Convergence of Multiple Autophagy and Cytoplasm to Vacuole Targeting Components to a Perivacuolar Membrane Compartment Prior tode Novo Vesicle Formation
2002; Elsevier BV; Volume: 277; Issue: 1 Linguagem: Inglês
10.1074/jbc.m109134200
ISSN1083-351X
AutoresJohn Kim, Wei‐Pang Huang, Per E. Strømhaug, Daniel J. Klionsky,
Tópico(s)Endoplasmic Reticulum Stress and Disease
ResumoUnder starvation conditions, the majority of intracellular degradation occurs at the lysosome or vacuole by the autophagy pathway. The cytoplasmic substrates destined for degradation are packaged inside unique double-membrane transport vesicles called autophagosomes and are targeted to the lysosome/vacuole for subsequent breakdown and recycling. Genetic analyses of yeast autophagy mutants,apg and aut, have begun to identify the molecular machinery as well as indicate a substantial overlap with the biosynthetic cytoplasm to vacuole targeting (Cvt) pathway. Transport vesicle formation is a key regulatory step of both pathways. In this study, we characterize the putative compartment from which both autophagosomes and the analogous Cvt vesicles may originate. Microscopy analyses identified a perivacuolar membrane as the resident compartment for both the Apg1-Cvt9 signaling complex, which mediates the switching between autophagic and Cvt transport, and the autophagy/Cvt-specific phosphatidylinositol 3-kinase complex. Furthermore, the perivacuolar compartment designates the initial site of membrane binding by the Apg/Cvt vesicle component Aut7, the Cvt cargo receptor Cvt19, and the Apg conjugation machinery, which functions in the de novoformation of vesicles. Biochemical isolation of the vesicle component Aut7 and density gradient analyses recapitulate the microscopy findings although also supporting the paradigm that components required for vesicle formation and packaging concentrate at subdomains within the donor membrane compartment. Under starvation conditions, the majority of intracellular degradation occurs at the lysosome or vacuole by the autophagy pathway. The cytoplasmic substrates destined for degradation are packaged inside unique double-membrane transport vesicles called autophagosomes and are targeted to the lysosome/vacuole for subsequent breakdown and recycling. Genetic analyses of yeast autophagy mutants,apg and aut, have begun to identify the molecular machinery as well as indicate a substantial overlap with the biosynthetic cytoplasm to vacuole targeting (Cvt) pathway. Transport vesicle formation is a key regulatory step of both pathways. In this study, we characterize the putative compartment from which both autophagosomes and the analogous Cvt vesicles may originate. Microscopy analyses identified a perivacuolar membrane as the resident compartment for both the Apg1-Cvt9 signaling complex, which mediates the switching between autophagic and Cvt transport, and the autophagy/Cvt-specific phosphatidylinositol 3-kinase complex. Furthermore, the perivacuolar compartment designates the initial site of membrane binding by the Apg/Cvt vesicle component Aut7, the Cvt cargo receptor Cvt19, and the Apg conjugation machinery, which functions in the de novoformation of vesicles. Biochemical isolation of the vesicle component Aut7 and density gradient analyses recapitulate the microscopy findings although also supporting the paradigm that components required for vesicle formation and packaging concentrate at subdomains within the donor membrane compartment. cytoplasm to vacuole targeting autophagy cyan fluorescent protein dolichol-phosphate mannose synthase green fluorescent protein open reading frame phosphatidylethanolamine alkaline phosphatase phosphatidylinositol carboxypeptidase Y yellow fluorescent protein enhanced CFP enhanced YFP endoplasmic reticulum aminopeptidase I hemagglutinin precursor Ape1 prevacuolar compartment Cellular homeostasis requires the regulated balance of biosynthetic and degradative activities. Autophagy functions as the major degradative pathway by which proteins and organelles are delivered to the lysosome or vacuole for breakdown and recycling. In addition to normal homeostatic function, autophagy is induced in response to certain environmental stress conditions such as serum deprivation, amino acid starvation, radiation damage (1Paglin S. Hollister T. Delohery T. Hackett N. McMahill M. Sphicas E. Domingo D. Yahalom J. Cancer Res. 2001; 61: 439-444PubMed Google Scholar), and in response to anti-estrogen agents (2Bursch W. Ellinger A. Kienzl H. Török L. Pandey S. Sikorska M. Walker R. Hermann R.S. Carcinogenesis. 1996; 17: 1595-1607Crossref PubMed Scopus (460) Google Scholar). Autophagy-mediated degradation may be important during mammalian embryogenesis, differentiation, and aging (3Cavallini G. Donati A. Gori Z. Pollera M. Bergamini E. Exp. Gerontol. 2001; 36: 497-506Crossref PubMed Scopus (61) Google Scholar, 4Klionsky D.J. Emr S.D. Science. 2000; 290: 1717-1721Crossref PubMed Scopus (2928) Google Scholar) and plays a critical role during type II non-apoptotic programmed cell death (reviewed in Ref. 5Bursch W. Cell Death Differ. 2001; 8: 569-581Crossref PubMed Scopus (554) Google Scholar). Dysfunction of autophagy has been implicated in the etiology of an increasing number of genetic diseases, and cancer in particular (6Liang X.H. Jackson S. Seaman M. Brown K. Kempkes B. Hibshoosh H. Levine B. Nature. 1999; 402: 672-676Crossref PubMed Scopus (2698) Google Scholar). Autophagy also appears to be associated with neurodegenerative conditions such as Parkinson's and Alzheimer's diseases (7Anglade P. Vyas S. Javoy-Agid F. Herrero M.T. Michel P.P. Marquez J. Mouatt-Prigent A. Ruberg M. Hirsch E.C. Agid Y. Histol. Histopathol. 1997; 12: 25-31PubMed Google Scholar, 8Cataldo A.M. Barnett J.L. Berman S.A. Li J. Quarless S. Bursztajn S. Lippa C. Nixon R.A. Neuron. 1995; 14: 671-680Abstract Full Text PDF PubMed Scopus (301) Google Scholar). Mutations in lysosomal associated membrane protein 2 result in cardiomyopathy associated with Danon's disease, and lysosomal associated membrane protein 2-deficient mice show defects in the autophagy pathway (reviewed in Ref. 9Saftig P. Tanaka Y. Lullmann-Rauch R. von Figura K. Trends Mol. Med. 2001; 7: 37-39Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). Therefore, understanding the basic mechanisms of autophagy may have wide therapeutic relevance.The use of pharmacological agents in mammalian systems has identified the role of signaling components including various kinases, phosphatases, and heterotrimeric G proteins, in the regulation of autophagy (reviewed in Refs. 4Klionsky D.J. Emr S.D. Science. 2000; 290: 1717-1721Crossref PubMed Scopus (2928) Google Scholar and 10Kim J. Klionsky D.J. Annu. Rev. Biochem. 2000; 69: 303-342Crossref PubMed Scopus (319) Google Scholar). The morphology of mammalian autophagy has also been described in detail (reviewed in Ref. 11Dunn Jr., W.A. Trends Cell Biol. 1994; 4: 139-143Abstract Full Text PDF PubMed Scopus (442) Google Scholar). In the past few years, a classical genetic approach in Saccharomyces cerevisiae has transformed our understanding of the autophagy pathway by identifying autophagy-specific protein machinery (reviewed in Refs. 12Stromhaug P.E. Klionsky D.J. Traffic. 2001; 2: 524-531Crossref PubMed Scopus (138) Google Scholar and 13Abeliovich H. Klionsky D.J. Microbiol. Mol. Biol. Rev. 2001; 65: 463-479Crossref PubMed Scopus (142) Google Scholar). Genetic screens based on starvation sensitivity or defects in the degradation of specific cytosolic proteins resulted in the isolation of the apg and aut mutants that are defective in autophagy (14Tsukada M. Ohsumi Y. FEBS Lett. 1993; 333: 169-174Crossref PubMed Scopus (1377) Google Scholar, 15Thumm M. Egner R. Koch M. Schlumpberger M. Straub M. Veenhuis M. Wolf D.H. FEBS Lett. 1994; 349: 275-280Crossref PubMed Scopus (479) Google Scholar). In addition, these mutants overlap with mutants in the cytoplasm to vacuole targeting (Cvt)1 pathway (16Harding T.M. Morano K.A. Scott S.V. Klionsky D.J. J. Cell Biol. 1995; 131: 591-602Crossref PubMed Scopus (394) Google Scholar, 17Harding T.M. Hefner-Gravink A. Thumm M. Klionsky D.J. J. Biol. Chem. 1996; 271: 17621-17624Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar) that were isolated from a screen based on defects in the proteolytic processing of the resident vacuolar hydrolase aminopeptidase I (Ape1). Genetic, biochemical, and morphological studies all demonstrate that autophagy and the Cvt pathway share largely the same targeting machinery (17Harding T.M. Hefner-Gravink A. Thumm M. Klionsky D.J. J. Biol. Chem. 1996; 271: 17621-17624Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar, 18Baba M. Osumi M. Scott S.V. Klionsky D.J. Ohsumi Y. J. Cell Biol. 1997; 139: 1687-1695Crossref PubMed Scopus (275) Google Scholar, 19Scott S.V. Baba M. Ohsumi Y. Klionsky D.J. J. Cell Biol. 1997; 138: 37-44Crossref PubMed Scopus (141) Google Scholar, 20Scott S.V. Hefner-Gravink A. Morano K.A. Noda T. Ohsumi Y. Klionsky D.J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12304-12308Crossref PubMed Scopus (213) Google Scholar). Both pathways employ a double membrane transport vesicle (termed the autophagosome and Cvt vesicle, respectively) to sequester cytoplasmic cargo, followed by docking and fusion of the outer vesicle membrane with the vacuole to release the cargo-containing inner vesicle (the autophagic and Cvt bodies). The smaller Cvt vesicles (diameter 100–150 nm) appear to exclude bulk cytoplasm, specifically transporting precursor Ape1 (prApe1) and α-mannosidase. Starvation conditions induce the elevated expression of autophagic proteins (21Kirisako T. Baba M. Ishihara N. Miyazawa K. Ohsumi M. Yoshimori T. Noda T. Ohsumi Y. J. Cell Biol. 1999; 147: 435-446Crossref PubMed Scopus (708) Google Scholar, 22Huang W.-P. Scott S.V. Kim J. Klionsky D.J. J. Biol. Chem. 2000; 275: 5845-5851Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar) to accommodate the formation of the much larger autophagosomes (diameter 300–900 nm) and the nonselective sequestration of cytoplasm in addition to resident vacuolar hydrolases (23Abeliovich H. Dunn Jr., W.A. Kim J. Klionsky D.J. J. Cell Biol. 2000; 151: 1025-1034Crossref PubMed Scopus (234) Google Scholar).The cloning and characterization of the gene products that complement the apg, aut, and cvt mutants have provided information about an increasing number of proteins required for the Cvt and autophagy pathways (reviewed in Refs. 10Kim J. Klionsky D.J. Annu. Rev. Biochem. 2000; 69: 303-342Crossref PubMed Scopus (319) Google Scholar, 12Stromhaug P.E. Klionsky D.J. Traffic. 2001; 2: 524-531Crossref PubMed Scopus (138) Google Scholar, and 13Abeliovich H. Klionsky D.J. Microbiol. Mol. Biol. Rev. 2001; 65: 463-479Crossref PubMed Scopus (142) Google Scholar). Mammalian orthologs for key autophagy genes have also been identified (reviewed in Refs. 12Stromhaug P.E. Klionsky D.J. Traffic. 2001; 2: 524-531Crossref PubMed Scopus (138) Google Scholar and 13Abeliovich H. Klionsky D.J. Microbiol. Mol. Biol. Rev. 2001; 65: 463-479Crossref PubMed Scopus (142) Google Scholar). Recent studies in yeast have begun to elucidate the molecular basis behind the mechanisms that mediate both the Cvt pathway and autophagy. However, the subcellular localization of many of the proteins that are involved in the complex membrane rearrangements that are the hallmark of these pathways has not been determined.The least understood and most complex step of the Cvt and autophagy pathways is the formation of the sequestering vesicle. Vesicle formation in the secretory pathway has been relatively well characterized and includes a requisite cargo concentration step in which cargo receptors bind their substrates and associate with general cytosolic coat proteins at the site of vesicle budding (reviewed in Refs. 24Springer S. Spang A. Schekman R. Cell. 1999; 97: 145-148Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar and 25Kirchhausen T. Nat. Rev. Mol. Cell. Biol. 2000; 1: 187-198Crossref PubMed Scopus (418) Google Scholar). Similar to the formation of vesicles in the secretory pathway, many proteins appear to be required at the vesicle formation step of the Cvt and autophagy pathways. These include a cargo receptor, Cvt19, that is required for transport of resident hydrolases (26Scott S.V. Guan J. Hutchins M.U. Kim J. Klionsky D.J. Mol. Cell. 2001; 7: 1131-1141Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar). In addition, a group of proteins involved in a novel conjugation reaction that covalently joins Apg12 to Apg5 are transiently associated with the forming vesicle (27Mizushima N. Noda T. Yoshimori T. Tanaka Y. Ishii T. George M.D. Klionsky D.J. Ohsumi M. Ohsumi Y. Nature. 1998; 395: 395-398Crossref PubMed Scopus (1263) Google Scholar, 28George M.D. Baba M. Scott S.V. Mizushima N. Garrison B.S. Ohsumi Y. Klionsky D.J. Mol. Biol. Cell. 2000; 11: 969-982Crossref PubMed Scopus (75) Google Scholar, 29Mizushima N. Yamamoto A. Hatano M. Kobayashi Y. Kabeya Y. Suzuki K. Tokuhisa T. Ohsumi Y. Yoshimori T. J. Cell Biol. 2001; 152: 657-667Crossref PubMed Scopus (1146) Google Scholar). Although the exact function of Apg conjugation is not well understood, this process is required for the membrane recruitment of Aut7, a protein that plays a role in vesicle formation (22Huang W.-P. Scott S.V. Kim J. Klionsky D.J. J. Biol. Chem. 2000; 275: 5845-5851Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar, 30Kirisako T. Ichimura Y. Okada H. Kabeya Y. Mizushima N. Yoshimori T. Ohsumi M. Takao T. Noda T. Ohsumi Y. J. Cell Biol. 2000; 151: 263-276Crossref PubMed Scopus (724) Google Scholar, 31Ichimura Y. Kirisako T. Takao T. Satomi Y. Shimonishi Y. Ishihara N. Mizushima N. Tanida I. Kominami E. Ohsumi M. Noda T. Ohsumi Y. Nature. 2000; 408: 488-492Crossref PubMed Scopus (1479) Google Scholar, 32Kim J. Huang W.-P. Klionsky D.J. J. Cell Biol. 2001; 152: 51-64Crossref PubMed Scopus (186) Google Scholar) and expansion of the autophagosomal membrane (23Abeliovich H. Dunn Jr., W.A. Kim J. Klionsky D.J. J. Cell Biol. 2000; 151: 1025-1034Crossref PubMed Scopus (234) Google Scholar). Aut7 is also unique in that it is the only characterized protein required for vesicle formation that remains associated with the completed vesicle. However, the initial site of Aut7 membrane binding, a location that may mark the origin of the membrane for the sequestering vesicle, has not been identified.Although much progress has been made in the characterization of yeast autophagy gene products, either individually or as part of a protein complex, an overall comparative examination placing these multiple autophagy and Cvt components and complexes at their physical site(s) of function has remained an important goal. In this study, we investigate the site of function of many of the recently characterized Apg/Cvt proteins and complexes. We demonstrate that the majority of these components co-localize to a distinct perivacuolar compartment. Surprisingly, isopycnic centrifugation of the membrane fraction of the cell divided the proteins co-localizing by microscopy into two separate populations peaking at different densities, the lighter peak containing the conjugation components as well as the prApe1 receptor Cvt19 and the vesicle marker Aut7. These results are supported by biochemical studies based on immunoisolation of Aut7. Together, the data suggest that although most autophagy and Cvt proteins co-localize to a unique perivacuolar compartment, they may display a unique spatial distribution within this compartment that may be essential to their function and the formation of the autophagosome/Cvt vesicle.DISCUSSIONThe transport of cargo to the vacuole by the Cvt and autophagy pathways differs significantly from classical modes of secretory traffic. Proteins within the secretory pathway that are destined for secretion or diverted to the vacuole are translocated into the endoplasmic reticulum and are packaged in a lumenal environment within single-membrane vesicles. In contrast, the Cvt and autophagy pathways capture cargo directly from the cytoplasm within double-membrane Cvt vesicles and autophagosomes. The key stage of regulation in this process occurs at the point of vesicle formation, as reflected by the finding that the majority of the genetically identified Cvt and Apg components function prior to, or at the step of, vesicle formation and completion (reviewed in Refs. 10Kim J. Klionsky D.J. Annu. Rev. Biochem. 2000; 69: 303-342Crossref PubMed Scopus (319) Google Scholar, 12Stromhaug P.E. Klionsky D.J. Traffic. 2001; 2: 524-531Crossref PubMed Scopus (138) Google Scholar, and 13Abeliovich H. Klionsky D.J. Microbiol. Mol. Biol. Rev. 2001; 65: 463-479Crossref PubMed Scopus (142) Google Scholar). Therefore, questions regarding the mechanisms of how Cvt vesicles and autophagosomes form remain a central focus of investigation in the field. In this study, we have demonstrated that a perivacuolar compartment provides residence for a remarkable array of specialized Cvt and Apg trafficking machinery that ultimately results in the formation of transport vesicles for the Cvt and autophagy pathways. These findings implicate the role of the Apg/Cvt perivacuolar compartment as the donor membrane for autophagosome and Cvt vesicle formation.The Apg1 Signaling ComplexBoth in vivo analyses using fluorescence microscopy and in vitro studies based on subcellular fractionation and density gradient separation demonstrate the co-localization of Apg1 and Cvt9 (Figs. 2 and 4). These results agree with previous studies that demonstrated an interaction between these two proteins based on two-hybrid interactions and co-immunoprecipitation (35Kim J. Kamada Y. Stromhaug P.E. Guan J. Hefner-Gravink A. Baba M. Scott S.V. Ohsumi Y. Dunn Jr., W.A. Klionsky D.J. J. Cell Biol. 2001; 153: 381-396Crossref PubMed Scopus (216) Google Scholar) and validate the approach taken in the current analysis. Furthermore, the present study clearly places the Apg1 kinase complex at the perivacuolar compartment and assigns an essential role for this membrane structure in the regulation of Cvt and autophagy transport. The kinase activity of Apg1 depends on upstream nutrient signals that are initially relayed through the Tor kinase (41Kamada Y. Funakoshi T. Shintani T. Nagano K. Ohsumi M. Ohsumi Y. J. Cell Biol. 2000; 150: 1507-1513Crossref PubMed Scopus (898) Google Scholar,52Noda T. Ohsumi Y. J. Biol. Chem. 1998; 273: 3963-3966Abstract Full Text Full Text PDF PubMed Scopus (1029) Google Scholar). Tor kinase repression leads to the induction of autophagy and increased Apg1 kinase activity by enhancing the binding of Apg13, an Apg1 activator, to Apg1 (41Kamada Y. Funakoshi T. Shintani T. Nagano K. Ohsumi M. Ohsumi Y. J. Cell Biol. 2000; 150: 1507-1513Crossref PubMed Scopus (898) Google Scholar). Growing evidence suggests that the Apg1 kinase complex plays a key role in switching between Cvt transport and autophagy. Apg1 physically interacts with Cvt9, a protein required for the Cvt pathway and selective peroxisome degradation but not autophagy (35Kim J. Kamada Y. Stromhaug P.E. Guan J. Hefner-Gravink A. Baba M. Scott S.V. Ohsumi Y. Dunn Jr., W.A. Klionsky D.J. J. Cell Biol. 2001; 153: 381-396Crossref PubMed Scopus (216) Google Scholar, 41Kamada Y. Funakoshi T. Shintani T. Nagano K. Ohsumi M. Ohsumi Y. J. Cell Biol. 2000; 150: 1507-1513Crossref PubMed Scopus (898) Google Scholar). In addition, an autophagy-specific component, Apg17, also physically interacts with Apg1, suggesting that Apg1, through its interactions with pathway-specific components, may dictate the initiation of either the Cvt or autophagy programs based on upstream nutrient signals (41Kamada Y. Funakoshi T. Shintani T. Nagano K. Ohsumi M. Ohsumi Y. J. Cell Biol. 2000; 150: 1507-1513Crossref PubMed Scopus (898) Google Scholar).Components Required for Cargo Packaging and Vesicle FormationAut7 is the only characterized protein required for the autophagy/Cvt pathways that is substantially induced under starvation conditions (21Kirisako T. Baba M. Ishihara N. Miyazawa K. Ohsumi M. Yoshimori T. Noda T. Ohsumi Y. J. Cell Biol. 1999; 147: 435-446Crossref PubMed Scopus (708) Google Scholar, 22Huang W.-P. Scott S.V. Kim J. Klionsky D.J. J. Biol. Chem. 2000; 275: 5845-5851Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar). Aut7 remains associated with the completed autophagosome/Cvt vesicle (21Kirisako T. Baba M. Ishihara N. Miyazawa K. Ohsumi M. Yoshimori T. Noda T. Ohsumi Y. J. Cell Biol. 1999; 147: 435-446Crossref PubMed Scopus (708) Google Scholar, 22Huang W.-P. Scott S.V. Kim J. Klionsky D.J. J. Biol. Chem. 2000; 275: 5845-5851Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar), and the increase in the level of Aut7 is critical for the expansion of the autophagosomal membrane (23Abeliovich H. Dunn Jr., W.A. Kim J. Klionsky D.J. J. Cell Biol. 2000; 151: 1025-1034Crossref PubMed Scopus (234) Google Scholar). Aut7 and a set of proteins involved in the conjugation of Apg12 to Apg5 function at the step of vesicle formation. The E1-like conjugation component, Apg7, activates Aut7 for subsequent reactions that lead to Aut7 lipidation (31Ichimura Y. Kirisako T. Takao T. Satomi Y. Shimonishi Y. Ishihara N. Mizushima N. Tanida I. Kominami E. Ohsumi M. Noda T. Ohsumi Y. Nature. 2000; 408: 488-492Crossref PubMed Scopus (1479) Google Scholar) and demonstrates that Aut7 and the Apg conjugation system function at the same stage in vesicle formation. Cvt19 is another component that remains associated with the completed transport vesicles, but unlike Aut7, Cvt19 does not appear to play a role in vesicle formation. Instead, Cvt19 appears to function as a specific receptor for concentrating biosynthetic cargo, including prApe1, that transits inside of autophagosomes/Cvt vesicles (26Scott S.V. Guan J. Hutchins M.U. Kim J. Klionsky D.J. Mol. Cell. 2001; 7: 1131-1141Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar). Perhaps concomitant with the Aut7 membrane recruitment events, the cargo-bound Cvt19 receptor presumably concentrates at the membrane sites of nascent vesicle formation so that efficient cargo packaging can occur. Our microscopy results place the Apg conjugation proteins, the Cvt19 cargo receptor, and the Aut7 vesicle component in the same perivacuolar compartment as Cvt9 and Apg1 (Figs. Figure 1, Figure 2, Figure 3). The co-localization of the cargo packaging and vesicle formation machinery to the same compartment as the signaling complex suggests that the transduction of the Tor kinase-dependent signals to the Apg1 kinase complex and then to downstream effectors all occurs at the perivacuolar Apg/Cvt compartment.The density gradient analyses revealed that while Aut7, Cvt19, and the Apg conjugation components co-fractionated with each other, they did not share the same density profile with Cvt9 and the Apg1 signaling complex (Figs. 5 and 6). Machinery involved in or associated with vesicle formation may be recruited to subdomains within the perivacuolar compartment. The segregation of Aut7, Cvt19, and Apg12/Apg5 into a separate subdomain is further suggested by biochemical immunoisolation experiments using protein A-Aut7ΔR (Fig.7). Cvt19, and to a lesser extent Apg12, could be recovered along with protein A-Aut7ΔR, whereas Cvt9 and Apg9 were not co-purified with this fusion protein. The biochemical isolation and manipulation of organelles tend to vesiculate membranes, resulting in a heterogeneous vesicle population with some fractions containing protein complexes that are concentrated to localized regions within the organelle. In that regard, the density gradients may be able to provide increased resolution of a given membrane compartment. There is increasing evidence for subdomains in the endomembrane system. For example, the transitional ER is a specialized region containing COPII components that are involved in packaging proteins for transport to the Golgi complex (53Kuehn M.J. Schekman R. Curr. Opin. Cell Biol. 1997; 9: 477-483Crossref PubMed Scopus (107) Google Scholar, 54Orci L. Ravazzola M. Meda P. Holcomb C. Moore H.P. Hicke L. Schekman R. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 8611-8615Crossref PubMed Scopus (146) Google Scholar). The Golgi complex has long been divided into separate cisternae based on biochemical and morphological criteria. However, recent data (reviewed in Ref. 55Glick B.S. Curr. Opin. Cell Biol. 2000; 12: 450-456Crossref PubMed Scopus (66) Google Scholar) have indicated that this is a highly dynamic organelle composed of constantly changing subdomains that are associated with specific protein components. Similarly, in mammalian cells, different endosomal populations are associated with distinct populations of Rab proteins (reviewed in Ref. 56Zerial M. McBride H. Nat. Rev. Mol. Cell. Biol. 2001; 2: 107-117Crossref PubMed Scopus (2683) Google Scholar).Alternatively, the peak membrane fractions to which Aut7, Cvt19, and the Apg conjugation components all co-localize by density gradients may constitute a distinct compartment that substantially overlaps, both in density and physical proximity, with the Apg1/Cvt9-localized membrane. According to this model, the close proximity of the two perivacuolar compartments would make resolving them by conventional fluorescence microscopy unlikely but instead would require immunoelectron microscopy analysis.Canonical cargo receptors recycle between the donor and the acceptor compartments to ensure continued rounds of cargo binding at the donor and release at the acceptor membranes. For example, the cargo receptor Vps10 transports Prc1 from the late Golgi to the PVC. The steady-state, subcellular distribution of Vps10 appears predominantly at the late Golgi donor compartment, where Prc1 binding and packaging into transport vesicles occurs (57Marcusson E.G. Horazdovsky B.F. Cereghino J.L. Gharakhanian E. Emr S.D. Cell. 1994; 77: 579-586Abstract Full Text PDF PubMed Scopus (397) Google Scholar, 58Cooper A.A. Stevens T.H. J. Cell Biol. 1996; 133: 529-541Crossref PubMed Scopus (239) Google Scholar). Analogous to Vps10, Cvt19 appeared distributed to two subcellular locations (26Scott S.V. Guan J. Hutchins M.U. Kim J. Klionsky D.J. Mol. Cell. 2001; 7: 1131-1141Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar) (Fig. 6B). The predominant pool of Cvt19 resides at the perivacuolar Apg/Cvt compartment, whereas a much smaller level can be detected accumulated in the vacuole of pep4Δ cells. Extrapolating from the donor/acceptor compartment distribution of Vps10, the localization pattern for the Cvt19 cargo receptor suggests that the perivacuolar Apg/Cvt compartment may serve as the donor membrane compartment for Cvt vesicles and autophagosomes.A recent study (29Mizushima N. Yamamoto A. Hatano M. Kobayashi Y. Kabeya Y. Suzuki K. Tokuhisa T. Ohsumi Y. Yoshimori T. J. Cell Biol. 2001; 152: 657-667Crossref PubMed Scopus (1146) Google Scholar) of the Apg5 conjugation component in mouse embryonic stem cells indicated that Apg5 is localized to forming vesicles but dissociates at a step just prior to vesicle completion. Importantly, the major species of Apg5 was detected in the Apg12-conjugated form. We also detected the Apg12-Apg5 conjugate by density gradients (data not shown). However, unlike the free monomers of Apg5 and Apg12, the Apg12-Apg5 conjugate did not co-fractionate with other components that function during vesicle formation (e.g. Aut7 and Cvt19) but instead peaked in fraction 2 of the density gradients (data not shown), in agreement with previously published density reports of the conjugate (27Mizushima N. Noda T. Yoshimori T. Tanaka Y. Ishii T. George M.D. Klionsky D.J. Ohsumi M. Ohsumi Y. Nature. 1998; 395: 395-398Crossref PubMed Scopus (1263) Google Scholar). The study in mouse embryonic stem cells localized the Apg12-Apg5 conjugate to a membrane structure that may represent an intermediate, incomplete transport vesicle, termed the isolation membrane. If an equivalent mechanism exists in yeast, then the free forms of Apg5 and Apg12 that reside at the perivacuolar compartment would exit the donor membrane in the conjugated form and associate with the pre-vesicular, isolation membranes. Subsequent vesicle completion and dissociation of the Apg12-Apg5 conjugate would then be followed by vesicle targeting to the vacuole. Further studies will be needed to clarify this issue.A PI 3-Kinase Complex Required for the Apg/Cvt Pathways Is Localized to the Perivacuolar CompartmentIn this study, we co-localized Apg14, a putative specificity factor for the Cvt and autophagy-specific PI 3-kinase complex, to the Cvt9-associated perivacuolar compartment. The core PI 3-kinase complex (Vps15, -30, and -34) may function at different membrane sites as has been suggested recently (51Kihara A. Noda T. Ishihara N. Ohsumi Y. J. Cell Biol. 2001; 152: 519-530Crossref PubMed Scopus (797) Google Scholar). The PI 3-kinase complex containing Vps38 may function primarily at the Golgi to mediate Prc1 targeting to the PVC, whereas the Apg14-associated PI 3-kinase complex may function at the perivacuolar, endosomal compartment to mediate Cvt and autophagy membrane traffic to the vacuole.The localization of Apg14, and by extension, the Apg/Cvt-specific PI 3-kinase complex, to the perivacuolar compartment (Fig. 2) has significant mechanistic consequences for the role of this compartment in membrane movement and vesicle formation. Phosphoinositide modifications by their kinases can be concentrated to membrane microdomains that serve to spatially restrict signaling to a localized region of a membrane compartment. A null mutation in APG14prevents vesicle formation in both the autophagy and Cvt pathways, suggesting a key role for the Apg/Cvt PI 3-kinase complex in the formation of these transport vesicles and implicating the Apg/Cvt perivacuolar compartment as the potential donor compartment for vesicle formation.The Perivacuolar Compartment Is the Donor Site for Vesicle FormationTo illustrate the physiological relevance of the Apg/Cvt perivacuolar compartment, we followed the steps in G
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