Molecular Mechanisms and Regulation of Specific and Nonspecific Autophagy Pathways in Yeast

The normal growth and development of eukaryotic cells requires a constant balance between biosynthetic and degradative processes. There are two highly conserved mechanisms for degradation in eukaryotes that are responsible for the majority of protein turnover: one is the ubiquitin-proteasome system and the other is autophagy. Under conditions of normal growth, the bulk of protein degradation occurs via the proteasomal machinery (reviewed in Ref. 1Hershko A. Ciechanover A. Annu. Rev. Biochem. 1998; 67: 425-479Crossref PubMed Scopus (6736) Google Scholar). Proteins that are to be degraded are post-translationally tagged with polyubiquitin and are subsequently broken down via the multisubunit 26 S proteasome present in the cytosol and nucleus. In contrast, autophagy is largely a nonspecific process that leads to the vacuolar/lysosomal degradation not just of proteins but also of other cytosolic macromolecular components. Importantly, autophagy is the only degradative pathway that has the capacity to degrade large aggregates and even entire organelles. Autophagy plays a role in normal development and differentiation; however, it is also involved in defense against pathogens, life span extension, and the prevention of certain types of cancer and neurodegeneration (reviewed in Ref. 2Levine B. Klionsky D.J. Dev. Cell. 2004; 6: 463-477Abstract Full Text Full Text PDF PubMed Scopus (3103) Google Scholar). In macroautophagy (hereafter referred to as autophagy), double membrane vesicles called autophagosomes surround the cargo intended for degradation (Fig. 1). Once the autophagosomes are fully formed, the outer membrane fuses with the lysosomal/vacuolar membrane releasing the inner vesicle, which is subsequently broken down. The cargo is released into the lumen of these organelles for degradation, and the resulting macromolecules are then made available for reuse. Two other forms of autophagy, called chaperone-mediated autophagy and microautophagy, have been reviewed recently elsewhere (3Farre J.C. Subramani S. Trends Cell Biol. 2004; 14: 515-523Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 4Massey A. Kiffin R. Cuervo A.M. Int. J. Biochem. Cell Biol. 2004; 36: 2420-2434Crossref PubMed Scopus (149) Google Scholar) and will not be covered here. There are several comprehensive reviews on the molecular mechanism of autophagy (3Farre J.C. Subramani S. Trends Cell Biol. 2004; 14: 515-523Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 5Klionsky D.J. J. Cell Sci. 2005; 118: 7-18Crossref PubMed Scopus (754) Google Scholar, 6Meijer A.J. Codogno P. Int. J. Biochem. Cell Biol. 2004; 36: 2445-2462Crossref PubMed Scopus (532) Google Scholar). Nonetheless, the functions of most of the autophagy proteins are not known. For example, Atg1 was the first protein component of this pathway identified and one of the very few with an obvious functional motif; it appears to be a serine/threonine-protein kinase (7Matsuura A. Tsukada M. Wada Y. Ohsumi Y. Gene (Amst.). 1997; 192: 245-250Crossref PubMed Scopus (376) Google Scholar). Although Atg1 is thought to play a central role in autophagy, almost a decade after its discovery the substrate of Atg1, aside from autocatalytic activity, has not been identified. Because a substantial amount of information is known about this protein, this article focuses on our current knowledge of the mechanism of autophagy with an emphasis on the role(s) of Atg1 in different steps of this degradative transport route in Saccharomyces cerevisiae. Although autophagy is a dynamic process, it can be conceptually divided into seven discrete steps: induction or a cue to start the formation of vesicles, selection and packaging of cargo, initiation of vesicle formation (nucleation), vesicle expansion and completion, retrieval of certain autophagy proteins during or after vesicle completion, fusion of the completed vesicle with the lysosome/vacuole, and the breakdown of the inner membrane of the vesicle and enclosed cargo within the lysosomal/vacuolar lumen (5Klionsky D.J. J. Cell Sci. 2005; 118: 7-18Crossref PubMed Scopus (754) Google Scholar). Mutants affecting different steps of this pathway have been isolated in yeast, and so far 27 different ATG (autophagy-related) genes specifically affecting various steps of this process have been identified. It is interesting to note that even though most of these genes were first identified in yeast, many of them are functionally conserved in higher eukaryotes (2Levine B. Klionsky D.J. Dev. Cell. 2004; 6: 463-477Abstract Full Text Full Text PDF PubMed Scopus (3103) Google Scholar). Contrary to its general mode of bulk cargo degradation, there are some instances in which autophagy plays a biosynthetic role by mediating the specific vesicular trafficking of some vacuolar enzymes (Fig. 1) or performs a homeostatic role (controlling biogenesis versus degradation) by selectively degrading unwanted organelles. A well studied example of the former is that of a selective, biosynthetic pathway called the cytoplasm to vacuole targeting (Cvt) pathway that operates constitutively under growing conditions in S. cerevisiae (8Harding T.M. Morano K.A. Scott S.V. Klionsky D.J. J. Cell Biol. 1995; 131: 591-602Crossref PubMed Scopus (389) Google Scholar). The vacuolar enzymes aminopeptidase I (Ape1) and α-mannosidase (Ams1) are synthesized in the cytoplasm as inactive precursors that assemble to form large homo-oligomeric complexes (9Kim J. Scott S.V. Oda M.N. Klionsky D.J. J. Cell Biol. 1997; 137: 609-618Crossref PubMed Scopus (115) Google Scholar, 10Hutchins M.U. Klionsky D.J. J. Biol. Chem. 2001; 276: 20491-20498Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). The oligomeric enzyme complexes are sequestered within double membrane Cvt vesicles and delivered into the vacuolar lumen where they function as resident hydrolases (11Scott S.V. Baba M. Ohsumi Y. Klionsky D.J. J. Cell Biol. 1997; 138: 37-44Crossref PubMed Scopus (141) Google Scholar). Although Cvt vesicles are morphologically similar to autophagosomes, they are smaller and appear to exclude cytoplasmic contents other than their specific cargo. The molecular components involved in the selection of the Cvt cargo and the temporal sequence of their action have recently been clarified (Fig. 2). A peripheral protein Atg19 serves as a receptor for the Cvt cargo and directly binds to Ape1 or Ams1 oligomers (12Scott 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 (202) Google Scholar, 13Shintani T. Huang W.-P. Stromhaug P.E. Klionsky D.J. Dev. Cell. 2002; 3: 825-837Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar). Another protein, Atg11, then binds to the C terminus of Atg19 and tethers the Cvt complex to a peri-vacuolar site called the pre-autophagosomal structure (PAS) 2The abbreviations used are: PASpre-autophagosomal structurePEphosphatidylethanolaminePtdInsphosphatidylinositol. (13Shintani T. Huang W.-P. Stromhaug P.E. Klionsky D.J. Dev. Cell. 2002; 3: 825-837Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar). The PAS is thought to be the site from which both Cvt vesicles and autophagosomes originate (14Suzuki K. Kirisako T. Kamada Y. Mizushima N. Noda T. Ohsumi Y. EMBO J. 2001; 20: 5971-5981Crossref PubMed Scopus (781) Google Scholar, 15Kim J. Huang W.-P. Stromhaug P.E. Klionsky D.J. J. Biol. Chem. 2002; 277: 763-773Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar), and the expansion of the intermediate structures is thought to occur by subsequent membrane addition. The source of membrane for the formation and expansion of these vesicles is still unknown, although recent data suggest a role for the early secretory pathway (16Ishihara N. Hamasaki M. Yokota S. Suzuki K. Kamada Y. Kihara A. Yoshimori T. Noda T. Ohsumi Y. Mol. Biol. Cell. 2001; 12: 3690-3702Crossref PubMed Scopus (289) Google Scholar, 17Hamasaki M. Noda T. Ohsumi Y. Cell Struct. Funct. 2003; 28: 49-54Crossref PubMed Scopus (78) Google Scholar, 18Reggiori F. Wang C.-W. Nair U. Shintani T. Abeliovich H. Klionsky D.J. Mol. Biol. Cell. 2004; 15: 2189-2204Crossref PubMed Scopus (113) Google Scholar) and possibly the mitochondria (19Reggiori F. Klionsky D.J. Curr. Opin. Cell Biol. 2005; 17: 415-422Crossref PubMed Scopus (230) Google Scholar). Three proteins, prApe1, Atg19, and Atg11, are required for formation of the PAS in nutrient-rich conditions (20Shintani T. Klionsky D.J. J. Biol. Chem. 2004; 279: 29889-29894Abstract Full Text Full Text PDF PubMed Scopus (286) Google Scholar, 21Yorimitsu T. Klionsky D.J. Mol. Biol. Cell. 2005; 16: 1593-1605Crossref PubMed Scopus (207) Google Scholar). Once the cargo complex arrives at the PAS, Atg19 interacts with a phosphatidylethanolamine (PE)-conjugated protein, Atg8–PE, which is initially present on both the inner and outer membrane of the growing vesicle (13Shintani T. Huang W.-P. Stromhaug P.E. Klionsky D.J. Dev. Cell. 2002; 3: 825-837Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar). The interaction between Atg19 and Atg8–PE may mediate the completion of the Cvt vesicle. When the membrane sequestration event is completed, the Cvt vesicle fuses with the vacuole releasing its inner membrane and cargo into the vacuolar lumen. Atg19 and the lumenally oriented Atg8–PE remain inside the vesicle and are degraded, whereas Atg11 is retrieved at some stage prior to vesicle completion and recycled (22Kim 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). The Atg8–PE on the outer vesicle surface is released from the vesicle by an Atg4-dependent cleavage event prior to fusion. pre-autophagosomal structure phosphatidylethanolamine phosphatidylinositol. In addition to binding Atg19, Atg11 appears to be a part of the putative Atg1 regulatory complex (Fig. 3). We refer to this complex as putative because a holocomplex containing all of these proteins has not been demonstrated to exist. In addition to Atg1 and Atg11, the proteins that interact as part of this complex include Atg13 (23Funakoshi T. Matsuura A. Noda T. Ohsumi Y. Gene (Amst.). 1997; 192: 207-213Crossref PubMed Scopus (135) Google Scholar), which appears to modulate Atg1 kinase activity, Atg17 (24Cheong H. Yorimitsu T. Reggiori F. Legakis J.E. Wang C.-W. Klionsky D.J. Mol. Biol. Cell. 2005; 16: 3438-3453Crossref PubMed Scopus (178) Google Scholar, 25Kabeya Y. Kamada Y. Baba M. Takikawa H. Sasaki M. Ohsumi Y. Mol. Biol. Cell. 2005; 16: 2544-2553Crossref PubMed Scopus (265) Google Scholar), a protein that is specific for autophagy, and three proteins of unknown function that are relatively specific for the Cvt pathway, Atg20, Atg24, and Vac8 (26Scott S.V. Nice III, D.C. Nau J.J. Weisman L.S. Kamada Y. Keizer-Gunnink I. Funakoshi T. Veenhuis M. Ohsumi Y. Klionsky D.J. J. Biol. Chem. 2000; 275: 25840-25849Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar). Deletion of the ATG1 gene results in a block in both the Cvt pathway and autophagy (7Matsuura A. Tsukada M. Wada Y. Ohsumi Y. 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Peroxisomes accumulate in the methylotropic yeasts Hansenula polymorpha and Pichia pastoris when these species are grown in media containing methanol as the sole carbon source. When cells are shifted to media containing preferred carbon sources, peroxisomes become superfluous and are selectively degraded via pexophagy (reviewed in Ref. 3Farre J.C. Subramani S. Trends Cell Biol. 2004; 14: 515-523Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). Homologs of S. cerevisiae Atg1 and Atg11 are present in both species, and both gene products are required for pexophagy (28Stromhaug P.E. Bevan A. Dunn Jr., W.A. J. Biol. Chem. 2001; 276: 42422-42435Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 29Mukaiyama H. Oku M. Baba M. Samizo T. Hammond A.T. Glick B.S. Kato N. Sakai Y. Genes Cells. 2002; 7: 75-90Crossref PubMed Scopus (100) Google Scholar). 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A. 2002; PubMed Scopus Google Scholar, Giddings Jr., T.H. S. M. Kirkegaard K. Biol. 2005; 3: PubMed Scopus Google Scholar, M. M. T. 2005; PubMed Scopus Google Scholar). The on the surface of these pathogens that them specific for autophagy have not been identified. In yeast, autophagy appears to at a in conditions and is during The induction of autophagic capacity is for because that are in autophagy during are not large Cvt not only they from other in and the of their but also in the components needed for vesicle as several components of the Atg1 complex that to be required for vesicle formation are specific for the Cvt pathway or autophagy. A is the cell the formation of of the for the induction of autophagy results in the of two highly serine/threonine-protein and (reviewed in Ref. M. Curr. Opin. Cell Biol. 2005; 17: PubMed Scopus Google Scholar). Under growing these autophagy several of cell The of these because of or with the specific even in leads to a cell in the and the induction of autophagy. our knowledge of the mechanism that leads to formation is very In growing the directly or a component of the Atg1 complex (Fig. 3). Atg13 has for Atg1, and this interaction is thought to the induction of autophagy Y. Funakoshi T. Shintani T. K. Ohsumi M. Ohsumi Y. J. Cell Biol. 2000; PubMed Scopus Google Scholar). Under conditions of Atg13 is largely by an unknown its for Atg1 Y. Funakoshi T. Shintani T. K. Ohsumi M. Ohsumi Y. J. Cell Biol. 2000; PubMed Scopus Google Scholar, H. Dunn Jr., W.A. Klionsky D.J. Mol. Biol. Cell. 2003; 14: PubMed Scopus Google Scholar). The interaction between these two proteins as a molecular that a from the Cvt pathway to autophagy. The role of Atg1 in the formation of autophagosomes is on the that they are the only proteins in the putative Atg1 complex that are for both the Cvt pathway and autophagy. the role of Atg1 kinase is currently under two different have to about its role in versus Cvt vesicle Because the of Atg1 is currently unknown, an substrate was used to its kinase in Under the of Atg1 to an substrate Y. Funakoshi T. Shintani T. K. Ohsumi M. Ohsumi Y. J. Cell Biol. 2000; PubMed Scopus Google Scholar), whereas its (7Matsuura A. Tsukada M. Wada Y. Ohsumi Y. Gene (Amst.). 1997; 192: 245-250Crossref PubMed Scopus (376) Google Scholar). is on and on the Atg1 Atg13 and and to a on Atg11 Y. Funakoshi T. Shintani T. K. Ohsumi M. Ohsumi Y. J. Cell Biol. 2000; PubMed Scopus Google Scholar). A was to the for Atg1 kinase in The is to only in the of Y. Kamada Y. Baba M. Takikawa H. Sasaki M. Ohsumi Y. Mol. Biol. Cell. 2005; 16: 2544-2553Crossref PubMed Scopus (265) Google Scholar, H. Dunn Jr., W.A. Klionsky D.J. Mol. Biol. Cell. 2003; 14: PubMed Scopus Google Scholar). In this of kinase a block in the Cvt Similarly, an ATG1 containing a in the kinase is not for the vacuolar of Ape1 through autophagy H. Dunn Jr., W.A. Klionsky D.J. Mol. Biol. Cell. 2003; 14: PubMed Scopus Google Scholar). The of these has been by the that both in the of and appear to a of all of the data that kinase is needed for both the Cvt pathway and autophagy, although a higher of appears to be more for the In Atg1 may play a role at in autophagy H. Dunn Jr., W.A. Klionsky D.J. Mol. Biol. Cell. 2003; 14: PubMed Scopus Google Scholar). these results on the role of Atg1 kinase in the initiation of formation and suggest that it may have a role in vesicle similar to Atg17 (24Cheong H. Yorimitsu T. Reggiori F. Legakis J.E. Wang C.-W. Klionsky D.J. Mol. Biol. Cell. 2005; 16: 3438-3453Crossref PubMed Scopus (178) Google Scholar, 25Kabeya Y. Kamada Y. Baba M. Takikawa H. Sasaki M. Ohsumi Y. Mol. Biol. Cell. 2005; 16: 2544-2553Crossref PubMed Scopus (265) Google Scholar). because Atg1 is not required for the formation of the PAS the localization of some proteins to this it is that it is involved in a event to autophagy (14Suzuki K. Kirisako T. Kamada Y. Mizushima N. Noda T. Ohsumi Y. EMBO J. 2001; 20: 5971-5981Crossref PubMed Scopus (781) Google Scholar). in higher in S. the of autophagosomes not appear to during conditions Jr., W.A. J. Cell Biol. PubMed Scopus Google Scholar). the of the of the degrading vesicle appears to be a to S. cerevisiae and possibly other It has been that in S. the but not the of autophagosomes requires protein during H. Dunn Jr., W.A. Kim J. Klionsky D.J. J. Cell Biol. 2000; PubMed Scopus Google Scholar). will be required to the that and to they modulate Atg1 kinase or interact with other components of the Atg1 the proteins that are directly involved in the Cvt pathway or autophagy, only and Atg19, remain with the completed vesicles and are degraded in the The other including an membrane protein, are retrieved for later at a step prior to the completion of vesicle formation or fusion of the completed vesicle with the Because most of the proteins are it is to that they can from the completed vesicle and remain in the cytosol they are however, the retrieval of membrane proteins include a vesicular is for both Cvt and autophagy and functions at an early stage of both T. Kim J. Huang W.-P. Baba M. Ohsumi Y. Klionsky D.J. J. Cell Biol. 2000; PubMed Scopus Google Scholar). Although most other proteins to the PAS in a and a peripheral membrane protein K.A. Reggiori F. Dunn Jr., W.A. Klionsky D.J. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar) to several cytoplasmic as in addition to the PAS, in both growing and conditions F. K.A. Stromhaug P.E. Klionsky D.J. Dev. Cell. 2004; 6: Full Text Full Text PDF PubMed Scopus Google Scholar). for this was to be in part the mitochondria F. Shintani T. Nair U. Klionsky D.J. 2005; PubMed Scopus (202) Google Scholar), but the organelle to which is not known. results suggest that the of between the PAS and the mitochondria may have a role in membrane from that organelle to the growing Indeed, that both the and the of are with structures F. Shintani T. Nair U. Klionsky D.J. 2005; PubMed Scopus (202) Google Scholar). A retrieval mechanism Atg1, and the protein has been demonstrated recently for F. K.A. Stromhaug P.E. Klionsky D.J. Dev. Cell. 2004; 6: Full Text Full Text PDF PubMed Scopus Google Scholar) (Fig. The data suggest that membrane vesicles containing and from the mitochondria at the PAS that is with and (14Suzuki K. Kirisako T. Kamada Y. Mizushima N. Noda T. Ohsumi Y. EMBO J. 2001; 20: 5971-5981Crossref PubMed Scopus (781) Google Scholar). The Atg1-Atg13 complex arrives at the PAS at a later in a mechanism of the complex (14Suzuki K. Kirisako T. Kamada Y. Mizushima N. Noda T. Ohsumi Y. EMBO J. 2001; 20: 5971-5981Crossref PubMed Scopus (781) Google Scholar, F. K.A. Stromhaug P.E. Klionsky D.J. Dev. Cell. 2004; 6: Full Text Full Text PDF PubMed Scopus Google Scholar). The of the with appears to be the that the of the growing vesicle. and form large oligomeric complexes that are to the growing with Atg8–PE and Atg1 and Atg13 mediate the retrieval of from the PAS, its interaction with and in the of Atg1, is not to the PAS T. Suzuki K. Kamada Y. Noda T. Ohsumi Y. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). When the is completed, the as well as from the vesicle, and Atg8–PE on the outer membrane of the is by and released into the In contrast, Atg8–PE, which the inner of the double membrane Cvt vesicle or is not In the of this protein interacts with and to the PAS in an K.A. Reggiori F. Dunn Jr., W.A. Klionsky D.J. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). It is that similar to also membrane to the growing vesicle. Under growing the retrieval of is only on the kinase of by contrast, the kinase of Atg1 is not needed for the transport of F. K.A. Stromhaug P.E. Klionsky D.J. Dev. Cell. 2004; 6: Full Text Full Text PDF PubMed Scopus Google Scholar). that the kinase of Atg1 is more in the Cvt pathway is only for the Cvt pathway, and Atg1 kinase is required for the retrieval of only in growing Under is within the cell in the for Atg1 kinase for its retrieval F. K.A. Stromhaug P.E. Klionsky D.J. Dev. Cell. 2004; 6: Full Text Full Text PDF PubMed Scopus Google Scholar), although the mechanism involved is not known. because Atg1 at a stage of vesicle formation and has a role in proteins as Atg11, and it is that it as a of vesicle Genes serine/threonine-protein similar to S. cerevisiae ATG1 have recently been in other as and and have been to function in autophagy N. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar, Dev. Cell. 2004; 7: Full Text Full Text PDF PubMed Scopus (754) Google Scholar, A. Z. M. B. 2003; PubMed Scopus Google Scholar). There is a of sequence between the kinase of these proteins and that of the S. cerevisiae however, their are The in these other not only have in autophagy but also in for example, development in formation in and and in The mechanism of action of Atg1 in these is to from their because components of the putative Atg1 complex are in these For example, Atg11 and Atg17 are not conserved of and Atg13 is only in It will be interesting to an Atg1 holocomplex in S. cerevisiae or Atg1 functions by smaller with its in a in the will the role of S. cerevisiae Atg1 in autophagy, as well as the and function of its We Reggiori and the of the Klionsky for