Aut7p, a Soluble Autophagic Factor, Participates in Multiple Membrane Trafficking Processes
2000; Elsevier BV; Volume: 275; Issue: 42 Linguagem: Inglês
10.1074/jbc.m000917200
ISSN1083-351X
AutoresAster Legesse-Miller, Yuval Sagiv, Rina Glozman, Zvulun Elazar,
Tópico(s)Calcium signaling and nucleotide metabolism
ResumoAut7p, a protein recently implicated in autophagic events in the yeast Saccharomyces cerevisiae, exhibits significant homology to a mammalian protein, p16, herein termed GATE-16 (Golgi-associated ATPaseEnhancer of 16 kDa), a novel intra-Golgi transport factor. Here we provide evidence for the involvement of Aut7p in different membrane trafficking processes. Aut7p largely substitutes for the activity of GATE-16 in mammalian intra-Golgi transport in vitro. In vivo, AUT7interacts genetically with endoplasmic reticulum to Golgi SNAREs, specifically withBET1and SEC22. Aut7p interacts physically with the following two v-SNAREs: Bet1p, which is involved in endoplasmic reticulum to Golgi vesicular transport, and Nyv1p, implicated in vacuolar inheritance. We suggest that, in addition to its role in autophagocytosis, Aut7p has pleiotropic effects and participates in at least two membrane traffic events. Aut7p, a protein recently implicated in autophagic events in the yeast Saccharomyces cerevisiae, exhibits significant homology to a mammalian protein, p16, herein termed GATE-16 (Golgi-associated ATPaseEnhancer of 16 kDa), a novel intra-Golgi transport factor. Here we provide evidence for the involvement of Aut7p in different membrane trafficking processes. Aut7p largely substitutes for the activity of GATE-16 in mammalian intra-Golgi transport in vitro. In vivo, AUT7interacts genetically with endoplasmic reticulum to Golgi SNAREs, specifically withBET1and SEC22. Aut7p interacts physically with the following two v-SNAREs: Bet1p, which is involved in endoplasmic reticulum to Golgi vesicular transport, and Nyv1p, implicated in vacuolar inheritance. We suggest that, in addition to its role in autophagocytosis, Aut7p has pleiotropic effects and participates in at least two membrane traffic events. endoplasmic reticulum N-ethylmaleimide-sensitive fusion protein NSF-attachment protein SNAP receptor carboxypeptidase Y cytoplasm to vacuole targeting polymerase chain reaction polyacrylamide gel electrophoresis wild type Membrane trafficking in eukaryotic cells is a highly regulated process that is essential for secretion of macromolecules, as well as for the maintenance of distinct subcellular compartments (1Palade G. Science. 1975; 189: 347-358Crossref PubMed Scopus (2323) Google Scholar, 2Rothman J.E. Nature. 1994; 372: 55-63Crossref PubMed Scopus (1995) Google Scholar). This process encompasses a series of highly regulated events, including cargo selection and vesicle budding at the donor membrane, followed by transport, docking, and fusion of the transport vesicle with the target organelle. We previously identified a cytosolic factor, GATE-16, which participates in intra-Golgi transport (3Legesse-Miller A. Sagiv Y. Porat A. Elazar Z. J. Biol. Chem. 1998; 273: 3105-3109Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 4Sagiv Y. Legesse-Miller A. Porat A. Elazar Z. EMBO J. 2000; 19: 1494-1504Crossref PubMed Scopus (205) Google Scholar). However, the yeast homologue of GATE-16, Aut7p, was recently shown to participate in autophagy (5Lang T. Schaeffeler E. Bernreuther D. Bredschneider M. Wolf D.H. Thumm M. EMBO J. 1998; 17: 3597-3607Crossref PubMed Scopus (230) Google Scholar, 6Kirisako 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). In this study, we have questioned whether Aut7p plays a role in constitutive protein transport, in addition to its involvement in autophagy.Vesicular transport between the ER1 and the Golgi apparatus in the yeast Saccharomyces cerevisiae has been extensively studied. The first step in this process, vesicle budding, involves the assembly of the COPII coat, composed of the Sec13p·Sec31p complex (7Barlowe C. Orci L. Yeung T. Hosobuchi M. Hamamoto S. Salama N. Rexach M.F. Ravazzola M. Amherdt M. Schekman R. Cell. 1994; 77: 895-907Abstract Full Text PDF PubMed Scopus (1033) Google Scholar, 8Pryer N.K. Salama N.R. Schekman R. Kaiser C.A. J. Cell Biol. 1993; 120: 865-875Crossref PubMed Scopus (120) Google Scholar, 9Salama N.R. Schekman R.W. Curr. Opin. Cell Biol. 1995; 7: 536-543Crossref PubMed Scopus (66) Google Scholar), the Sec23p·Sec24p heterodimer (10Hicke L. Yoshihisa T. Schekman R. Mol. Biol. Cell. 1992; 3: 667-676Crossref PubMed Scopus (90) Google Scholar), as well as a small GTPase, Sar1p (11Nakano A. Muramatsu M. J. Cell Biol. 1989; 109: 2677-2691Crossref PubMed Scopus (336) Google Scholar), and the multidomain protein Sec16p (12Espenshade P. Gimeno R.E. Holzmacher E. Teung P. Kaiser C.A. J. Cell Biol. 1995; 131: 311-324Crossref PubMed Scopus (145) Google Scholar, 13Shaywitz D.A. Espenshade P.J. Gimeno R.E. Kaiser C.A. J. Biol. Chem. 1997; 272: 25413-25416Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). Docking of an ER-derived COPII vesicle with the cis-Golgi compartment takes place just after, or concurrently with, a tethering event mediated by Uso1p (14Cao X. Ballew N. Barlowe C. EMBO J. 1998; 17: 2156-2165Crossref PubMed Scopus (291) Google Scholar), the yeast homologue of p115 (15Barroso M. Nelson D.S. Sztul E. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 527-531Crossref PubMed Scopus (110) Google Scholar, 16Sapperstein S.K. Walter D.M. Grosvenor A.R. Heuser J.E. Waters M.G. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 522-526Crossref PubMed Scopus (171) Google Scholar). It has been further suggested that docking involves the interaction of ER to Golgi v-SNAREs, Bet1p, Bos1p, Sec22p, and Ykt6p (17Newman A.P. Groesch M.E. Ferro-Novick S. EMBO J. 1992; 11: 3609-3617Crossref PubMed Scopus (64) Google Scholar, 18Lian J.P. Ferro N.S. Cell. 1993; 73: 735-745Abstract Full Text PDF PubMed Scopus (121) Google Scholar, 19Søgaard M. Tani K. Ye R.R. Geromanos S. Tempst P. Kirchhaousen T. Rothman J.E. Söllner T. Cell. 1994; 78: 937-948Abstract Full Text PDF PubMed Scopus (441) Google Scholar, 20McNew J.A. Søgaard M. Lampen N.M. Machida S. Ye R.R. Lacomis L. Tempst P. Rothman J.E. Sollner T.H. J. Biol. Chem. 1997; 272: 17776-17783Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar), with Sed5, the cognate t-SNARE on the Golgi (21Hardwick K.G. Pelham H.R. J. Cell Biol. 1992; 119: 513-521Crossref PubMed Scopus (255) Google Scholar) to form the v·t-SNARE complex. This complex binds the yeast SNAP (Sec17p) and NSF (Sec18p), which in turn catalyze its disassembly (19Søgaard M. Tani K. Ye R.R. Geromanos S. Tempst P. Kirchhaousen T. Rothman J.E. Söllner T. Cell. 1994; 78: 937-948Abstract Full Text PDF PubMed Scopus (441) Google Scholar) after a round of fusion, thus allowing a new round to take place (22Mayer A. Wickner W. Haas A. Cell. 1996; 85: 83-94Abstract Full Text Full Text PDF PubMed Scopus (478) Google Scholar, 23Otto H. Hanson P.I. Jahn R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6197-6201Crossref PubMed Scopus (230) Google Scholar).Homotypic vacuolar fusion is the last step in yeast vacuole inheritance. Like many membrane trafficking processes, it is mediated by a number of membrane and soluble factors, including Vam3p (a t-SNARE), Nyv1p (a v-SNARE), Ypt7p (a Rab protein), Sec17p, Sec18p, and a low molecular weight factor, LMA1 (24Darsow T. Rieder S.E. Emr S.D. J. Cell Biol. 1997; 138: 517-529Crossref PubMed Scopus (296) Google Scholar, 25Nichols B.J. Ungermann C. Pelham H.R. Wickner W.T. Haas A. Nature. 1997; 387: 199-202Crossref PubMed Scopus (379) Google Scholar, 26Haas A. Scheglmann D. Lazar T. Gallwitz D. Wickner W. EMBO J. 1995; 14: 5258-5270Crossref PubMed Scopus (231) Google Scholar, 27Haas A. Wickner W. EMBO J. 1996; 15: 3296-3305Crossref PubMed Scopus (151) Google Scholar, 28Xu Z. Mayer A. Muller E. Wickner W. J. Cell Biol. 1997; 136: 299-306Crossref PubMed Scopus (63) Google Scholar). Vacuolar homotypic fusion has been divided into the following three distinct subset reactions: priming, docking, and fusion. Priming of SNARE molecules for a new fusion event is mediated by Sec17p, Sec18p, and LMA1 (22Mayer A. Wickner W. Haas A. Cell. 1996; 85: 83-94Abstract Full Text Full Text PDF PubMed Scopus (478) Google Scholar, 29Ungermann C. Wickner W. Xu Z. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11194-11199Crossref PubMed Scopus (88) Google Scholar). Based on a cell-free system reconstituting vacuolar homotypic fusion, it appears that the formation of the SNARE complex is only an intermediate step in the overall fusion reaction (30Ungermann C. Nichols B.J. Pelham H.R. Wickner W. J. Cell Biol. 1998; 140: 61-69Crossref PubMed Scopus (210) Google Scholar). Accordingly, SNARE molecules are involved in docking between donor and acceptor membranes, whereas another set of proteins participates in subsequent stages of the fusion process. This model for the course of events is supported by Peters and Mayer (31Peters C. Mayer A. Nature. 1998; 396: 575-580Crossref PubMed Scopus (322) Google Scholar), who have suggested that calmodulin and other yet-unidentified factors are involved in mediating late stages of vacuolar fusion.The yeast vacuole takes in membrane-bound traffic through at least the following five different transport pathways: the carboxypeptidase Y (CPY) pathway, the alkaline phosphatase I pathway, the endocytic pathway, autophagy, and the cytoplasm to vacuole targeting (Cvt) pathway (32Bryant N.J. Stevens T.H. Microbiol. Mol. Biol. Rev. 1998; 62: 230-247Crossref PubMed Google Scholar). Each of these pathways has different cargo, transport intermediates, and genetic requirements. Autophagy is a bulk protein degradation process by which cytoplasmic components, including organelles, become enclosed in double membrane structures (autophagosomes), which are then delivered to the vacuole for degradation (33Dunn W.A.J. Trends Cell Biol. 1994; 4: 139-143Abstract Full Text PDF PubMed Scopus (442) Google Scholar). Recent studies have revealed that the autophagic process in the budding yeast S. cerevisiae is similar to that of higher eukaryotes (34Baba M. Osumi M. Ohsumi Y. Cell Struct. Funct. 1995; 20: 465-471Crossref PubMed Scopus (126) Google Scholar, 35Mizushima N. Sugita H. Yoshimori T. Ohsumi Y. J. Biol. Chem. 1998; 273: 33889-33892Abstract Full Text Full Text PDF PubMed Scopus (408) Google Scholar, 36Mizushima 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). Autophagy in yeast may be dissected into the following series of subreactions: starvation signaling, formation of autophagosomes, targeting of autophagosomes to the vacuole, docking and fusion with the vacuolar membrane, and degradation of the autophagosome body within the vacuole (37Mizushima N. Noda T. Ohsumi Y. EMBO J. 1999; 18: 3888-3896Crossref PubMed Scopus (338) Google Scholar, 38Noda T. Ohsumi Y. J. Biol. Chem. 1998; 273: 3963-3966Abstract Full Text Full Text PDF PubMed Scopus (1029) Google Scholar). Transport of autophagosomes to lysosomes or vacuoles should therefore be regarded as a membrane traffic process. The Cvt pathway is a constitutive biosynthetic process that shares many common transport components with autophagy (39Harding 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, 40Baba M. Osumi M. Scott S.V. Klionsky D.J. Ohsumi Y. J. Cell Biol. 1997; 139: 1687-1695Crossref PubMed Scopus (276) Google Scholar). Both Cvt and the autophagy pathways involve at least two membrane fusion events, which are dependent on Sec18p and on the vacuolar t-SNARE Vam3p; the latter acts as a multispecific receptor for heterotypic membrane docking and fusion reactions (24Darsow T. Rieder S.E. Emr S.D. J. Cell Biol. 1997; 138: 517-529Crossref PubMed Scopus (296) Google Scholar). Additionally, Tlg2p, a member of the syntaxin family of t-SNARE proteins, and Vps45p, a Sec1p homologue, are reported to be required for the constitutive Cvt pathway (41Abeliovich H. Darsow T. Emr S.D. EMBO J. 1999; 18: 6005-6016Crossref PubMed Scopus (106) Google Scholar).In this study, we demonstrate that Aut7p can largely replace GATE-16 activity in vitro, indicating that the two proteins share a similar, conserved function. Aut7p is a peripheral membrane protein localized predominantly on the Golgi complex and vacuolar membrane. It interacts genetically and physically with Bet1p, a v-SNARE involved in ER to Golgi protein transport. Aut7p also interacts physically with the vacuolar v-SNARE Nyv1p. We hypothesize that Aut7p participates in several transport steps by interacting with the docking and fusion machinery.DISCUSSIONAut7p, previously identified as an autophagic factor (5Lang T. Schaeffeler E. Bernreuther D. Bredschneider M. Wolf D.H. Thumm M. EMBO J. 1998; 17: 3597-3607Crossref PubMed Scopus (230) Google Scholar, 6Kirisako 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), is shown here to take part in multiple intracellular membrane trafficking processes. We have demonstrated that Aut7p specifically interacts with different v-SNARE molecules involved in ER to Golgi transport and in vacuolar fusion. Our findings imply that in addition to its involvement in transport to the vacuole, Aut7p also participates in membrane fusion events that take place in the early secretory pathway.That Aut7p is essential for autophagocytosis (5Lang T. Schaeffeler E. Bernreuther D. Bredschneider M. Wolf D.H. Thumm M. EMBO J. 1998; 17: 3597-3607Crossref PubMed Scopus (230) Google Scholar, 6Kirisako 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) is consistent with the notion that it participates in membrane traffic. Lang et al. (5Lang T. Schaeffeler E. Bernreuther D. Bredschneider M. Wolf D.H. Thumm M. EMBO J. 1998; 17: 3597-3607Crossref PubMed Scopus (230) Google Scholar) proposed that Aut7p and Aut2p are involved in the delivery of autophagosomes to the vacuole along microtubules. Kirisako et al. (6Kirisako 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) suggested that Aut7p plays an important role in autophagosome formation. We propose that the formation of autophagosomes and/or their fusion with the vacuolar membrane are SNARE-dependent and that Aut7p is essential for the fusion process. The t-SNARE Vam3p, a protein known to participate along with other vacuolar protein sorting gene products in directing endosomes to vacuole transport, has also been shown to be involved in autophagy (24Darsow T. Rieder S.E. Emr S.D. J. Cell Biol. 1997; 138: 517-529Crossref PubMed Scopus (296) Google Scholar). Vam3p interacts primarily with Nyv1p, the vacuolar v-SNARE involved in homotypic fusion (25Nichols B.J. Ungermann C. Pelham H.R. Wickner W.T. Haas A. Nature. 1997; 387: 199-202Crossref PubMed Scopus (379) Google Scholar). As we show here, Aut7p and Nyv1p form a complex, suggesting that Aut7p may play a role in the fusion between autophagosomes and vacuoles by interacting with the docking/fusion machinery. It was reported that Nyv1p is not involved in transport of AP1 to the vacuole; in contrast, Vti1p, a v-SNARE found in Golgi-derived vesicles and known to be involved in many transport events, is required for this transport step (52von Mollard G.F. Stevens T.H. Mol. Biol. Cell. 1999; 10: 1719-17232Crossref PubMed Scopus (133) Google Scholar). Additionally, Tlg2p, a member of the syntaxin family of t-SNARE proteins, and Vps45p, a Sec1p homologue, are required in the constitutive Cvt pathway but not in inducible macroautophagy (41Abeliovich H. Darsow T. Emr S.D. EMBO J. 1999; 18: 6005-6016Crossref PubMed Scopus (106) Google Scholar). It is therefore likely that Aut7p may interact with an as yet unidentified related v-SNARE molecule that mediates fusion between autophagosomes and the vacuole. Defining the origin of the donor membrane required for the formation of autophagic vesicle, and isolation of these vesicles using Aut7p as a marker, will allow a better understanding of the roles of the various proteins involved in this pathway.It appears that Aut7p interacts with v-SNAREs and thereby affects their activity. Several regulatory proteins that interact with SNAREs have been reported. Sec1p in yeast and its homologues nSec1p, Munc18, and unc18 in higher eukaryotes have been shown to regulate exocytosis (53Aalto M.K. Ruohonen L. Hosono K. Keranen S. Yeast. 1991; 7: 643-650Crossref PubMed Scopus (60) Google Scholar, 54Garcia E.P. Gatti E. Butler M. Burton J. De Camilli P. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2003-2007Crossref PubMed Scopus (222) Google Scholar, 55Gengyo-Ando K. Kamiya Y. Yamakawa A. Kodaira K. Nishiwaki K. Miwa J. Hori I. Hosono R. Neuron. 1993; 11: 703-711Abstract Full Text PDF PubMed Scopus (95) Google Scholar, 56Hata Y. Slaughter C.A. Sudhof T.C. Nature. 1993; 366: 347-351Crossref PubMed Scopus (584) Google Scholar, 57Salzberg A. Cohen N. Halachmi N. Kimchie Z. Lev Z. Development. 1993; 117: 1309-1319PubMed Google Scholar, 58Pevsner J. Hsu S.C. Scheller R.H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1445-1449Crossref PubMed Scopus (350) Google Scholar). This protein family acts directly on the syntaxin t-SNAREs (54Garcia E.P. Gatti E. Butler M. Burton J. De Camilli P. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2003-2007Crossref PubMed Scopus (222) Google Scholar, 59Aalto M.K. Ronne H. Keranen S. EMBO J. 1993; 12: 4095-4104Crossref PubMed Scopus (343) Google Scholar, 60Pevsner J. Hsu S.C. Braun J.E. Calakos N. Ting A.E. Bennett M.K. Scheller R.H. Neuron. 1994; 13: 353-361Abstract Full Text PDF PubMed Scopus (524) Google Scholar, 61Aalto M.K. Jantti J. Ostling J. Keranen S. Ronne H. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7331-7336Crossref PubMed Scopus (30) Google Scholar). Sec1p also inhibits the assembly of a v·t-SNARE complexin vitro and dissociates from syntaxin upon formation of the v·t-complex (54Garcia E.P. Gatti E. Butler M. Burton J. De Camilli P. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2003-2007Crossref PubMed Scopus (222) Google Scholar, 56Hata Y. Slaughter C.A. Sudhof T.C. Nature. 1993; 366: 347-351Crossref PubMed Scopus (584) Google Scholar, 60Pevsner J. Hsu S.C. Braun J.E. Calakos N. Ting A.E. Bennett M.K. Scheller R.H. Neuron. 1994; 13: 353-361Abstract Full Text PDF PubMed Scopus (524) Google Scholar). Furthermore, experiments in vivoin Drosophila melanogaster and C. elegans showed that overexpression of Sec1p homologues inhibits neurotransmitter release (62Harrison S.D. Broadie K. van de Goor J. Rubin G.M. Neuron. 1994; 13: 555-566Abstract Full Text PDF PubMed Scopus (207) Google Scholar, 63Schulze K.L. Littleton J.T. Salzberg A. Halachmi N. Stern M. Lev Z. Bellen H.J. Neuron. 1994; 13: 1099-1108Abstract Full Text PDF PubMed Scopus (160) Google Scholar). It has therefore been suggested that these proteins act as negative regulators of fusion. Vsm1p, another factor that interacts with v-SNARE (Snc2p), was recently suggested to regulate Snc2p entry to the SNARE complex (64Lustgarten V. Gerst J.E. Mol. Cell. Biol. 1999; 19: 4480-4494Crossref PubMed Scopus (50) Google Scholar). Finally, LMA1, a low molecular weight heterodimer composed of thioredoxin and the protease B inhibitor IB2, originally identified as a protein required for in vitro vacuolar homotypic fusion (28Xu Z. Mayer A. Muller E. Wickner W. J. Cell Biol. 1997; 136: 299-306Crossref PubMed Scopus (63) Google Scholar, 65Xu Z. Wickner W. J. Cell Biol. 1996; 132: 787-794Crossref PubMed Scopus (66) Google Scholar), as well as for ER to Golgi transport (14Cao X. Ballew N. Barlowe C. EMBO J. 1998; 17: 2156-2165Crossref PubMed Scopus (291) Google Scholar, 66Barlowe C. J. Cell Biol. 1997; 139: 1097-1108Crossref PubMed Scopus (155) Google Scholar), was shown to interact with the vacuolar t-SNARE Vam3p in a Sec18p-dependent manner (67Xu Z. Sato K. Wickner W. Cell. 1998; 93: 1125-1134Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). Possibly, Aut7p acts on v-SNAREs in a similar manner to that by which LMA1 acts on the t-SNARE Vam3p.Bet1p, Bos1p, and Sec22p, three v-SNAREs, have been implicated in transport between the ER and the Golgi. It has been recently proposed that Bos1p participates exclusively in anterograde transport from the ER to the Golgi; Sec22p is involved in retrograde transport from the Golgi to the ER, whereas Bet1p acts in both directions (68Spang A. Schekman R. J. Cell Biol. 1998; 143: 589-599Crossref PubMed Scopus (109) Google Scholar). We show here that Aut7p specifically interacts with Bet1p but not with Bos1p, suggesting that it is not involved in anterograde transport but rather in retrograde transport from the Golgi to the ER. This is supported by the suppression effect of AUT7on the retrograde mutant sec22–2. The viability of theaut7 null strain and the lack of a detectable transport defect in this strain suggest that Aut7p plays a regulatory role in this process or that other factors may substitute its function. Because under starvation conditions Aut7p is essential for autophagy, we speculate that Bet1p or Sec22p may participate in the formation of the autophagic membrane or that under starvation Aut7p interacts with other SNARE molecules mediating autophagy.We have recently found that GATE-16, the mammalian homologue of Aut7p, interacts specifically with a Golgi v-SNARE in an NSF- and SNAP-dependent manner (4Sagiv Y. Legesse-Miller A. Porat A. Elazar Z. EMBO J. 2000; 19: 1494-1504Crossref PubMed Scopus (205) Google Scholar). Although the precise mechanism for the function of Aut7p in membrane traffic is uncertain, we propose that it may act as a positive regulator of v-SNAREs. The data presented in the present study support the notion that the function of Aut7p is closely related to the activity of SNAREs.Our findings are summarized by the model described in Fig.6. Accordingly, under normal growth conditions Aut7p functions in early steps of the secretory pathway by interacting with v-SNARE molecules such as Bet1p. This Aut7p activity can probably be replaced by other unidentified factor(s). Under constitutive steady-state conditions, Aut7p is essential for the Cvt pathway. Upon nitrogen starvation, Aut7p expression levels are significantly increased, and it becomes involved in autophagy. Based on the fact that Aut7p interacts with the vacuolar v-SNARE, Nyv1p, we propose that Aut7p may be involved in vacuolar fusion or that Nyv1p is involved in autophagy triggered upon nitrogen starvation. Further experiments are required to resolve this issue. Membrane trafficking in eukaryotic cells is a highly regulated process that is essential for secretion of macromolecules, as well as for the maintenance of distinct subcellular compartments (1Palade G. Science. 1975; 189: 347-358Crossref PubMed Scopus (2323) Google Scholar, 2Rothman J.E. Nature. 1994; 372: 55-63Crossref PubMed Scopus (1995) Google Scholar). This process encompasses a series of highly regulated events, including cargo selection and vesicle budding at the donor membrane, followed by transport, docking, and fusion of the transport vesicle with the target organelle. We previously identified a cytosolic factor, GATE-16, which participates in intra-Golgi transport (3Legesse-Miller A. Sagiv Y. Porat A. Elazar Z. J. Biol. Chem. 1998; 273: 3105-3109Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 4Sagiv Y. Legesse-Miller A. Porat A. Elazar Z. EMBO J. 2000; 19: 1494-1504Crossref PubMed Scopus (205) Google Scholar). However, the yeast homologue of GATE-16, Aut7p, was recently shown to participate in autophagy (5Lang T. Schaeffeler E. Bernreuther D. Bredschneider M. Wolf D.H. Thumm M. EMBO J. 1998; 17: 3597-3607Crossref PubMed Scopus (230) Google Scholar, 6Kirisako 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). In this study, we have questioned whether Aut7p plays a role in constitutive protein transport, in addition to its involvement in autophagy. Vesicular transport between the ER1 and the Golgi apparatus in the yeast Saccharomyces cerevisiae has been extensively studied. The first step in this process, vesicle budding, involves the assembly of the COPII coat, composed of the Sec13p·Sec31p complex (7Barlowe C. Orci L. Yeung T. Hosobuchi M. Hamamoto S. Salama N. Rexach M.F. Ravazzola M. Amherdt M. Schekman R. Cell. 1994; 77: 895-907Abstract Full Text PDF PubMed Scopus (1033) Google Scholar, 8Pryer N.K. Salama N.R. Schekman R. Kaiser C.A. J. Cell Biol. 1993; 120: 865-875Crossref PubMed Scopus (120) Google Scholar, 9Salama N.R. Schekman R.W. Curr. Opin. Cell Biol. 1995; 7: 536-543Crossref PubMed Scopus (66) Google Scholar), the Sec23p·Sec24p heterodimer (10Hicke L. Yoshihisa T. Schekman R. Mol. Biol. Cell. 1992; 3: 667-676Crossref PubMed Scopus (90) Google Scholar), as well as a small GTPase, Sar1p (11Nakano A. Muramatsu M. J. Cell Biol. 1989; 109: 2677-2691Crossref PubMed Scopus (336) Google Scholar), and the multidomain protein Sec16p (12Espenshade P. Gimeno R.E. Holzmacher E. Teung P. Kaiser C.A. J. Cell Biol. 1995; 131: 311-324Crossref PubMed Scopus (145) Google Scholar, 13Shaywitz D.A. Espenshade P.J. Gimeno R.E. Kaiser C.A. J. Biol. Chem. 1997; 272: 25413-25416Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). Docking of an ER-derived COPII vesicle with the cis-Golgi compartment takes place just after, or concurrently with, a tethering event mediated by Uso1p (14Cao X. Ballew N. Barlowe C. EMBO J. 1998; 17: 2156-2165Crossref PubMed Scopus (291) Google Scholar), the yeast homologue of p115 (15Barroso M. Nelson D.S. Sztul E. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 527-531Crossref PubMed Scopus (110) Google Scholar, 16Sapperstein S.K. Walter D.M. Grosvenor A.R. Heuser J.E. Waters M.G. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 522-526Crossref PubMed Scopus (171) Google Scholar). It has been further suggested that docking involves the interaction of ER to Golgi v-SNAREs, Bet1p, Bos1p, Sec22p, and Ykt6p (17Newman A.P. Groesch M.E. Ferro-Novick S. EMBO J. 1992; 11: 3609-3617Crossref PubMed Scopus (64) Google Scholar, 18Lian J.P. Ferro N.S. Cell. 1993; 73: 735-745Abstract Full Text PDF PubMed Scopus (121) Google Scholar, 19Søgaard M. Tani K. Ye R.R. Geromanos S. Tempst P. Kirchhaousen T. 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