from a melanoma complementary DNA library. Cancer Res 60:3584–3591

    PubMed  Google Scholar 

  18. Fujikawa K, Satoh AK, Kawamura S, Ozaki K (2002) Molecular and functional characterization of a unique Rab protein, RABRP1, containing the WDIAGQE sequence in a GTPase motif. Zool Sci 19:981–993

    Article  PubMed  CAS  Google Scholar 

  19. Shimizu F, Katagiri T, Suzuki M, Watanabe TK, Okuno S, Kuga Y, Nagata M, Fujiwara T, Nakamura Y, Takahashi E (1997) Cloning and chromosome assignment to 1q32 of a human cDNA (RAB7L1) encoding a small GTP-binding protein, a member of the RAS superfamily Cytogenet. Cell Genet 77:261–263

    Article  CAS  Google Scholar 

  20. Norian L, Dragoi IA, O’Halloran T (1999) Molecular characterization of rabE, developmentally regulated dictyostelium homolog of mammalian rab GTPases. DNA Cell Biol 8:59–64

    Article  Google Scholar 

  21. Wasmeier C, Romao M, Plowright L, Bennett DC, Raposo G, Seabra MC (2006) Rab38 and Rab32 control post-Golgi trafficking of melanogenic enzymes. J Cell Biol 175:271–281

    Article  PubMed  CAS  Google Scholar 

  22. Park M, Serpinskaya AS, Papalopulu N, Gelfand VI (2007) Rab32 regulates melanosome transport in Xenopus melanophores by protein kinase A recruitment. Curr Biol 17:1–5

    Article  CAS  Google Scholar 

  23. Bao X, Faris AE, Jang EK, Haslam RJ (2002) Molecular cloning, bacterial expression and properties of Rab31 and Rab32. Eur J Biochem 269:259–271

    Article  PubMed  CAS  Google Scholar 

  24. Lee MH, Lee SW, Lee EJ, Choi SJ, Chung SS, Lee JI, Cho JM, Seol JH, Baek SH, Kim KI, Chiba T, Tanaka K, Bang OS, Chung CH (2006) SUMO-specific protease SUSP4 positively regulates p53 by promoting Mdm2 self-ubiquitination. Nat Cell Biol 8:1424–1431

    Article  PubMed  CAS  Google Scholar 

  25. Hirota Y, Kuronita T, Fujita H, Tanaka Y (2007) A role for Rab5 activity in the biogenesis of endosomal and lysosomal compartments. Biochem Biophys Res Commun 364:40–47

    Article  PubMed  CAS  Google Scholar 

  26. Asanuma K, Tanida I, Shirato I, Ueno T, Takahara H, Nishitani T, Kominami E, Tomino Y (2003) MAP-LC3, a promising autophagosomal marker, is processed during the differentiation and recovery of podocytes from PAN nephrosis. FASEB J 17:1165–1167

    PubMed  CAS  Google Scholar 

  27. Saliba RS, Munro PMG, Luthert PJ, Cheetham ME (2002) The cellular fate of mutant rhodopsin: quality control, degradation and aggresome formation. J Cell Sci 15:2907–2918

    Google Scholar 

  28. Wang C, Tan JMM, Ho MWL, Zaiden N, Wong SH, Chew CLC, Eng PW, Lim TM, Dawson TM, Lim KL (2005) Alterations in the solubility and intracellular localization of parkin by several familial Parkinson’s disease-linked point mutations. J Neurochem 93:422–431

    Article  PubMed  CAS  Google Scholar 

  29. Wang Q, Mosser DD, Bag J (2005) Induction of HSP70 expression and recruitment of HSC70 in the nucleus reduce aggregation of a polyalanine expansion mutant of PABPN1 in HeLa cells. Hum Mol Genet 14:3673–3684

    Article  PubMed  CAS  Google Scholar 

  30. Sawada MT, Morinaga C, Izumi K, Sawada H (1999) The 26S proteasome assembly is regulated by a maturation-inducing hormone in Starfish Oocytes. Biochem Biophys Res Commun 254:338–344

    Article  PubMed  CAS  Google Scholar 

  31. Kabeya Y, Mizushima N, Ueno T, Yamamoto A, Kirisako T, Noda T, Kominami E, Ohsumi Y, Yoshimori T (2000) LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing. EMBO J 19:5720–5728

    Article  PubMed  CAS  Google Scholar 

  32. Alto NM, Soderling J, Scott JD (2002) Rab32 is an A-kinase anchoring protein and participates in mitochondrial dynamics. J Cell Biol 158:659–668

    Article  PubMed  CAS  Google Scholar 

  33. Ardley HC, Scott GB, Rose SA, Tan NGS, Robinson PA (2004) UCH-L1 aggresome formation in response to proteasome impairment indicates a role in inclusion formation in Parkinson’s disease. J Neurochem 90:379–391

    Article  PubMed  CAS  Google Scholar 

  34. Namekata K, Nishimura N, Kimura H (2002) Presenilin-binding protein forms aggresomes in monkey kidney COS-7 cells. J Neurochem 82:819–827

    Article  PubMed  CAS  Google Scholar 

  35. Ardley HC, Scott GB, Rose SA, Tan NGS, Markham AF, Robinson PA (2003) Inhibition of proteasomal activity causes inclusion formation in neuronal and non-neuronal cells overexpressing parkin. Mol Biol Cell 14:4541–4556

    Article  PubMed  CAS  Google Scholar 

  36. Fu L, Gao Y, Tousson A, Shah A, Chen T-LL, Vertel BB, Sztul E (2005) Nuclear aggresomes form by fusion of PML-associated aggregates. Mol Biol Cell 16:4905–4917

    Article  PubMed  CAS  Google Scholar 

  37. Johnston JA, Ward CL, Kopito RR (1998) Aggresomes: a cellular response to misfolded proteins. J Cell Biol 143:1883–1898

    Article  PubMed  CAS  Google Scholar 

  38. Imai Y, Soda M, Inoue H, Hattori N, Mizuno Y, Takahashi R (2001) An unfolded putative transmembrane polypeptide, which can lead to endoplasmic reticulum stress, is a substrate of parkin. Cell 105:891–902

    Article  PubMed  CAS  Google Scholar 

  39. Rubinsztein DC (2006) The roles of intracellular protein-degradation pathways in neurodegeneration. Nature 443:780–786

    Article  PubMed  CAS  Google Scholar 

  40. Komatsu M, Waguri S, Koike M, Sou Y, Ueno T, Hara T, Mizushima N, Iwata J, Ezaki J, Murata S, Hamasaki J, Nishito Y, Iemura S, Natsume T, Yanagawa T, Uwayama J, Warabi E, Yoshida H, Ishii T, Kobayashi A, Yamamoto M, Yue Z, Uchiyama Y, Kominami E, Tanaka K (2007) Homeostatic levels of p62 control cytoplasmic inclusion body formation in autophagy-deficient mice. Cell 131:1149–1163

    Article  PubMed  CAS  Google Scholar 

  41. Pankiv S, Høyvarde T, Lamark T, Brech A, Bruun J-A, Outzen H, Øvervatn A, Bjørkøy G, Johansen T (2007) p/62/SQSTM1 binds directly to Atg8/LC3 to facilitate degradation of ubiquitinated protein aggregates by autophagy. J Biol Chem 282:24131–24145

    Article  PubMed  CAS  Google Scholar 

  42. Bjørkøy G, Lamark T, Brech A, Outzen H, Perander M, Øvervatn A, Stenmark H, Johansen T (2005) p62/SQSTM1 forms protein aggregates degraded by autophagy and has a protective effect on huntingtin-induced cell death. J Cell Biol 171:603–614

    Article  PubMed  CAS  Google Scholar 

  43. Levine B, Klionsky DJ (2004) Development by self-digestion: molecular mechanisms and biological functions of autophagy. Dev Cell 6:463–477

    Article  PubMed  CAS  Google Scholar 

  44. Ogier-Denis E, Codogno P (2003) Autophagy: a barrier or an adaptive response to cancer. Biochim Biophys Acta 1603:113–128

    PubMed  CAS  Google Scholar 

  45. Gagliardi S, Gatti PA, Belfiore P, Zocchetti A, Clarke GD, Farina C (1998) Synthesis and structure-activity relationships of bafilomycin A1 derivatives as inhibitors of vacuolar H+-ATPase. J Med Chem 41:1883–1893

    Article  PubMed  CAS  Google Scholar 

  46. Yamamoto A, Tagawa Y, Yoshimori T, Moriyama Y, Masaki R, Tashiro Y (1998) Bafilomycin A1 prevents maturation of autophagic vacuoles by inhibiting fusion between autophagosomes and lysosomes in rat hepatoma cell line, H-4-II-E cells. Cell Struct Funct 23:33–42

    Article  PubMed  CAS  Google Scholar 

  47. Tanida I, Minematsu-Ikeguchi N, Ueno T, Kominami E (2005) Lysosomal turnover, but not a cellular level, of endogenous LC3 is a marker for autophagy. Autophagy 1:e2–e9

    Google Scholar 

  48. Zhang L, Yu J, Pan H, Hu P, Hao Y, Cai W, Zhu H, Yu AD, Xie X, Ma D, Yuan J (2007) Small molecule regulators of autophagy identified by an image-based high-throughput screen. Proc Natl Acad Sci USA 104:19023–19028

    Article  PubMed  CAS  Google Scholar 

  49. Blommaart EFC, Luiken JJFP, Blommaart PJE, van Woerkom GM, Meijer AJ (1995) Phosphorylation of ribosomal protein S6 is inhibitory for autophagy in isolated rat hepatocytes. J Biol Chem 270:2320–2326

    Article  PubMed  CAS  Google Scholar 

  50. Codogono P, Meijer AJ (2005) Autophagy and signaling: their role in cell survival and cell death. Cell Death Differ 12:1509–1518

    Article  CAS  Google Scholar 

  51. Suzuki T, Nakagawa M, Yoshikawa A, Sasagawa N, Yoshimori T, Ohsumi Y, Nishino I, Ishiura S, Nonaka I (2002) The first molecular evidence that autophagy relates rimmed vacuole formation in chloroquine myopathy. J Biochem 131:647–651

    PubMed  CAS  Google Scholar 

  52. Kimura N, Kumamoto T, Kawamura Y, Himeno T, Nakamura K, Ueyama H, Arakawa R (2007) Expression of autophagy-associated genes in skeletal muscle: an experimental model of chloroquine-induced myopathy. Pathobiology 72:169–176

    Article  CAS  Google Scholar 

  53. Høyer-Hansen M, Bastholm L, Szyniarowski P, Campanella M, Szabadkai G, Farkas T, Bianchi K, Fehrenbacher N, Elling F, Rizzuto R, Mathiasen IS, Jäättelä M (2007) Control of macroautophagy by calcium, calmodulin-dependent kinase kinase-β, and Bcl-2. Mol Cell 25:193–205

    Article  PubMed  CAS  Google Scholar 

  54. Juhasz G, Neufeld TP (2006) Autophagy: a forty-year search for a missing membrane source. PLoS Biol 4:e36

    Article  PubMed  CAS  Google Scholar 

  55. Budovskaya YV, Stephan JS, Reggiori F, Klionsky DJ, Herman PK (2004) The Ras/cAMP-dependent protein kinase signaling pathway regulates an early step of autophagy process in Saccharomyces cerevisiae. J Biol Chem 279:20663–20671

    Article  PubMed  CAS  Google Scholar 

  56. Mavrakis M, Lippincott-Schwartz J, Stratakis CA, Bossis I (2006) Depletion of type IA regulatory subunit (RIα) of protein kinase A (PKA) in mammalian cells and tissues activates mTOR and causes autophagic deficiency. Hum Mol Genet 15:2962–2971

    Article  PubMed  CAS  Google Scholar 

  57. Schmelzle T, Beck T, Martin DE, Hall MN (2004) Activation of the RAS/cyclic AMP pathway suppresses a TOR deficiency in yeast. Mol Cell Biol 24:338–351

    Article  PubMed  CAS  Google Scholar 

  58. Yorimitsu T, Zaman S, Broach JR, Klionsky DJ (2007) Protein kinase A and Sch9 cooperatively regulate induction of autophagy in Saccharomyces cerevisiae. Mol Biol Cell 18:4180–4189

    Article  PubMed  CAS  Google Scholar 

  59. Mavrakis M, Lippincott-Schwartz J, Stratakis CA, Bossis I (2007) mTOR kinase and the regulatory subunit of protein kinase A (PRKAR1A) spatially and functionally interact during autophagosome maturation. Autophagy 3:151–153

    PubMed  CAS  Google Scholar 

  60. Maltese W, Soule G, Gunning W, Calomeni E, Alexander B (2002) Mutant Rab24 GTPase is targeted to nuclear inclusions. BMC Cell Biol 3:25

    Article  PubMed  Google Scholar 

  61. Wu M, Yin G, Zhao X, Ji C, Gu S, Tang R, Dong H, Xie Y, Mao Y (2006) Human Rab24, interestingly and predominantly distributed in the nuclei of COS-7 cells, is colocalized with cyclophilin A and GABARAP. Int J Mol Med 17:749–754

    PubMed  CAS  Google Scholar 

  62. Waelter S, Boeddrich A, Lurz R, Scherzinger E, Lueder G, Lehrach H, Wanker EE (2001) Accumulation of mutant huntingtin fragments in aggresome-like inclusion bodies as a result of insufficient protein degradation. Mol Biol Cell 12:1393–1407

    PubMed  CAS  Google Scholar 

  63. Webb JL, Ravikumar B, Atkins J, Skeppers JN, Rubinsztein DC (2003) α-Synuclein is degraded by both autophagy and the proteasome. J Biol Chem 278:25009–25013

    Article  PubMed  CAS  Google Scholar 

  64. Berger Z, Ravikumar B, Menzies FM, Oroz LG, Underwood BR, Pangalos MN, Schmitt I, Wullner U, Evert BO, O’Kane CJ, Rubinsztein DC (2006) Rapamycin alleviates toxicity of different aggregate-prone proteins. Hum Mol Genet 15:433–442

    Article  PubMed  CAS  Google Scholar 

  65. Iwata A, Christianson JC, Bucci M, Ellerby LM, Nukina N, Forno LS, Kopito RR (2005) Increased susceptibility of cytoplasmic over nuclear polyglutamine aggregates to autophagic degradation. Proc Natl Acad Sci USA 102:13135–13140

    Article  PubMed  CAS  Google Scholar 

  66. Qin Z-H, Wang Y, Kegel KB, Kazantsev A, Apostol BL, Thompson LM, Yoder J, Aronin N, DiFiglia M (2003) Autophagy regulates the processing of amino terminal huntingtin fragments. Hum Mol Genet 12:3231–3244

    Article  PubMed  CAS  Google Scholar 

  67. Ravikumar B, Duden R, Rubinsztei DC (2002) Aggregate-prone proteins with polyglutamine and polyalanine expansions are degraded by autophagy. Hum Mol Genet 11:1107–1117

    Article  PubMed  CAS  Google Scholar 

  68. Ravikumar B, Vacher C, Berger Z, Davies JE, Luo S, Oroz LG, Scaravilli F, Easton DF, Duden R, O’Kane CJ, Rubinsztein DC (2004) Inhibition of mTOR induces autophagy and reduces toxicity of polyglutamine expansions in fly and mouse models of Huntingtin disease. Nat Genet 36:585–595

    Article  PubMed  CAS  Google Scholar 

  69. Shibata D, Mori Y, Cai K, Zhang L, Yin J, Elahi A, Hamelin R, Wong YF, Lo WK, Chung TKH, Sato F, Karpeh MS Jr, Meltzer SJ (2006) RAB32 hypermethylation and microsatellite instability in gastric and endometrial adenocarcinomas. Int J Cancer 119:801–806

    Article  PubMed  CAS  Google Scholar 

  70. Kuma A, Matsui M, Mizushima N (2007) LC3, an autophagosome marker, can be incorporated into protein aggregates independent of autophagy: caution in the interpretation of LC3 localization. Autophagy 3:323–328

    PubMed  CAS  Google Scholar 

  71. Ceresa BP, Bahr SJ (2006) Rab7 activity affects epidermal growth factor: epidermal growth factor receptor degradation by regulating endocytic trafficking from the late endosome. J Biol Chem 281:1099–1106

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

We thank Drs. Ikuo Wada (Fukushima Medical University) and Tomoki Chiba (University of Tsukuba) for providing the ER-cherry and FLAG-tagged ubiquitin constructs, respectively. This study was supported in part by a grant-in-aid from the Ministry of Education, Culture, Sports, Sciences and Technology of Japan. Yuko Hirota is a Research Fellow of the Japan Society for the Promotion of Science.

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  1. Division of Pharmaceutical Cell Biology, Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Fukuoka, 812-8582, Japan

    Yuko Hirota & Yoshitaka Tanaka

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  1. Yuko Hirota
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Correspondence to Yoshitaka Tanaka.

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Hirota, Y., Tanaka, Y. A small GTPase, human Rab32, is required for the formation of autophagic vacuoles under basal conditions. Cell. Mol. Life Sci. 66, 2913–2932 (2009). https://doi.org/10.1007/s00018-009-0080-9

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