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W2023758154 abstract "The yeast vacuolar enzyme aminopeptidase I (API) is synthesized in the cytoplasm as a precursor (pAPI). Upon its assembly into dodecamers, pAPI is wrapped by double-membrane saccular structures for its further transport within vesicles that fuse with the vacuolar membrane and release their content in the vacuolar lumen. Targeting of API to the vacuole occurs by two alternative transport routes, the cvt and the autophagy pathways, which although mechanistically similar specifically operate under vegetative growth or nitrogen starvation conditions, respectively. We have studied the role of Yol082p, a protein identified by its ability to interact with API, in the transport of its precursor to the vacuole. We show that Yol082p interacts with mature API, an interaction that is strengthened by the amino extension of the API protein. Yol082p is required for targeting of pAPI to the vacuole, both under growing and short term nitrogen starvation conditions. Absence of Yol082p does not impede the assembly of pAPI into dodecamers, but precludes the enclosure of pAPI within transport vesicles. Microscopy studies show that during vegetative growth Yol082p is distributed between a cytoplasmic pool and a variable number of 0.13–0.27-µm round, mobile structures, which are no longer observed under conditions of nitrogen starvation, and become larger in cells expressing the inactive Yol082ΔC32p, or lacking Apg12p. In contrast to the autophagy mutants involved in API transport, a Δyol082 strain does not lose viability under nitrogen starvation conditions, indicating normal function of the autophagy pathway. The data are consistent with a role of Yol082p in an early step of the API transport, after its assembly into dodecamers. Because Yol082p fulfills the functional requisites that define the CVT proteins, we propose to name it Cvt19. The yeast vacuolar enzyme aminopeptidase I (API) is synthesized in the cytoplasm as a precursor (pAPI). Upon its assembly into dodecamers, pAPI is wrapped by double-membrane saccular structures for its further transport within vesicles that fuse with the vacuolar membrane and release their content in the vacuolar lumen. Targeting of API to the vacuole occurs by two alternative transport routes, the cvt and the autophagy pathways, which although mechanistically similar specifically operate under vegetative growth or nitrogen starvation conditions, respectively. We have studied the role of Yol082p, a protein identified by its ability to interact with API, in the transport of its precursor to the vacuole. We show that Yol082p interacts with mature API, an interaction that is strengthened by the amino extension of the API protein. Yol082p is required for targeting of pAPI to the vacuole, both under growing and short term nitrogen starvation conditions. Absence of Yol082p does not impede the assembly of pAPI into dodecamers, but precludes the enclosure of pAPI within transport vesicles. Microscopy studies show that during vegetative growth Yol082p is distributed between a cytoplasmic pool and a variable number of 0.13–0.27-µm round, mobile structures, which are no longer observed under conditions of nitrogen starvation, and become larger in cells expressing the inactive Yol082ΔC32p, or lacking Apg12p. In contrast to the autophagy mutants involved in API transport, a Δyol082 strain does not lose viability under nitrogen starvation conditions, indicating normal function of the autophagy pathway. The data are consistent with a role of Yol082p in an early step of the API transport, after its assembly into dodecamers. Because Yol082p fulfills the functional requisites that define the CVT proteins, we propose to name it Cvt19. yeast vacuolar leucine aminopeptidase I API precursor mature API autophagy cytoplasm to vacuole targeting green fluorescent protein Yol082p round structure polymerase chain reaction enhanced chemiluminescence 1,4-piperazinediethanesulfonic acid carboxypeptidase Y polyacrylamide gel electrophoresis In the yeast Saccharomyces cerevisiae the vacuolar hydrolase leucine aminopeptidase I (API)1 is synthesized in the cytoplasm as a precursor (pAPI) (1Klionsky D.J. Cueva R. Yaver D.S. J. Cell Biol. 1992; 119: 287-299Crossref PubMed Scopus (305) Google Scholar) and delivered to the vacuole by one of two alternative routes that operate under distinct physiological conditions: the cytoplasm to vacuole targeting (Cvt), in nutrient-rich conditions, and the autophagy (Apg) pathway, under starvation conditions (2Baba M. Osumi M. Scott S.V. Klionsky D.J. Ohsumi Y. J. Cell Biol. 1997; 139: 1687-1695Crossref PubMed Scopus (275) Google Scholar). The Cvt pathway is constitutive and biosynthetic, while autophagy is nonselective and degradative and is induced to survive periods of nutrient limitation (3Scott 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). However, the two pathways share many molecular components and both involve sequestration by double-membrane saccular structures of unknown origin that capture the load, close into vesicles, and then fuse with the vacuole (4Scott S.V. Baba M. Ohsumi Y. Klionsky D.J. J. Cell Biol. 1997; 138: 37-44Crossref PubMed Scopus (141) Google Scholar). A major difference between these pathways appears to be the size and content of the transport vesicles. The Cvt vesicles exclude cytoplasm and are smaller than autophagosomes that engulf bulk cytoplasm and even organelles (2Baba M. Osumi M. Scott S.V. Klionsky D.J. Ohsumi Y. J. Cell Biol. 1997; 139: 1687-1695Crossref PubMed Scopus (275) Google Scholar). Strikingly, despite all these differences, targeting of API to the vacuole is specific and saturable, both in vegetative growth conditions and under nitrogen deprivation (3Scott 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), although the molecular details of its selective recognition and capture remain essentially unknown. Previous studies have shown that pAPI recognition by the transport machinery involves its prepro-amino extension (5Seguı́-Real B. Martı́nez M. Sandoval I.V. EMBO J. 1995; 14: 5476-5484Crossref PubMed Scopus (31) Google Scholar, 6Oda M.N. Scott S.V. Hefner-Gravink A. Caffarelli A.D. Klionsky D.J. J. Cell Biol. 1996; 132: 999-1010Crossref PubMed Scopus (77) Google Scholar) and cytoplasmic chaperones of the Ssa family (7Silles E. Mazón M.J. Gevaert K. Goethals M. Vandekerckhove J. Leber R. Sandoval I.V. J. Biol. Chem. 2000; 275: 34054-34059Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar, 8Satyanarayana C. Schroder-Kohne S. Craig E.A. Schu P.V. Horst M. FEBS Lett. 2000; 470: 232-238Crossref PubMed Scopus (19) Google Scholar). Furthermore, the amino extension is necessary and sufficient to target the reporter protein GFP to the vacuole (9Martı́nez E. Seguı́-Real B. Silles E. Mazón M.J. Sandoval I.V. Mol. Microbiol. 1999; 33: 52-62Crossref PubMed Scopus (12) Google Scholar). In this study we report that Yol082p, a protein shown to interact physically with pAPI in a two-hybrid screening performed with the whole yeast genome (10Uetz P. Giot L. Cagney G. Mansfield T.A. Judson R.S. Knight J.R. Lockshon D. Narayan V. Srinivasan M. Pochart P. Qureshi-Emili A. Li Y. Godwin B. Conover D. Kalbfleisch T. Vijayadamodar G. Yang M. Johnston M. Fields S. Rothberg J.M. Nature. 2000; 403: 623-627Crossref PubMed Scopus (3896) Google Scholar), mediates API loading into transport vesicles and targeting to the vacuole. We also show that Yol082p interacts with API by a process that does not only involve the prepro-amino extension but also the mature part of the API protein. Yol082p is distributed between the cytoplasm and distinct round mobile structures.DISCUSSIONIn this study we have shown that Yol082p, identified in a whole-genome analysis of protein-protein interactions as an API-interacting protein (10Uetz P. Giot L. Cagney G. Mansfield T.A. Judson R.S. Knight J.R. Lockshon D. Narayan V. Srinivasan M. Pochart P. Qureshi-Emili A. Li Y. Godwin B. Conover D. Kalbfleisch T. Vijayadamodar G. Yang M. Johnston M. Fields S. Rothberg J.M. Nature. 2000; 403: 623-627Crossref PubMed Scopus (3896) Google Scholar), is required for vacuolar targeting and conversion of pAPI into mAPI, both in vegetative growth and under short term nitrogen starvation conditions.The interaction of Yol082p with pAPI and mAPI, but not with the prepro-amino extension of the precursor, is particularly interesting given the role of the latter in the transport of pAPI to the vacuole and the observation that it is necessary and sufficient for the transport of the reporter protein GFP from the cytoplasm to the vacuole (9Martı́nez E. Seguı́-Real B. Silles E. Mazón M.J. Sandoval I.V. Mol. Microbiol. 1999; 33: 52-62Crossref PubMed Scopus (12) Google Scholar). This observation suggests that transport of API to the vacuole requires additional transport determinants localized in the mature part, outside its amino extension. In addition, the stronger two-hybrid interaction of Yol082p with pAPI, as compared with mAPI, suggests that either Yol082p interacts physically with the amino extension in the context of the native protein or, alternatively, that the extension is required for proper folding and exposure of the transport determinants contained in the mature part of API to Yol082p. Clearly, further research is required to determine whether the determinants involved in its interaction with Yol082p are specific of API.Our studies on the processing of pAPI in wild-type and Δyol082 cells show that Yol082p is required for targeting and conversion of pAPI into mAPI in the vacuole, both under vegetative growth and short periods of nitrogen starvation. The rescue of the API processing defect in Δyol082 cells transformed with Yol082p expressed from an inducible promoter unambiguously shows the involvement of Yol082p in the vacuolar import of pAPI.The block in API vacuolar processing shown byΔyol082 cells incubated for short periods in SD(−N) medium is in contrast to the ability of mutants with impeded API transport, such as apg13, vac8,cvt3, aut2, and aut7, to overcome the pAPI accumulation soon after their shift to medium without nitrogen (2Baba M. Osumi M. Scott S.V. Klionsky D.J. Ohsumi Y. J. Cell Biol. 1997; 139: 1687-1695Crossref PubMed Scopus (275) Google Scholar,24Scott S.V. Nice 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, 25Abeliovich H. Dunn W.A.J. Kim J. Klionsky D.J. J. Cell Biol. 2000; 151: 1025-1034Crossref PubMed Scopus (234) Google Scholar). Furthermore, it is interesting that the inhibition of the pAPI processing in Δyol082 cells is partially reversed by the extension of the nitrogen starvation period. Under these conditions, mAPI is detected after 12 h in SD(−N) medium, and maturation proceeds slowly to reach a plateau at ∼30% in 16 h. These observations strongly suggest that Yol082p is involved in the rapid and specific capture of pAPI by the autophagosomes developed after a short starvation period, but not in the slow and unspecific capture that occurs with the engulfment of large portions of the cytoplasm after prolonged starvation.Apg mutants (26Tsukada M. Ohsumi Y. FEBS Lett. 1993; 333: 169-174Crossref PubMed Scopus (1376) Google Scholar) often show a correlation between the effect of the mutation in autophagosome biogenesis and the loss in viability under nitrogen starvation, so that mutants with defective autophagosome nucleation die earlier upon nitrogen deprivation (25Abeliovich H. Dunn W.A.J. Kim J. Klionsky D.J. J. Cell Biol. 2000; 151: 1025-1034Crossref PubMed Scopus (234) Google Scholar). In this context it is, therefore, interesting that although Yol082p appears to function in an early step in the pathway of API transport, theΔyol082 mutant is completely starvation-resistant. This resistance to starvation suggests again that Yol082p is not essential for autophagosome biogenesis.Regarding the API transport step in which Yol082p is involved, we have shown that pAPI assembly into dodecamers takes place normally in the mutant cells. We also show that in the absence of Yol082p, the interaction of the oligomerized pAPI with the sequestering double-membrane sacs appears to be affected. The proteinase K protection assay performed with metabolically labeled protein reveals that, in the mutant strain, the newly synthesized pAPI remains unprotected in the cytoplasm after 2 h of its synthesis, which is in contrast to the wild-type. This difference suggests that Yol082p may work in the recognition of pAPI by the wrapping membranes or, alternatively, by closing these into vesicles. The recovery of the protease-sensitive pAPI extracted from apg5,apg7, apg9, aut7/apg8, andcvt3 cells defective in biogenesis of the API transport vesicles (19Noda T. Kim J. Huang W.P. Baba M. Tokunaga C. Ohsumi Y. Klionsky D.J. J. Cell Biol. 2000; 148: 465-480Crossref PubMed Scopus (302) Google Scholar, 27George 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, 28Kim J. Dalton V.M. Eggerton K.P. Scott S.V. Klionsky D.J. Mol. Biol. Cell. 1999; 10: 1337-1351Crossref PubMed Scopus (177) Google Scholar, 29Huang W.P. Scott S.V. Kim J. Klionsky D.J. J. Biol. Chem. 2000; 275: 5845-5851Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar) with the membranes that float on Ficoll, and the exclusion of the bulk of the pAPI extracted fromΔyol082 cells from the membrane fraction, rather supports the first of the two above alternatives.Confocal fluorescence microscopy studies, performed using the fluorescent protein GFP-Yol082p, show the existence of a pool of protein homogeneously distributed throughout the cytoplasm in equilibrium with a second pool organized into one or more round-shaped structures, which we have called YR. Furthermore, the equilibrium is dramatically shifted by changes in the cell growing conditions, the ability of the cells to use the autophagy pathway, and the functional activity of GFP-Yol082p. Although we lack direct evidence on the meaning of the above changes in distribution, some of these observations suggest that the pool organized into YR structures could be actively engaged in the transport of pAPI to the vacuole by the Cvt pathway. With regard to this, the disappearance of the YR structures from cells incubated for 2 h in SD(−N) medium strongly suggests that they are not needed under conditions in which the autophagy pathway is activated. The difference between the rapid processing of pAPI in mutants with a defective Cvt pathway and the inability ofΔyol082 cells to process pAPI, when they were incubated in SD(−N) medium, strongly suggests that transport of pAPI by the autophagy pathway requires the disassembly of the YR structures. This may be related to the decrease in the cytoplasmic pool of GFP-Yol082p and the enhanced visibility of YR structures observed inΔapg12 cells incubated with SD or SD(−N) medium, changes that could reflect a blockage of the Cvt/Apg pathways after the transport step mediated by Yol082p. Also interesting is the disappearance of the cytoplasmic protein and the increase in size of YR structures observed in wild-type cells expressing the functionally inactive Yol082ΔC32p-GFP. This shift in equilibrium could again be consistent with our view that YR structures are involved in protein transport through the Cvt pathway, since the functionally inactive protein may cause the jam of the transport machinery and/or the accumulation of the transported material. A second alternative is the possibility that the deletion of the last 32 residues may shift the protein equilibrium toward the YR structures, which could also explain the insensitivity of these to the incubation of cells in SD(−N) medium. Obviously, these two possibilities are not mutually exclusive.Because of its behavior as a Cvt protein we propose to rename Yol082p as Cvt19p.Further research is required to characterize the transport step mediated by Yol082p, a step that appears to lay after the assembly of pAPI into dodecamers and before the wrapping of Cvt complexes by the saccular structures. In the yeast Saccharomyces cerevisiae the vacuolar hydrolase leucine aminopeptidase I (API)1 is synthesized in the cytoplasm as a precursor (pAPI) (1Klionsky D.J. Cueva R. Yaver D.S. J. Cell Biol. 1992; 119: 287-299Crossref PubMed Scopus (305) Google Scholar) and delivered to the vacuole by one of two alternative routes that operate under distinct physiological conditions: the cytoplasm to vacuole targeting (Cvt), in nutrient-rich conditions, and the autophagy (Apg) pathway, under starvation conditions (2Baba M. Osumi M. Scott S.V. Klionsky D.J. Ohsumi Y. J. Cell Biol. 1997; 139: 1687-1695Crossref PubMed Scopus (275) Google Scholar). The Cvt pathway is constitutive and biosynthetic, while autophagy is nonselective and degradative and is induced to survive periods of nutrient limitation (3Scott 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). However, the two pathways share many molecular components and both involve sequestration by double-membrane saccular structures of unknown origin that capture the load, close into vesicles, and then fuse with the vacuole (4Scott S.V. Baba M. Ohsumi Y. Klionsky D.J. J. Cell Biol. 1997; 138: 37-44Crossref PubMed Scopus (141) Google Scholar). A major difference between these pathways appears to be the size and content of the transport vesicles. The Cvt vesicles exclude cytoplasm and are smaller than autophagosomes that engulf bulk cytoplasm and even organelles (2Baba M. Osumi M. Scott S.V. Klionsky D.J. Ohsumi Y. J. Cell Biol. 1997; 139: 1687-1695Crossref PubMed Scopus (275) Google Scholar). Strikingly, despite all these differences, targeting of API to the vacuole is specific and saturable, both in vegetative growth conditions and under nitrogen deprivation (3Scott 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), although the molecular details of its selective recognition and capture remain essentially unknown. Previous studies have shown that pAPI recognition by the transport machinery involves its prepro-amino extension (5Seguı́-Real B. Martı́nez M. Sandoval I.V. EMBO J. 1995; 14: 5476-5484Crossref PubMed Scopus (31) Google Scholar, 6Oda M.N. Scott S.V. Hefner-Gravink A. Caffarelli A.D. Klionsky D.J. J. Cell Biol. 1996; 132: 999-1010Crossref PubMed Scopus (77) Google Scholar) and cytoplasmic chaperones of the Ssa family (7Silles E. Mazón M.J. Gevaert K. Goethals M. Vandekerckhove J. Leber R. Sandoval I.V. J. Biol. Chem. 2000; 275: 34054-34059Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar, 8Satyanarayana C. Schroder-Kohne S. Craig E.A. Schu P.V. Horst M. FEBS Lett. 2000; 470: 232-238Crossref PubMed Scopus (19) Google Scholar). Furthermore, the amino extension is necessary and sufficient to target the reporter protein GFP to the vacuole (9Martı́nez E. Seguı́-Real B. Silles E. Mazón M.J. Sandoval I.V. Mol. Microbiol. 1999; 33: 52-62Crossref PubMed Scopus (12) Google Scholar). In this study we report that Yol082p, a protein shown to interact physically with pAPI in a two-hybrid screening performed with the whole yeast genome (10Uetz P. Giot L. Cagney G. Mansfield T.A. Judson R.S. Knight J.R. Lockshon D. Narayan V. Srinivasan M. Pochart P. Qureshi-Emili A. Li Y. Godwin B. Conover D. Kalbfleisch T. Vijayadamodar G. Yang M. Johnston M. Fields S. Rothberg J.M. Nature. 2000; 403: 623-627Crossref PubMed Scopus (3896) Google Scholar), mediates API loading into transport vesicles and targeting to the vacuole. We also show that Yol082p interacts with API by a process that does not only involve the prepro-amino extension but also the mature part of the API protein. Yol082p is distributed between the cytoplasm and distinct round mobile structures. DISCUSSIONIn this study we have shown that Yol082p, identified in a whole-genome analysis of protein-protein interactions as an API-interacting protein (10Uetz P. Giot L. Cagney G. Mansfield T.A. Judson R.S. Knight J.R. Lockshon D. Narayan V. Srinivasan M. Pochart P. Qureshi-Emili A. Li Y. Godwin B. Conover D. Kalbfleisch T. Vijayadamodar G. Yang M. Johnston M. Fields S. Rothberg J.M. Nature. 2000; 403: 623-627Crossref PubMed Scopus (3896) Google Scholar), is required for vacuolar targeting and conversion of pAPI into mAPI, both in vegetative growth and under short term nitrogen starvation conditions.The interaction of Yol082p with pAPI and mAPI, but not with the prepro-amino extension of the precursor, is particularly interesting given the role of the latter in the transport of pAPI to the vacuole and the observation that it is necessary and sufficient for the transport of the reporter protein GFP from the cytoplasm to the vacuole (9Martı́nez E. Seguı́-Real B. Silles E. Mazón M.J. Sandoval I.V. Mol. Microbiol. 1999; 33: 52-62Crossref PubMed Scopus (12) Google Scholar). This observation suggests that transport of API to the vacuole requires additional transport determinants localized in the mature part, outside its amino extension. In addition, the stronger two-hybrid interaction of Yol082p with pAPI, as compared with mAPI, suggests that either Yol082p interacts physically with the amino extension in the context of the native protein or, alternatively, that the extension is required for proper folding and exposure of the transport determinants contained in the mature part of API to Yol082p. Clearly, further research is required to determine whether the determinants involved in its interaction with Yol082p are specific of API.Our studies on the processing of pAPI in wild-type and Δyol082 cells show that Yol082p is required for targeting and conversion of pAPI into mAPI in the vacuole, both under vegetative growth and short periods of nitrogen starvation. The rescue of the API processing defect in Δyol082 cells transformed with Yol082p expressed from an inducible promoter unambiguously shows the involvement of Yol082p in the vacuolar import of pAPI.The block in API vacuolar processing shown byΔyol082 cells incubated for short periods in SD(−N) medium is in contrast to the ability of mutants with impeded API transport, such as apg13, vac8,cvt3, aut2, and aut7, to overcome the pAPI accumulation soon after their shift to medium without nitrogen (2Baba M. Osumi M. Scott S.V. Klionsky D.J. Ohsumi Y. J. Cell Biol. 1997; 139: 1687-1695Crossref PubMed Scopus (275) Google Scholar,24Scott S.V. Nice 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, 25Abeliovich H. Dunn W.A.J. Kim J. Klionsky D.J. J. Cell Biol. 2000; 151: 1025-1034Crossref PubMed Scopus (234) Google Scholar). Furthermore, it is interesting that the inhibition of the pAPI processing in Δyol082 cells is partially reversed by the extension of the nitrogen starvation period. Under these conditions, mAPI is detected after 12 h in SD(−N) medium, and maturation proceeds slowly to reach a plateau at ∼30% in 16 h. These observations strongly suggest that Yol082p is involved in the rapid and specific capture of pAPI by the autophagosomes developed after a short starvation period, but not in the slow and unspecific capture that occurs with the engulfment of large portions of the cytoplasm after prolonged starvation.Apg mutants (26Tsukada M. Ohsumi Y. FEBS Lett. 1993; 333: 169-174Crossref PubMed Scopus (1376) Google Scholar) often show a correlation between the effect of the mutation in autophagosome biogenesis and the loss in viability under nitrogen starvation, so that mutants with defective autophagosome nucleation die earlier upon nitrogen deprivation (25Abeliovich H. Dunn W.A.J. Kim J. Klionsky D.J. J. Cell Biol. 2000; 151: 1025-1034Crossref PubMed Scopus (234) Google Scholar). In this context it is, therefore, interesting that although Yol082p appears to function in an early step in the pathway of API transport, theΔyol082 mutant is completely starvation-resistant. This resistance to starvation suggests again that Yol082p is not essential for autophagosome biogenesis.Regarding the API transport step in which Yol082p is involved, we have shown that pAPI assembly into dodecamers takes place normally in the mutant cells. We also show that in the absence of Yol082p, the interaction of the oligomerized pAPI with the sequestering double-membrane sacs appears to be affected. The proteinase K protection assay performed with metabolically labeled protein reveals that, in the mutant strain, the newly synthesized pAPI remains unprotected in the cytoplasm after 2 h of its synthesis, which is in contrast to the wild-type. This difference suggests that Yol082p may work in the recognition of pAPI by the wrapping membranes or, alternatively, by closing these into vesicles. The recovery of the protease-sensitive pAPI extracted from apg5,apg7, apg9, aut7/apg8, andcvt3 cells defective in biogenesis of the API transport vesicles (19Noda T. Kim J. Huang W.P. Baba M. Tokunaga C. Ohsumi Y. Klionsky D.J. J. Cell Biol. 2000; 148: 465-480Crossref PubMed Scopus (302) Google Scholar, 27George 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, 28Kim J. Dalton V.M. Eggerton K.P. Scott S.V. Klionsky D.J. Mol. Biol. Cell. 1999; 10: 1337-1351Crossref PubMed Scopus (177) Google Scholar, 29Huang W.P. Scott S.V. Kim J. Klionsky D.J. J. Biol. Chem. 2000; 275: 5845-5851Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar) with the membranes that float on Ficoll, and the exclusion of the bulk of the pAPI extracted fromΔyol082 cells from the membrane fraction, rather supports the first of the two above alternatives.Confocal fluorescence microscopy studies, performed using the fluorescent protein GFP-Yol082p, show the existence of a pool of protein homogeneously distributed throughout the cytoplasm in equilibrium with a second pool organized into one or more round-shaped structures, which we have called YR. Furthermore, the equilibrium is dramatically shifted by changes in the cell growing conditions, the ability of the cells to use the autophagy pathway, and the functional activity of GFP-Yol082p. Although we lack direct evidence on the meaning of the above changes in distribution, some of these observations suggest that the pool organized into YR structures could be actively engaged in the transport of pAPI to the vacuole by the Cvt pathway. With regard to this, the disappearance of the YR structures from cells incubated for 2 h in SD(−N) medium strongly suggests that they are not needed under conditions in which the autophagy pathway is activated. The difference between the rapid processing of pAPI in mutants with a defective Cvt pathway and the inability ofΔyol082 cells to process pAPI, when they were incubated in SD(−N) medium, strongly suggests that transport of pAPI by the autophagy pathway requires the disassembly of the YR structures. This may be related to the decrease in the cytoplasmic pool of GFP-Yol082p and the enhanced visibility of YR structures observed inΔapg12 cells incubated with SD or SD(−N) medium, changes that could reflect a blockage of the Cvt/Apg pathways after the transport step mediated by Yol082p. Also interesting is the disappearance of the cytoplasmic protein and the increase in size of YR structures observed in wild-type cells expressing the functionally inactive Yol082ΔC32p-GFP. This shift in equilibrium could again be consistent with our view that YR structures are involved in protein transport through the Cvt pathway, since the functionally inactive protein may cause the jam of the transport machinery and/or the accumulation of the transported material. A second alternative is the possibility that the deletion of the last 32 residues may shift the protein equilibrium toward the YR structures, which could also explain the insensitivity of these to the incubation of cells in SD(−N) medium. Obviously, these two possibilities are not mutually exclusive.Because of its behavior as a Cvt protein we propose to rename Yol082p as Cvt19p.Further research is required to characterize the transport step mediated by Yol082p, a step that appears to lay after the assembly of pAPI into dodecamers and before the wrapping of Cvt complexes by the saccular structures. In this study we have shown that Yol082p, identified in a whole-genome analysis of protein-protein interactions as an API-interacting protein (10Uetz P. Giot L. Cagney G. Mansfield T.A. Judson R.S. Knight J.R. Lockshon D. Narayan V. Srinivasan M. Pochart P. Qureshi-Emili A. Li Y. Godwin B. Conover D. Kalbfleisch T. Vijayadamodar G. Yang M. Johnston M. Fields S. Rothberg J.M. Nature. 2000; 403: 623-627Crossref PubMed Scopus (3896) Google Scholar), is required for vacuolar targeting and conversion of pAPI into mAPI, both in vegetative growth and under short term nitrogen starvation conditions. The interaction of Yol082p with pAPI and mAPI, but not with the prepro-amino extension of the precursor, is particularly interesting given the role of the latter in the transport of pAPI to the vacuole and the observation that it is necessary and sufficient for the transport of the reporter protein GFP from the cytoplasm to the vacuole (9Martı́nez E. Seguı́-Real B. Silles E. Mazón M.J. Sandoval I.V. Mol. Microbiol. 1999; 33: 52-62Crossref PubMed Scopus (12) Google Scholar). This observation suggests that transport of API to the vacuole requires additional transport determinants localized in the mature part, outside its amino extension. In addition, the stronger two-hybrid interaction of Yol082p with pAPI, as compared with mAPI, suggests that either Yol082p interacts physically with the amino extension in the context of the native protein or, alternatively, that the extension is required for proper folding and exposure of the transport determinants contained in the mature part of API to Yol082p. Clearly, further research is required to determine whether the determinants involved in its interaction with Yol082p are specific of API. Our studies on the processing of pAPI in wild-type and Δyol082 cells show that Yol082p is required for targeting and conversion of pAPI into mAPI in the vacuole, both under vegetative growth and short periods of nitrogen starvation. The rescue of the API processing defect in Δyol082 cells transformed with Yol082p expressed from an inducible promoter unambiguously shows the involvement of Yol082p in the vacuolar import of pAPI. The block in API vacuolar processing shown byΔyol082 cells incubated for short periods in SD(−N) medium is in contrast to the ability of mutants with impeded API transport, such as apg13, vac8,cvt3, aut2, and aut7, to overcome the pAPI accumulation soon after their shift to medium without nitrogen (2Baba M. Osumi M. Scott S.V. Klionsky D.J. Ohsumi Y. J. Cell Biol. 1997; 139: 1687-1695Crossref PubMed Scopus (275) Google Scholar,24Scott S.V. Nice 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, 25Abeliovich H. Dunn W.A.J. Kim J. Klionsky D.J. J. Cell Biol. 2000; 151: 1025-1034Crossref PubMed Scopus (234) Google Scholar). Furthermore, it is interesting that the inhibition of the pAPI processing in Δyol082 cells is partially reversed by the extension of the nitrogen starvation period. Under these conditions, mAPI is detected after 12 h in SD(−N) medium, and maturation proceeds slowly to reach a plateau at ∼30% in 16 h. These observations strongly suggest that Yol082p is involved in the rapid and specific capture of pAPI by the autophagosomes developed after a short starvation period, but not in the slow and unspecific capture that occurs with the engulfment of large portions of the cytoplasm after prolonged starvation. Apg mutants (26Tsukada M. Ohsumi Y. FEBS Lett. 1993; 333: 169-174Crossref PubMed Scopus (1376) Google Scholar) often show a correlation between the effect of the mutation in autophagosome biogenesis and the loss in viability under nitrogen starvation, so that mutants with defective autophagosome nucleation die earlier upon nitrogen deprivation (25Abeliovich H. Dunn W.A.J. Kim J. Klionsky D.J. J. Cell Biol. 2000; 151: 1025-1034Crossref PubMed Scopus (234) Google Scholar). In this context it is, therefore, interesting that although Yol082p appears to function in an early step in the pathway of API transport, theΔyol082 mutant is completely starvation-resistant. This resistance to starvation suggests again that Yol082p is not essential for autophagosome biogenesis. Regarding the API transport step in which Yol082p is involved, we have shown that pAPI assembly into dodecamers takes place normally in the mutant cells. We also show that in the absence of Yol082p, the interaction of the oligomerized pAPI with the sequestering double-membrane sacs appears to be affected. The proteinase K protection assay performed with metabolically labeled protein reveals that, in the mutant strain, the newly synthesized pAPI remains unprotected in the cytoplasm after 2 h of its synthesis, which is in contrast to the wild-type. This difference suggests that Yol082p may work in the recognition of pAPI by the wrapping membranes or, alternatively, by closing these into vesicles. The recovery of the protease-sensitive pAPI extracted from apg5,apg7, apg9, aut7/apg8, andcvt3 cells defective in biogenesis of the API transport vesicles (19Noda T. Kim J. Huang W.P. Baba M. Tokunaga C. Ohsumi Y. Klionsky D.J. J. Cell Biol. 2000; 148: 465-480Crossref PubMed Scopus (302) Google Scholar, 27George 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, 28Kim J. Dalton V.M. Eggerton K.P. Scott S.V. Klionsky D.J. Mol. Biol. Cell. 1999; 10: 1337-1351Crossref PubMed Scopus (177) Google Scholar, 29Huang W.P. Scott S.V. Kim J. Klionsky D.J. J. Biol. Chem. 2000; 275: 5845-5851Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar) with the membranes that float on Ficoll, and the exclusion of the bulk of the pAPI extracted fromΔyol082 cells from the membrane fraction, rather supports the first of the two above alternatives. Confocal fluorescence microscopy studies, performed using the fluorescent protein GFP-Yol082p, show the existence of a pool of protein homogeneously distributed throughout the cytoplasm in equilibrium with a second pool organized into one or more round-shaped structures, which we have called YR. Furthermore, the equilibrium is dramatically shifted by changes in the cell growing conditions, the ability of the cells to use the autophagy pathway, and the functional activity of GFP-Yol082p. Although we lack direct evidence on the meaning of the above changes in distribution, some of these observations suggest that the pool organized into YR structures could be actively engaged in the transport of pAPI to the vacuole by the Cvt pathway. With regard to this, the disappearance of the YR structures from cells incubated for 2 h in SD(−N) medium strongly suggests that they are not needed under conditions in which the autophagy pathway is activated. The difference between the rapid processing of pAPI in mutants with a defective Cvt pathway and the inability ofΔyol082 cells to process pAPI, when they were incubated in SD(−N) medium, strongly suggests that transport of pAPI by the autophagy pathway requires the disassembly of the YR structures. This may be related to the decrease in the cytoplasmic pool of GFP-Yol082p and the enhanced visibility of YR structures observed inΔapg12 cells incubated with SD or SD(−N) medium, changes that could reflect a blockage of the Cvt/Apg pathways after the transport step mediated by Yol082p. Also interesting is the disappearance of the cytoplasmic protein and the increase in size of YR structures observed in wild-type cells expressing the functionally inactive Yol082ΔC32p-GFP. This shift in equilibrium could again be consistent with our view that YR structures are involved in protein transport through the Cvt pathway, since the functionally inactive protein may cause the jam of the transport machinery and/or the accumulation of the transported material. A second alternative is the possibility that the deletion of the last 32 residues may shift the protein equilibrium toward the YR structures, which could also explain the insensitivity of these to the incubation of cells in SD(−N) medium. Obviously, these two possibilities are not mutually exclusive. Because of its behavior as a Cvt protein we propose to rename Yol082p as Cvt19p. Further research is required to characterize the transport step mediated by Yol082p, a step that appears to lay after the assembly of pAPI into dodecamers and before the wrapping of Cvt complexes by the saccular structures. We thank Dr. G. Högenauer for kindly supplying the anti-CPY antibody, Eulalia Morgado for construction of the plasmids containing wild-type and truncated forms ofYOL082 and measurement of β-galactosidase, and O. Zaragoza for helpful discussions. We also thank Carlos Sánchez, from the Servicio de Microscopı́a Óptica y Confocal, Centro de Biologı́a Molecular Severo Ochoa, for his help with the confocal images." @default.
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W2023758154 title "Yol082p, a Novel CVT Protein Involved in the Selective Targeting of Aminopeptidase I to the Yeast Vacuole" @default.
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W2023758154 cites W2133013060 @default.
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