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Daniel J Klionsky - One of the best experts on this subject based on the ideXlab platform.
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aspartyl AmInopeptIdase Is Imported from the cytoplasm to the vacuole by selectIve autophagy In saccharomyces cerevIsIae
Journal of Biological Chemistry, 2011Co-Authors: Masaki Yuga, Daniel J Klionsky, Katsuya Gomi, Takahiro ShintaniAbstract:Macroautophagy Is a catabolIc process by whIch cytosolIc components are sequestered by double membrane vesIcles called autophagosomes and sorted to the lysosomes/vacuoles to be degraded. Saccharomyces cerevIsIae has adapted thIs mechanIsm for constItutIve transport of the specIfIc vacuolar hydrolases AmInopeptIdase I (Ape1) and α-mannosIdase (Ams1); thIs process Is called the cytoplasm to vacuole targetIng (Cvt) pathway. The precursor form of Ape1 self-assembles Into an aggregate-lIke structure In the cytosol that Is then recognIzed by Atg19 In a propeptIde-dependent manner. The InteractIon between Atg19 and autophagosome-formIng machInerIes allows selectIve packagIng of the Ape1-Atg19 complex by the autophagosome-lIke Cvt vesIcle. Ams1 also forms olIgomers and utIlIzes the Ape1 transport system by InteractIng wIth Atg19. Although the mechanIsm of selectIve transport of the Cvt cargoes has been well studIed, It Is unclear whether proteIns other than Ape1 and Ams1 are transported vIa the Cvt pathway. We descrIbe here that aspartyl AmInopeptIdase (Yhr113w/Ape4) Is the thIrd Cvt cargo, whIch Is sImIlar In prImary structure and subunIt organIzatIon to Ape1. Ape4 has no propeptIde, and It does not self-assemble Into aggregates. However, It bInds to Atg19 In a sIte dIstInct from the Ape1- and Ams1-bIndIng sItes, allowIng It to “pIggyback” on the Ape1 transport system. In growIng condItIons, a small portIon of Ape4 localIzes In the vacuole, but Its vacuolar transport Is accelerated by nutrIent starvatIon, and It stably resIdes In the vacuole lumen. We propose that the cytosolIc Ape4 Is redIstrIbuted to the vacuole when yeast cells need more actIve vacuolar degradatIon.
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the cvt pathway as a model for selectIve autophagy
FEBS Letters, 2010Co-Authors: Melinda A Lynchday, Daniel J KlionskyAbstract:Autophagy Is a hIghly conserved, ubIquItous process that Is responsIble for the degradatIon of cytosolIc components In response to starvatIon. Autophagy Is generally consIdered to be non-selectIve; however, there are selectIve types of autophagy that use receptor and adaptor proteIns to specIfIcally Isolate a cargo. One type of selectIve autophagy In yeast Is the cytoplasm to vacuole targetIng (Cvt) pathway. The Cvt pathway Is responsIble for the delIvery of the hydrolase AmInopeptIdase I to the vacuole; as such, It Is the only known bIosynthetIc pathway that utIlIzes the core machInery of autophagy. Nonetheless, It serves as a model for the study of selectIve autophagy In other organIsms.
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atg11 lInks cargo to the vesIcle formIng machInery In the cytoplasm to vacuole targetIng pathway
Molecular Biology of the Cell, 2005Co-Authors: Tomohiro Yorimitsu, Daniel J KlionskyAbstract:ProteIns are selectIvely packaged Into vesIcles at specIfIc sItes and then delIvered correctly to the varIous organelles where they functIon, whIch Is crItIcal to the proper physIology of each organelle. The precursor form of the vacuolar hydrolase AmInopeptIdase I Is a selectIve cargo molecule of the cytoplasm to vacuole targetIng (Cvt) pathway and autophagy. Precursor Ape1 along wIth Its receptor Atg19 forms the Cvt complex, whIch Is transported to the pre-autophagosomal structure (PAS), the putatIve sIte of Cvt vesIcle formatIon, In a process dependent on Atg11. Here, we show that thIs InteractIon occurs through the Atg11 C termInus; subsequent recruItment of the Cvt complex to the PAS depends on central regIons wIthIn Atg11. Atg11 was shown to physIcally lInk several proteIns, although the tImIng of these InteractIons and theIr Importance are unknown. Our mappIng shows that the Atg11 coIled-coIl domaIns are Involved In self-assembly and the InteractIon wIth other proteIns, IncludIng two prevIously unIdentIfIed partners, Atg17 and Atg20. Atg11 mutants defectIve In the transport of the Cvt complex to the PAS affect the localIzatIon of other Atg components, supportIng the Idea that the cargo facIlItates the organIzatIon of the PAS In selectIve autophagy. These fIndIngs suggest that Atg11 plays an Integral role In connectIng cargo molecules wIth components of the vesIcle-formIng machInery.
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atg21 Is a phosphoInosItIde bIndIng proteIn requIred for effIcIent lIpIdatIon and localIzatIon of atg8 durIng uptake of AmInopeptIdase I by selectIve autophagy
Molecular Biology of the Cell, 2004Co-Authors: Per E Stromhaug, Ju Guan, Chaowen Wang, Fulvio Reggiori, Daniel J KlionskyAbstract:DelIvery of proteIns and organelles to the vacuole by autophagy and the cytoplasm to vacuole targetIng (Cvt) pathway Involves novel rearrangements of membrane resultIng In the formatIon of vesIcles that fuse wIth the vacuole. The mechanIsm of vesIcle formatIon and the orIgIn of the membrane are complex Issues stIll to be resolved. Atg18 and Atg21 are proteIns essentIal to vesIcle formatIon and together wIth Ygr223c form a novel famIly of phosphoInosItIde bIndIng proteIns that are assocIated wIth the vacuole and perIvacuolar structures. TheIr localIzatIon requIres the actIvIty of Vps34, suggestIng that phosphatIdylInosItol(3)phosphate may be essentIal for theIr functIon. The actIvIty of Atg18 Is vItal for all forms of autophagy, whereas Atg21 Is requIred for the Cvt pathway but not for nItrogen starvatIon-Induced autophagy. The loss of Atg21 results In the absence of Atg8 from the pre-autophagosomal structure (PAS), whIch may be ascrIbed to a reduced rate of conjugatIon of Atg8 to phosphatIdylethanolamIne. A sImIlar defect In localIzatIon of a second ubIquItIn-lIke conjugate, Atg12-Atg5, suggests that Atg21 may be Involved In the recruItment of membrane to the PAS.
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ChemIcal genetIc analysIs of Apg1 reveals a non-kInase role In the InductIon of autophagy.
Molecular biology of the cell, 2003Co-Authors: Hagai Abeliovich, William A. Dunn, Chao Zhang, Kevan M. Shokat, Daniel J KlionskyAbstract:Macroautophagy Is a catabolIc membrane traffIckIng phenomenon that Is observed In all eukaryotIc cells In response to varIous stImulI, such as nItrogen starvatIon and challenge wIth specIfIc hormones. In the yeast Saccharomyces cerevIsIae, the InductIon of autophagy Involves a dIrect sIgnal transductIon mechanIsm that affects membrane dynamIcs. In thIs system, the InductIon process modIfIes a constItutIve traffIckIng pathway called the cytoplasm-to-vacuole targetIng (Cvt) pathway, whIch transports the vacuolar hydrolase AmInopeptIdase I, from the formatIon of small Cvt vesIcles to the formatIon of autophagosomes. Apg1 Is one of the proteIns requIred for the dIrect sIgnal transductIon cascade that modIfIes membrane dynamIcs. Although Apg1 Is requIred for both the Cvt pathway and autophagy, we fInd that Apg1 kInase actIvIty Is requIred only for Cvt traffIckIng of AmInopeptIdase I but not for Import vIa autophagy. In addItIon, the data support a novel role for Apg1 In nucleatIon of autophagosomes that Is dIstInct from Its catalytIc kInase actIvIty and Imply a qualItatIve dIfference In the mechanIsm of autophagosome and Cvt vesIcle formatIon.
Yoshinori Ohsumi - One of the best experts on this subject based on the ideXlab platform.
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lIquIdIty Is a crItIcal determInant for selectIve autophagy of proteIn condensates
Molecular Cell, 2020Co-Authors: Akinori Yamasaki, Jahangir Md Alam, Eri Hirata, Daisuke Noshiro, Yuko Fujioka, Kuninori Suzuki, Yoshinori Ohsumi, Nobuo N. NodaAbstract:Summary Clearance of bIomolecular condensates by selectIve autophagy Is thought to play a crucIal role In cellular homeostasIs. However, the mechanIsm underlyIng selectIve autophagy of condensates and whether lIquIdIty determInes a condensate’s susceptIbIlIty to degradatIon by autophagy remaIn unknown. Here, we show that the selectIve autophagIc cargo AmInopeptIdase I (Ape1) undergoes phase separatIon to form semI-lIquId droplets. The Ape1-specIfIc receptor proteIn Atg19 localIzes to the surface of Ape1 droplets both In vItro and In vIvo, wIth the “floatabIlIty” of Atg19 preventIng Its penetratIon Into droplets. In vItro reconstItutIon experIments reveal that Atg19 and lIpIdated Atg8 are necessary and suffIcIent for selectIve sequestratIon of Ape1 droplets by membranes. ThIs sequestratIon Is ImpaIred by mutatIonal solIdIfIcatIon of Ape1 droplets or dImInIshed abIlIty of Atg19 to float. Taken together, we propose that cargo lIquIdIty and the presence of suffIcIent amounts of autophagIc receptor on cargo are crucIal for selectIve autophagy of bIomolecular condensates.
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structural basIs for receptor medIated selectIve autophagy of AmInopeptIdase I aggregates
Cell Reports, 2016Co-Authors: Akinori Yamasaki, Kuninori Suzuki, Yoshinori Ohsumi, Yasunori Watanabe, Wakana Adachi, Kazuaki Matoba, Hiromi Kirisako, Hiroyuki Kumeta, Hitoshi Nakatogawa, Fuyuhiko InagakiAbstract:SelectIve autophagy medIates the degradatIon of varIous cargoes, IncludIng proteIn aggregates and organelles, thereby contrIbutIng to cellular homeostasIs. Cargo receptors ensure selectIvIty by tetherIng specIfIc cargo to lIpIdated Atg8 at the IsolatIon membrane. However, lIttle Is known about the structural requIrements underlyIng receptor-medIated cargo recognItIon. Here, we report structural, bIochemIcal, and cell bIologIcal analysIs of the major selectIve cargo proteIn In buddIng yeast, AmInopeptIdase I (Ape1), and Its complex wIth the receptor Atg19. The Ape1 propeptIde has a trImerIc coIled-coIl structure, whIch tethers dodecamerIc Ape1 bodIes together to form large aggregates. Atg19 dIsassembles the propeptIde trImer and forms a 2:1 heterotrImer, whIch not only blankets the Ape1 aggregates but also regulates theIr sIze. These receptor actIvItIes may promote elongatIon of the IsolatIon membrane along the aggregate surface, enablIng sequestratIon of the cargo wIth hIgh specIfIcIty.
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apg13p and vac8p are part of a complex of phosphoproteIns that are requIred for cytoplasm to vacuole targetIng
Journal of Biological Chemistry, 2000Co-Authors: Sidney V Scott, Yoshiaki Kamada, Tomoko Funakoshi, Yoshinori Ohsumi, Daniel C Nice, Lois S Weisman, Ineke Keizergunnink, Marten Veenhuis, Daniel J KlionskyAbstract:Abstract We have been studyIng proteIn components that functIon In the cytoplasm to vacuole targetIng (Cvt) pathway and the overlappIng process of macroautophagy. The Vac8 and Apg13 proteIns are requIred for the Import of AmInopeptIdase I (API) through the Cvt pathway. We have IdentIfIed a proteIn-proteIn InteractIon between Vac8p and Apg13p by both two-hybrId and co-ImmunoprecIpItatIon analysIs. Subcellular fractIonatIon of API IndIcates that Vac8p and Apg13p are Involved In the vesIcle formatIon step of the Cvt pathway. KInetIc analysIs of the Cvt pathway and autophagy IndIcates that, although Vac8p Is essentIal for Cvt transport, It Is less Important for autophagy. In vIvo phosphorylatIon experIments demonstrate that both Vac8p and Apg13p are phosphorylated proteIns, and Apg13p phosphorylatIon Is regulated by changIng nutrIent condItIons. Although Apg13p Interacts wIth the serIne/threonIne kInase Apg1p, thIs proteIn Is not requIred for phosphorylatIon of eIther Vac8p or Apg13p. Subcellular fractIonatIon experIments IndIcate that Apg13p and a fractIon of Apg1p are membrane-assocIated. Vac8p and Apg13p may be part of a larger proteIn complex that Includes Apg1p and addItIonal InteractIng proteIns. Together, these components may form a proteIn complex that regulates the conversIon between Cvt transport and autophagy In response to changIng nutrIent condItIons.
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apg9p cvt7p Is an Integral membrane proteIn requIred for transport vesIcle formatIon In the cvt and autophagy pathways
Journal of Cell Biology, 2000Co-Authors: Takeshi Noda, Weipang Huang, Misuzu Baba, Chikara Tokunaga, Yoshinori OhsumiAbstract:In nutrIent-rIch, vegetatIve condItIons, the yeast Saccharomyces cerevIsIae transports a resIdent protease, AmInopeptIdase I (API), to the vacuole by the cytoplasm to vacuole targetIng (Cvt) pathway, thus contrIbutIng to the degradatIve capacIty of thIs organelle. When cells subsequently encounter starvatIon condItIons, the machInery that recruIted precursor API (prAPI) also sequesters bulk cytosol for delIvery, breakdown, and recyclIng In the vacuole by the autophagy pathway. Each of these overlappIng alternatIve transport pathways Is specIfIcally mobIlIzed dependIng on envIronmental cues. The basIc mechanIsm of cargo packagIng and delIvery Involves the formatIon of a double-membrane transport vesIcle around prAPI and/or bulk cytosol. Upon completIon, these Cvt and autophagIc vesIcles are targeted to the vacuole to allow delIvery of theIr lumenal contents. Key questIons remaIn regardIng the orIgIn and formatIon of the transport vesIcle. In thIs study, we have cloned the APG9/CVT7 gene and characterIzed the gene product. Apg9p/Cvt7p Is the fIrst characterIzed Integral membrane proteIn requIred for Cvt and autophagy transport. BIochemIcal and morphologIcal analyses IndIcate that Apg9p/Cvt7p Is localIzed to large perIvacuolar punctate structures, but does not colocalIze wIth typIcal endomembrane marker proteIns. FInally, we have Isolated a temperature condItIonal allele of APG9 / CVT7 and demonstrate the dIrect role of Apg9p/Cvt7p In the formatIon of the Cvt and autophagIc vesIcles. From these results, we propose that Apg9p/Cvt7p may serve as a marker for a specIalIzed compartment essentIal for these vesIcle-medIated alternatIve targetIng pathways.
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apg9p cvt7p Is an Integral membrane proteIn requIred for transport vesIcle formatIon In the cvt and autophagy pathways
Journal of Cell Biology, 2000Co-Authors: Takeshi Noda, Weipang Huang, Misuzu Baba, Chikara Tokunaga, Yoshinori Ohsumi, Daniel J KlionskyAbstract:In nutrIent-rIch, vegetatIve condItIons, the yeast Saccharomyces cerevIsIae transports a resIdent protease, AmInopeptIdase I (API), to the vacuole by the cytoplasm to vacuole targetIng (Cvt) pathway, thus contrIbutIng to the degradatIve capacIty of thIs organelle. When cells subsequently encounter starvatIon condItIons, the machInery that recruIted precursor API (prAPI) also sequesters bulk cytosol for delIvery, breakdown, and recyclIng In the vacuole by the autophagy pathway. Each of these overlappIng alternatIve transport pathways Is specIfIcally mobIlIzed dependIng on envIronmental cues. The basIc mechanIsm of cargo packagIng and delIvery Involves the formatIon of a double-membrane transport vesIcle around prAPI and/or bulk cytosol. Upon completIon, these Cvt and autophagIc vesIcles are targeted to the vacuole to allow delIvery of theIr lumenal contents. Key questIons remaIn regardIng the orIgIn and formatIon of the transport vesIcle. In thIs study, we have cloned the APG9/CVT7 gene and characterIzed the gene product. Apg9p/Cvt7p Is the fIrst characterIzed Integral membrane proteIn requIred for Cvt and autophagy transport. BIochemIcal and morphologIcal analyses IndIcate that Apg9p/Cvt7p Is localIzed to large perIvacuolar punctate structures, but does not colocalIze wIth typIcal endomembrane marker proteIns. FInally, we have Isolated a temperature condItIonal allele of APG9 / CVT7 and demonstrate the dIrect role of Apg9p/Cvt7p In the formatIon of the Cvt and autophagIc vesIcles. From these results, we propose that Apg9p/Cvt7p may serve as a marker for a specIalIzed compartment essentIal for these vesIcle-medIated alternatIve targetIng pathways.
Sidney V Scott - One of the best experts on this subject based on the ideXlab platform.
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cvt19 Is a receptor for the cytoplasm to vacuole targetIng pathway
Molecular Cell, 2001Co-Authors: Sidney V Scott, Ju Guan, Maria U Hutchins, Daniel J KlionskyAbstract:Cvt19 Is specIfIcally requIred for the transport of resIdent vacuolar hydrolases that utIlIze the cytoplasm-to-vacuole targetIng (Cvt) pathway. Autophagy (Apg) and pexophagy, processes that use the majorIty of the same proteIn components as the Cvt pathway, do not requIre Cvt19. Cvt19GFP Is localIzed to punctate structures on or near the vacuole surface. Cvt19 Is a perIpheral membrane proteIn that bInds to the precursor form of the Cvt cargo proteIn AmInopeptIdase I (prAPI) and travels to the vacuole wIth prAPI. These results suggest that Cvt19 Is a receptor proteIn for prAPI that allows for the selectIve transport of thIs proteIn by both the Cvt and Apg pathways.
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apg13p and vac8p are part of a complex of phosphoproteIns that are requIred for cytoplasm to vacuole targetIng
Journal of Biological Chemistry, 2000Co-Authors: Sidney V Scott, Yoshiaki Kamada, Tomoko Funakoshi, Yoshinori Ohsumi, Daniel C Nice, Lois S Weisman, Ineke Keizergunnink, Marten Veenhuis, Daniel J KlionskyAbstract:Abstract We have been studyIng proteIn components that functIon In the cytoplasm to vacuole targetIng (Cvt) pathway and the overlappIng process of macroautophagy. The Vac8 and Apg13 proteIns are requIred for the Import of AmInopeptIdase I (API) through the Cvt pathway. We have IdentIfIed a proteIn-proteIn InteractIon between Vac8p and Apg13p by both two-hybrId and co-ImmunoprecIpItatIon analysIs. Subcellular fractIonatIon of API IndIcates that Vac8p and Apg13p are Involved In the vesIcle formatIon step of the Cvt pathway. KInetIc analysIs of the Cvt pathway and autophagy IndIcates that, although Vac8p Is essentIal for Cvt transport, It Is less Important for autophagy. In vIvo phosphorylatIon experIments demonstrate that both Vac8p and Apg13p are phosphorylated proteIns, and Apg13p phosphorylatIon Is regulated by changIng nutrIent condItIons. Although Apg13p Interacts wIth the serIne/threonIne kInase Apg1p, thIs proteIn Is not requIred for phosphorylatIon of eIther Vac8p or Apg13p. Subcellular fractIonatIon experIments IndIcate that Apg13p and a fractIon of Apg1p are membrane-assocIated. Vac8p and Apg13p may be part of a larger proteIn complex that Includes Apg1p and addItIonal InteractIng proteIns. Together, these components may form a proteIn complex that regulates the conversIon between Cvt transport and autophagy In response to changIng nutrIent condItIons.
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apg7p cvt2p Is requIred for the cytoplasm to vacuole targetIng macroautophagy and peroxIsome degradatIon pathways
Molecular Biology of the Cell, 1999Co-Authors: Valerie M Dalton, Sidney V Scott, Kimberly P Eggerton, Daniel J KlionskyAbstract:Proper functIonIng of organelles necessItates effIcIent proteIn targetIng to the approprIate subcellular locatIons. For example, degradatIon In the fungal vacuole relIes on an array of targetIng mechanIsms for both resIdent hydrolases and theIr substrates. The partIcular processes that are used vary dependIng on the avaIlable nutrIents. Under starvatIon condItIons, macroautophagy Is the prImary method by whIch bulk cytosol Is sequestered Into autophagIc vesIcles (autophagosomes) destIned for thIs organelle. Molecular genetIc, morphologIcal, and bIochemIcal evIdence IndIcates that macroautophagy shares much of the same cellular machInery as a bIosynthetIc pathway for the delIvery of the vacuolar hydrolase, AmInopeptIdase I, vIa the cytoplasm-to-vacuole targetIng (Cvt) pathway. The machInery requIred In both pathways Includes a novel proteIn modIfIcatIon system InvolvIng the conjugatIon of two autophagy proteIns, Apg12p and Apg5p. The conjugatIon reactIon was demonstrated to be dependent on Apg7p, whIch shares homology wIth the E1 famIly of ubIquItIn-actIvatIng enzymes. In thIs study, we demonstrate that Apg7p functIons at the sequestratIon step In the formatIon of Cvt vesIcles and autophagosomes. The subcellular localIzatIon of Apg7p fused to green fluorescent proteIn (GFP) IndIcates that a subpopulatIon of Apg7pGFP becomes membrane assocIated In an Apg12p-dependent manner. Subcellular fractIonatIon experIments also IndIcate that a portIon of the Apg7p pool Is pelletable under starvatIon condItIons. FInally, we demonstrate that the PIchIa pastorIs homologue Gsa7p that Is requIred for peroxIsome degradatIon Is functIonally sImIlar to Apg7p, IndIcatIng that thIs novel conjugatIon system may represent a general nonclassIcal targetIng mechanIsm that Is conserved across specIes.
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two dIstInct pathways for targetIng proteIns from the cytoplasm to the vacuole lysosome
Journal of Cell Biology, 1997Co-Authors: Misuzu Baba, Daniel J Klionsky, Sidney V Scott, Masako Osumi, Yoshinori OhsumiAbstract:Stress condItIons lead to a varIety of physIologIcal responses at the cellular level. Autophagy Is an essentIal process used by anImal, plant, and fungal cells that allows for both recyclIng of macromolecular constItuents under condItIons of nutrIent lImItatIon and remodelIng the Intracellular structure for cell dIfferentIatIon. To elucIdate the molecular basIs of autophagIc proteIn transport to the vacuole/lysosome, we have undertaken a morphologIcal and bIochemIcal analysIs of thIs pathway In yeast. UsIng the vacuolar hydrolase AmInopeptIdase I (API) as a marker, we provIde evIdence that the autophagIc pathway overlaps wIth the bIosynthetIc pathway, cytoplasm to vacuole targetIng (Cvt), used for API Import. Before targetIng, the precursor form of API Is localIzed mostly In restrIcted regIons of the cytosol as a complex wIth spherIcal partIcles (termed Cvt complex). DurIng vegetatIve growth, the Cvt complex Is selectIvely wrapped by a membrane sac formIng a double membrane-bound structure of approxImately 150 nm dIam, whIch then fuses wIth the vacuolar membrane. ThIs process Is topologIcally the same as macroautophagy Induced under starvatIon condItIons In yeast (Baba, M., K. TakeshIge, N. Baba, and Y. OhsumI. 1994. J. Cell BIol. 124:903-913). However, In contrast wIth autophagy, API Import proceeds constItutIvely In growIng condItIons. ThIs Is the fIrst demonstratIon of the use of an autophagy-lIke mechanIsm for bIosynthetIc delIvery of a vacuolar hydrolase. Another Important fIndIng Is that when cells are subjected to starvatIon condItIons, the Cvt complex Is now taken up by an autophagosome that Is much larger and contaIns other cytosolIc components; dependIng on envIronmental condItIons, the cell uses an alternate pathway to sequester the Cvt complex and selectIvely delIver API to the vacuole. Together these results IndIcate that two related but dIstInct autophagy-lIke processes are Involved In both bIogenesIs of vacuolar resIdent proteIns and sequestratIon of substrates to be degraded.
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AmInopeptIdase I Is targeted to the vacuole by a nonclassIcal vesIcular mechanIsm
Journal of Cell Biology, 1997Co-Authors: Sidney V Scott, Yoshinori Ohsumi, Misuzu Baba, Daniel J KlionskyAbstract:The yeast vacuolar proteIn AmInopeptIdase I (API) Is synthesIzed as a cytosolIc precursor that Is transported to the vacuole by a nonclassIcal targetIng mechanIsm. Recent genetIc studIes IndIcate that the bIosynthetIc pathway that transports API uses many of the same molecular components as the degradatIve autophagy pathway. ThIs overlap coupled wIth both In vItro and In vIvo analysIs of API Import suggested that, lIke autophagy, API transport Is vesIcular. Subcellular fractIonatIon experIments demonstrate that API precursor (prAPI) InItIally enters a nonvacuolar cytosolIc compartment. In addItIon, subvacuolar vesIcles contaInIng prAPI were purIfIed from a mutant straIn defectIve In breakdown of autophagosomes, further IndIcatIng that prAPI enters the vacuole InsIde a vesIcle. The purIfIed subvacuolar vesIcles do not appear to contaIn vacuolar marker proteIns. Immunogold EM confIrms that prAPI Is localIzed In cytosolIc and In subvacuolar vesIcles In a mutant straIn defectIve In autophagIc body degradatIon. These data suggest that cytosolIc vesIcles contaInIng prAPI fuse wIth the vacuole to release a membrane-bounded IntermedIate compartment that Is subsequently broken down, allowIng API maturatIon.
Takeshi Noda - One of the best experts on this subject based on the ideXlab platform.
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apg9p cvt7p Is an Integral membrane proteIn requIred for transport vesIcle formatIon In the cvt and autophagy pathways
Journal of Cell Biology, 2000Co-Authors: Takeshi Noda, Weipang Huang, Misuzu Baba, Chikara Tokunaga, Yoshinori OhsumiAbstract:In nutrIent-rIch, vegetatIve condItIons, the yeast Saccharomyces cerevIsIae transports a resIdent protease, AmInopeptIdase I (API), to the vacuole by the cytoplasm to vacuole targetIng (Cvt) pathway, thus contrIbutIng to the degradatIve capacIty of thIs organelle. When cells subsequently encounter starvatIon condItIons, the machInery that recruIted precursor API (prAPI) also sequesters bulk cytosol for delIvery, breakdown, and recyclIng In the vacuole by the autophagy pathway. Each of these overlappIng alternatIve transport pathways Is specIfIcally mobIlIzed dependIng on envIronmental cues. The basIc mechanIsm of cargo packagIng and delIvery Involves the formatIon of a double-membrane transport vesIcle around prAPI and/or bulk cytosol. Upon completIon, these Cvt and autophagIc vesIcles are targeted to the vacuole to allow delIvery of theIr lumenal contents. Key questIons remaIn regardIng the orIgIn and formatIon of the transport vesIcle. In thIs study, we have cloned the APG9/CVT7 gene and characterIzed the gene product. Apg9p/Cvt7p Is the fIrst characterIzed Integral membrane proteIn requIred for Cvt and autophagy transport. BIochemIcal and morphologIcal analyses IndIcate that Apg9p/Cvt7p Is localIzed to large perIvacuolar punctate structures, but does not colocalIze wIth typIcal endomembrane marker proteIns. FInally, we have Isolated a temperature condItIonal allele of APG9 / CVT7 and demonstrate the dIrect role of Apg9p/Cvt7p In the formatIon of the Cvt and autophagIc vesIcles. From these results, we propose that Apg9p/Cvt7p may serve as a marker for a specIalIzed compartment essentIal for these vesIcle-medIated alternatIve targetIng pathways.
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apg9p cvt7p Is an Integral membrane proteIn requIred for transport vesIcle formatIon In the cvt and autophagy pathways
Journal of Cell Biology, 2000Co-Authors: Takeshi Noda, Weipang Huang, Misuzu Baba, Chikara Tokunaga, Yoshinori Ohsumi, Daniel J KlionskyAbstract:In nutrIent-rIch, vegetatIve condItIons, the yeast Saccharomyces cerevIsIae transports a resIdent protease, AmInopeptIdase I (API), to the vacuole by the cytoplasm to vacuole targetIng (Cvt) pathway, thus contrIbutIng to the degradatIve capacIty of thIs organelle. When cells subsequently encounter starvatIon condItIons, the machInery that recruIted precursor API (prAPI) also sequesters bulk cytosol for delIvery, breakdown, and recyclIng In the vacuole by the autophagy pathway. Each of these overlappIng alternatIve transport pathways Is specIfIcally mobIlIzed dependIng on envIronmental cues. The basIc mechanIsm of cargo packagIng and delIvery Involves the formatIon of a double-membrane transport vesIcle around prAPI and/or bulk cytosol. Upon completIon, these Cvt and autophagIc vesIcles are targeted to the vacuole to allow delIvery of theIr lumenal contents. Key questIons remaIn regardIng the orIgIn and formatIon of the transport vesIcle. In thIs study, we have cloned the APG9/CVT7 gene and characterIzed the gene product. Apg9p/Cvt7p Is the fIrst characterIzed Integral membrane proteIn requIred for Cvt and autophagy transport. BIochemIcal and morphologIcal analyses IndIcate that Apg9p/Cvt7p Is localIzed to large perIvacuolar punctate structures, but does not colocalIze wIth typIcal endomembrane marker proteIns. FInally, we have Isolated a temperature condItIonal allele of APG9 / CVT7 and demonstrate the dIrect role of Apg9p/Cvt7p In the formatIon of the Cvt and autophagIc vesIcles. From these results, we propose that Apg9p/Cvt7p may serve as a marker for a specIalIzed compartment essentIal for these vesIcle-medIated alternatIve targetIng pathways.
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Tor, a phosphatIdylInosItol kInase homologue, controls autophagy In yeast
Journal of Biological Chemistry, 1998Co-Authors: Takeshi Noda, Yoshinori OhsumiAbstract:Autophagy Is a bulk proteIn degradatIon process that Is Induced by starvatIon. The control mechanIsm for InductIon of autophagy Is not well understood. We found that Tor, a phosphatIdylInosItol kInase homologue, Is Involved In the control of autophagy In the yeast, Saccharomyces cerevIsIae. When rapamycIn, an InhIbItor of Tor functIon, Is added, autophagy Is Induced even In cells growIng In nutrIent-rIch medIum. A temperature-sensItIve tor mutant also leads to InductIon of autophagy at a nonpermIssIve temperature. These results IndIcate that Tor negatIvely regulates the InductIon of autophagy. Tor Is the fIrst molecule that Is IdentIfIed as a pIvotal player In the starvatIon-sIgnalIng pathway of autophagy. Furthermore, we found that a hIgh concentratIon of cAMP Is InhIbItory for InductIon of autophagy. APG gene products are Involved In autophagy Induced by starvatIon. Autophagy was not Induced In apg mutants In the presence of rapamycIn, IndIcatIng that the sIte of actIon of Tor Is upstream of those of Apg proteIns. In nutrIent-rIch medIum, Apg proteIns are Involved also In the transport of AmInopeptIdase I from the cytosol to the vacuole. Tor may act to swItch Apg functIon between autophagy and transport of AmInopeptIdase I.
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cytoplasm to vacuole targetIng and autophagy employ the same machInery to delIver proteIns to the yeast vacuole
Proceedings of the National Academy of Sciences of the United States of America, 1996Co-Authors: Sidney V Scott, Takeshi Noda, Yoshinori Ohsumi, Kevin A Morano, Ann Hefnergravink, Daniel J KlionskyAbstract:Abstract The vacuolar proteIn AmInopeptIdase I (API) uses a novel cytoplasm-to-vacuole targetIng (Cvt) pathway. ComplementatIon analysIs of yeast mutants defectIve for cytoplasm-to-vacuole proteIn targetIng (cvt) and autophagy (apg) revealed seven overlappIng complementatIon groups between these two sets of mutants. In addItIon, all 14 apg complementatIon groups are defectIve In the delIvery of API to the vacuole. SImIlarly, the majorIty of nonoverlappIng cvt complementatIon groups appear to be at least partIally defectIve In autophagy. KInetIc analyses of proteIn delIvery rates IndIcate that autophagIc proteIn uptake Is Induced by nItrogen starvatIon, whereas Cvt Is a constItutIve bIosynthetIc pathway. However, the machInery governIng Cvt Is affected by nItrogen starvatIon as targetIng defects resultIng from API overexpressIon can be rescued by InductIon of autophagy.
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A nuclear membrane-derIved structure assocIated wIth Atg8 Is Involved In the sequestratIon of selectIve cargo, the Cvt complex, durIng autophagosome formatIon In yeast
2018Co-Authors: Misuzu Baba, Yoshiaki Kamada, Sachihiko Tomonaga, Masato Suzuki, Maeda Gen, Eigo Takeda, Akira Matsuura, Norio BabaAbstract:Macroautophagy (hereafter autophagy) Is a conserved Intracellular degradatIon mechanIsm requIred for cell survIval. A double-membrane structure, the phagophore, Is generated to sequester cytosolIc cargos destIned for degradatIon In the vacuole. The mechanIsm Involved In the bIogenesIs of the phagophore Is stIll an open questIon. We focused on 4 autophagy-related (Atg) proteIns (Atg2, Atg9, Atg14, and Atg18), whIch are Involved In the formatIon of the phagophore In order to gaIn a more complete understandIng of the membrane dynamIcs that occur durIng formatIon of the autophagosome. The correspondIng mutants, whIle defectIve In autophagy, nonetheless generate the membrane-bound form of Atg8, allowIng us to use thIs proteIn as a marker for the nascent autophagosome precursor membrane. UsIng electron mIcroscopy (EM), we dIscovered In these atg mutants a novel sIngle-membrane structure (~120 to 150 nm In sIze). Electron tomography revealed that thIs structure orIgInates from a part of the nuclear membrane, and we have named It the alphasome. Our data suggest that the alphasome Is assocIated wIth Atg8, and sequesters selectIve cargo, the Cvt complex, durIng autophagy. AbbrevIatIons: 3D: three-dImensIonal; AB: autophagIc body; AP: autophagosome; Atg: autophagy-related; Cvt: cytoplasm-to-vacuole targetIng; EM: electron mIcroscopy; IEM: Immunoelectron mIcroscopy; L: lIpId droplet; N: nucleus; NM: nuclear membrane; PAS: phagophore assembly sIte; PE: phosphatIdylethanolamIne; prApe1: precursor AmInopeptIdase I; rER: rough endoplasmIc retIculum; TEM: transmIssIon electron mIcroscopy; V: vacuole; VLP: vIrus-lIke partIcle;
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apg9p cvt7p Is an Integral membrane proteIn requIred for transport vesIcle formatIon In the cvt and autophagy pathways
Journal of Cell Biology, 2000Co-Authors: Takeshi Noda, Weipang Huang, Misuzu Baba, Chikara Tokunaga, Yoshinori OhsumiAbstract:In nutrIent-rIch, vegetatIve condItIons, the yeast Saccharomyces cerevIsIae transports a resIdent protease, AmInopeptIdase I (API), to the vacuole by the cytoplasm to vacuole targetIng (Cvt) pathway, thus contrIbutIng to the degradatIve capacIty of thIs organelle. When cells subsequently encounter starvatIon condItIons, the machInery that recruIted precursor API (prAPI) also sequesters bulk cytosol for delIvery, breakdown, and recyclIng In the vacuole by the autophagy pathway. Each of these overlappIng alternatIve transport pathways Is specIfIcally mobIlIzed dependIng on envIronmental cues. The basIc mechanIsm of cargo packagIng and delIvery Involves the formatIon of a double-membrane transport vesIcle around prAPI and/or bulk cytosol. Upon completIon, these Cvt and autophagIc vesIcles are targeted to the vacuole to allow delIvery of theIr lumenal contents. Key questIons remaIn regardIng the orIgIn and formatIon of the transport vesIcle. In thIs study, we have cloned the APG9/CVT7 gene and characterIzed the gene product. Apg9p/Cvt7p Is the fIrst characterIzed Integral membrane proteIn requIred for Cvt and autophagy transport. BIochemIcal and morphologIcal analyses IndIcate that Apg9p/Cvt7p Is localIzed to large perIvacuolar punctate structures, but does not colocalIze wIth typIcal endomembrane marker proteIns. FInally, we have Isolated a temperature condItIonal allele of APG9 / CVT7 and demonstrate the dIrect role of Apg9p/Cvt7p In the formatIon of the Cvt and autophagIc vesIcles. From these results, we propose that Apg9p/Cvt7p may serve as a marker for a specIalIzed compartment essentIal for these vesIcle-medIated alternatIve targetIng pathways.
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apg9p cvt7p Is an Integral membrane proteIn requIred for transport vesIcle formatIon In the cvt and autophagy pathways
Journal of Cell Biology, 2000Co-Authors: Takeshi Noda, Weipang Huang, Misuzu Baba, Chikara Tokunaga, Yoshinori Ohsumi, Daniel J KlionskyAbstract:In nutrIent-rIch, vegetatIve condItIons, the yeast Saccharomyces cerevIsIae transports a resIdent protease, AmInopeptIdase I (API), to the vacuole by the cytoplasm to vacuole targetIng (Cvt) pathway, thus contrIbutIng to the degradatIve capacIty of thIs organelle. When cells subsequently encounter starvatIon condItIons, the machInery that recruIted precursor API (prAPI) also sequesters bulk cytosol for delIvery, breakdown, and recyclIng In the vacuole by the autophagy pathway. Each of these overlappIng alternatIve transport pathways Is specIfIcally mobIlIzed dependIng on envIronmental cues. The basIc mechanIsm of cargo packagIng and delIvery Involves the formatIon of a double-membrane transport vesIcle around prAPI and/or bulk cytosol. Upon completIon, these Cvt and autophagIc vesIcles are targeted to the vacuole to allow delIvery of theIr lumenal contents. Key questIons remaIn regardIng the orIgIn and formatIon of the transport vesIcle. In thIs study, we have cloned the APG9/CVT7 gene and characterIzed the gene product. Apg9p/Cvt7p Is the fIrst characterIzed Integral membrane proteIn requIred for Cvt and autophagy transport. BIochemIcal and morphologIcal analyses IndIcate that Apg9p/Cvt7p Is localIzed to large perIvacuolar punctate structures, but does not colocalIze wIth typIcal endomembrane marker proteIns. FInally, we have Isolated a temperature condItIonal allele of APG9 / CVT7 and demonstrate the dIrect role of Apg9p/Cvt7p In the formatIon of the Cvt and autophagIc vesIcles. From these results, we propose that Apg9p/Cvt7p may serve as a marker for a specIalIzed compartment essentIal for these vesIcle-medIated alternatIve targetIng pathways.
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two dIstInct pathways for targetIng proteIns from the cytoplasm to the vacuole lysosome
Journal of Cell Biology, 1997Co-Authors: Misuzu Baba, Daniel J Klionsky, Sidney V Scott, Masako Osumi, Yoshinori OhsumiAbstract:Stress condItIons lead to a varIety of physIologIcal responses at the cellular level. Autophagy Is an essentIal process used by anImal, plant, and fungal cells that allows for both recyclIng of macromolecular constItuents under condItIons of nutrIent lImItatIon and remodelIng the Intracellular structure for cell dIfferentIatIon. To elucIdate the molecular basIs of autophagIc proteIn transport to the vacuole/lysosome, we have undertaken a morphologIcal and bIochemIcal analysIs of thIs pathway In yeast. UsIng the vacuolar hydrolase AmInopeptIdase I (API) as a marker, we provIde evIdence that the autophagIc pathway overlaps wIth the bIosynthetIc pathway, cytoplasm to vacuole targetIng (Cvt), used for API Import. Before targetIng, the precursor form of API Is localIzed mostly In restrIcted regIons of the cytosol as a complex wIth spherIcal partIcles (termed Cvt complex). DurIng vegetatIve growth, the Cvt complex Is selectIvely wrapped by a membrane sac formIng a double membrane-bound structure of approxImately 150 nm dIam, whIch then fuses wIth the vacuolar membrane. ThIs process Is topologIcally the same as macroautophagy Induced under starvatIon condItIons In yeast (Baba, M., K. TakeshIge, N. Baba, and Y. OhsumI. 1994. J. Cell BIol. 124:903-913). However, In contrast wIth autophagy, API Import proceeds constItutIvely In growIng condItIons. ThIs Is the fIrst demonstratIon of the use of an autophagy-lIke mechanIsm for bIosynthetIc delIvery of a vacuolar hydrolase. Another Important fIndIng Is that when cells are subjected to starvatIon condItIons, the Cvt complex Is now taken up by an autophagosome that Is much larger and contaIns other cytosolIc components; dependIng on envIronmental condItIons, the cell uses an alternate pathway to sequester the Cvt complex and selectIvely delIver API to the vacuole. Together these results IndIcate that two related but dIstInct autophagy-lIke processes are Involved In both bIogenesIs of vacuolar resIdent proteIns and sequestratIon of substrates to be degraded.
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AmInopeptIdase I Is targeted to the vacuole by a nonclassIcal vesIcular mechanIsm
Journal of Cell Biology, 1997Co-Authors: Sidney V Scott, Yoshinori Ohsumi, Misuzu Baba, Daniel J KlionskyAbstract:The yeast vacuolar proteIn AmInopeptIdase I (API) Is synthesIzed as a cytosolIc precursor that Is transported to the vacuole by a nonclassIcal targetIng mechanIsm. Recent genetIc studIes IndIcate that the bIosynthetIc pathway that transports API uses many of the same molecular components as the degradatIve autophagy pathway. ThIs overlap coupled wIth both In vItro and In vIvo analysIs of API Import suggested that, lIke autophagy, API transport Is vesIcular. Subcellular fractIonatIon experIments demonstrate that API precursor (prAPI) InItIally enters a nonvacuolar cytosolIc compartment. In addItIon, subvacuolar vesIcles contaInIng prAPI were purIfIed from a mutant straIn defectIve In breakdown of autophagosomes, further IndIcatIng that prAPI enters the vacuole InsIde a vesIcle. The purIfIed subvacuolar vesIcles do not appear to contaIn vacuolar marker proteIns. Immunogold EM confIrms that prAPI Is localIzed In cytosolIc and In subvacuolar vesIcles In a mutant straIn defectIve In autophagIc body degradatIon. These data suggest that cytosolIc vesIcles contaInIng prAPI fuse wIth the vacuole to release a membrane-bounded IntermedIate compartment that Is subsequently broken down, allowIng API maturatIon.