FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2131799247> ?p ?o ?g. }

• W2131799247 endingPage "25849" @default.
• W2131799247 startingPage "25840" @default.
• W2131799247 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. 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. prevacuolar compartment alkaline phosphatase aminopeptidase I armadillo repeat Cytoplasm to vacuole targeting phosphoglycerate kinase proteinase A synthetic minimal medium lacking nitrogen synthetic minimal medium containing nitrogen low phosphate medium precursor aminopeptidase I kilobase pair(s) green fluorescent protein polymerase chain reaction 1,4-piperazinediethanesulfonic acid The majority of intracellular degradation is carried out in the lysosome/vacuole of eukaryotic cells. In order to compartmentalize these reactions, substrates as well as degradative enzymes must be faithfully delivered to this organelle. In Saccharomyces cerevisiae, proteins are known to be delivered to the vacuole for degradation by several different pathways (1Scott S.V. Klionsky D.J. Curr. Opin. Cell Biol. 1998; 10: 523-529Crossref PubMed Scopus (80) Google Scholar). For example, cell surface proteins are transported by endocytosis; the cytosolic enzyme fructose-1,6-bisphosphatase is targeted to the vacuole by the vacuolar import and degradation pathway when changing nutrient conditions necessitate down-regulation; and during periods of starvation, bulk cytosol is packaged into vesicles and delivered to the vacuole by macroautophagy. Vacuole resident proteins are delivered by four characterized pathways. In the carboxypeptidase Y pathway, proteins travel through the secretory pathway and are diverted from the late Golgi to the vacuole via an endosomal intermediate or prevacuolar compartment (PVC).1 The multivesicular body sorting pathway also utilizes the PVC, but in this case proteins enter vesicles resulting from invagination of the organelle's limiting membrane. The alkaline phosphatase (ALP) pathway utilizes the early secretory compartments, but the proteins are transported from the late Golgi to the vacuole without passing through the PVC. In the cytoplasm to vacuole targeting (Cvt) pathway, cargo is packaged into cytosolic vesicles that are delivered to the vacuole. Under vegetative conditions the yeast vacuolar hydrolase aminopeptidase I (API) is targeted to the vacuole by the Cvt pathway. API is synthesized as a precursor in the cytosol where it rapidly oligomerizes into dodecamers (2Kim J. Scott S.V. Oda M.N. Klionsky D.J. J. Cell Biol. 1997; 137: 609-618Crossref PubMed Scopus (117) Google Scholar). Multiple API dodecamers then assemble into a membrane-bound complex called a Cvt complex (3Baba M. Osumi M. Scott S.V. Klionsky D.J. Ohsumi Y. J. Cell Biol. 1997; 139: 1687-1695Crossref PubMed Scopus (280) Google Scholar). Double membrane structures surround the Cvt complex resulting in the formation of Cvt vesicles. The outer membrane of these vesicles fuses with the vacuole resulting in the release of a still intact vesicle, called a Cvt body, into the vacuole lumen. These structures are broken down by resident hydrolases, and the released precursor API (prAPI) is processed to its mature size. Yeast mutants have been isolated that are defective in the Cvt pathway (4Harding T.M. Morano K.A. Scott S.V. Klionsky D.J. J. Cell Biol. 1995; 131: 591-602Crossref PubMed Scopus (401) Google Scholar, 5Scott 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 (216) Google Scholar). Analysis of these mutants revealed that many are allelic to mutants in macroautophagy (apg and aut; Refs.5Scott 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 (216) Google Scholar, 6Harding T.M. Hefner-Gravink A. Thumm M. Klionsky D.J. J. Biol. Chem. 1996; 271: 17621-17624Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar, 7Thumm M. Egner R. Koch M. Schlumpberger M. Straub M. Veenhuis M. Wolf D.H. FEBS Lett. 1994; 349: 275-280Crossref PubMed Scopus (483) Google Scholar, 8Tsukada M. Ohsumi Y. FEBS Lett. 1993; 333: 169-174Crossref PubMed Scopus (1427) Google Scholar). This was a surprising finding because the transport of prAPI is selective and constitutive, while delivery of soluble cytosolic proteins to the vacuole by macroautophagy is nonselective and is induced by stress conditions such as starvation (5Scott 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 (216) Google Scholar). However, it is worth noting that under certain conditions macroautophagy may be a selective process. For example, peroxisomes can be selectively degraded by a macroautophagic process in response to changing nutrient conditions (reviewed in Ref. 9Klionsky D.J. Ohsumi Y. Annu. Rev. Cell. Dev. Biol. 1999; 15: 1-32Crossref PubMed Scopus (391) Google Scholar). Biochemical and morphological studies confirm that the basic properties of targeting by the Cvt pathway are similar to those of macroautophagy (3Baba M. Osumi M. Scott S.V. Klionsky D.J. Ohsumi Y. J. Cell Biol. 1997; 139: 1687-1695Crossref PubMed Scopus (280) Google Scholar, 10Scott S.V. Baba M. Ohsumi Y. Klionsky D.J. J. Cell Biol. 1997; 138: 37-44Crossref PubMed Scopus (142) Google Scholar). Both pathways involve formation of double membrane vesicles that engulf cytosolic cargo and deliver it to the vacuole by fusion of the outer membrane with the vacuole surface. When cells are growing in rich media, the primary mode of prAPI delivery is via Cvt vesicles (3Baba M. Osumi M. Scott S.V. Klionsky D.J. Ohsumi Y. J. Cell Biol. 1997; 139: 1687-1695Crossref PubMed Scopus (280) Google Scholar). These vesicles are approximately 150 nm in diameter, relatively rare, and contain concentrated cargo that appears different than bulk cytosol. In contrast, in starvation conditions, the majority of prAPI appears to be targeted to the vacuole by autophagosomes. Compared with Cvt vesicles, autophagosomes are larger (approximately 300–900 nm), more abundant, and appear to contain bulk cytosolic components in addition to Cvt complexes. The mechanisms by which environmental changes are transduced to signal alterations in vesicle morphology and selectivity are not known. Recent cloning of the genes that complement mutants defective in the autophagy and Cvt pathways indicates that the majority of the molecular components involved in these pathways are shared (reviewed in Ref. 9Klionsky D.J. Ohsumi Y. Annu. Rev. Cell. Dev. Biol. 1999; 15: 1-32Crossref PubMed Scopus (391) Google Scholar). However, several mutants have been identified that appear to be defective primarily in one or the other of these pathways. These include aut4 (6Harding T.M. Hefner-Gravink A. Thumm M. Klionsky D.J. J. Biol. Chem. 1996; 271: 17621-17624Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar),apg17, 2Y. Kamada, T. Funakoshi, T. Shintani, K. Nagano, M. Ohsumi, and Y. Ohsumi, submitted for publication.,cvt3 (5Scott 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 (216) Google Scholar), cvt9, 3J. Kim, J. Guan, Y. Kamada, A. Hefner-Gravink, Y. Ohsumi, and D. J. Klionsky, manuscript in preparation. tlg2(11Abeliovich H. Darsow T. Emr S.D. EMBO J. 1999; 18: 6005-6016Crossref PubMed Scopus (106) Google Scholar), and vac8 (12Wang Y.X. Catlett N.L. Weisman L.S. J. Cell Biol. 1998; 140: 1063-1074Crossref PubMed Scopus (145) Google Scholar). The vac8 mutant was identified through a screen for strains defective in vacuole inheritance (13Wang Y.X. Zhao H. Harding T.M. Gomes de Mesquita D.S. Woldringh C.L. Klionsky D.J. Munn A.L. Weisman L.S. Mol. Biol. Cell. 1996; 7: 1375-1389Crossref PubMed Scopus (75) Google Scholar). In addition to a role in the migration of vacuoles from the mother to the bud during cell division, Vac8p is required for Cvt transport. Analysis of vac8mutants by a vesicle test that monitors the accumulation of subvacuolar vesicles under starvation conditions, however, provided preliminary evidence that this protein is not necessary for autophagy (12Wang Y.X. Catlett N.L. Weisman L.S. J. Cell Biol. 1998; 140: 1063-1074Crossref PubMed Scopus (145) Google Scholar). The majority of Vac8p consists of 11 armadillo repeats. These domains are contained within the Drosophila armadillo protein and the mammalian homologues β-catenin and plakoglobin. The armadillo domains in β-catenin and plakoglobin serve to link regions of the plasma membrane to actin, and Vac8p co-sediments with actin filaments in vitro (12Wang Y.X. Catlett N.L. Weisman L.S. J. Cell Biol. 1998; 140: 1063-1074Crossref PubMed Scopus (145) Google Scholar). Localization experiments indicate that Vac8p is found primarily on the vacuole membrane (12Wang Y.X. Catlett N.L. Weisman L.S. J. Cell Biol. 1998; 140: 1063-1074Crossref PubMed Scopus (145) Google Scholar, 14Fleckenstein D. Rohde M. Klionsky D.J. Rüdiger M. J. Cell Sci. 1998; 111: 3109-3118Crossref PubMed Google Scholar, 15Pan X. Goldfarb D.S. J. Cell Sci. 1998; 111: 2137-2147Crossref PubMed Google Scholar). In addition, Vac8p is both myristoylated and palmitoylated, and mutational analysis indicates that acylation is required both for vacuole localization of Vac8p and for vacuole inheritance. Interestingly, vac8 mutants defective in acylation are not defective in Cvt transport, suggesting that different cellular pools of Vac8p are utilized for inheritance and Cvt transport (12Wang Y.X. Catlett N.L. Weisman L.S. J. Cell Biol. 1998; 140: 1063-1074Crossref PubMed Scopus (145) Google Scholar). To identify proteins that interact with Vac8p, we have performed a two-hybrid screen using this protein as bait. We identified Apg13p as a Vac8p-interacting protein. Apg13p is predicted to be an 83-kDa protein, with no significant homology to other known proteins (16Funakoshi T. Matsuura A. Noda T. Ohsumi Y. Gene (Amst.). 1997; 192: 207-213Crossref PubMed Scopus (140) Google Scholar). Overexpression of APG1 suppresses the autophagy defect in the apg13–1 mutant, suggesting that these two proteins also interact (16Funakoshi T. Matsuura A. Noda T. Ohsumi Y. Gene (Amst.). 1997; 192: 207-213Crossref PubMed Scopus (140) Google Scholar). Mutants in APG13 are defective in both the Cvt and autophagy pathways (5Scott 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 (216) Google Scholar, 16Funakoshi T. Matsuura A. Noda T. Ohsumi Y. Gene (Amst.). 1997; 192: 207-213Crossref PubMed Scopus (140) Google Scholar). We propose that these proteins are part of a large protein complex that functions in the vesicle formation step that is required for sequestration of cargo during import from the cytoplasm to the vacuole. The S. cerevisiae strains used in this study are listed in Table I.Table IYeast strains used in this studyStrainGenotypeSource or referenceSEY6210MATα leu2–3,112 ura3–52 his3-Δ200 trp1-Δ901 lys2–801 suc2-Δ9 GAL(41Robinson J.S. Klionsky D.J. Banta L.M. Emr S.D. Mol. Cell. Biol. 1988; 8: 4936-4948Crossref PubMed Scopus (739) Google Scholar)BJ2168MATa prb1–1122 prc1–407 pep4–3 leu2 trp1 ura3–52 gal2(42Jones E.W. Methods Enzymol. 1991; 194: 428-453Crossref PubMed Scopus (367) Google Scholar)LWY7235MATa leu2–3,112 ura3–52 his3-Δ200 trp1-Δ901 lys2–801 suc2-Δ9(12Wang Y.X. Catlett N.L. Weisman L.S. J. Cell Biol. 1998; 140: 1063-1074Crossref PubMed Scopus (145) Google Scholar)TN121MATa leu2–3,112 trp1 ura3–52 pho8∷pho8Δ60 pho13∷URA3(36Noda T. Matsuura A. Wada Y. Ohsumi Y. Biochem. Biophys. Res. Commun. 1995; 210: 126-132Crossref PubMed Scopus (295) Google Scholar)TN124MATa leu2–3,112 trp1 ura3–52 pho8::pho8Δ60 pho13::LEU2(36Noda T. Matsuura A. Wada Y. Ohsumi Y. Biochem. Biophys. Res. Commun. 1995; 210: 126-132Crossref PubMed Scopus (295) Google Scholar)NNY20MATa ura3 trp1 leu2 apg1Δ∷LEU2(28Matsuura A. Tsukada M. Wada Y. Ohsumi Y. Gene (Amst.). 1997; 192: 245-250Crossref PubMed Scopus (390) Google Scholar)TVY1MATα leu2–3,112 ura3–52 his3-Δ200 trp1-Δ901 lys2–801 suc2-Δ9 GAL pep4Δ∷LEU2(43Gerhardt B. Kordas T.J. Thompson C.M. Patel P. Vida T. J. Biol. Chem. 1998; 273: 15818-15829Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar)AHY001SEY6210 cvt9Δ∷HIS3This studyD3Y101LWY7235 apg13Δ∷LEU2This studyD3Y102SEY6210 vac8Δ∷TRP1This studyD3Y103TN124 apg13Δ∷URA3This studyD3Y104TN121 vac8Δ∷TRP1This studyD3Y105D3Y101 pep4Δ∷URA3This studyD3Y106TVY1 vac8Δ∷TRP1This study Open table in a new tab Yeast strains were grown in the indicated media: synthetic minimal medium (SMD; 0.67% yeast nitrogen base, 2% glucose, and auxotrophic amino acids and vitamins as needed), YPD (1% yeast extract, 2% peptone, 2% glucose), synthetic minimal medium containing 2% glucose without ammonium sulfate or amino acids (SD-N), and low phosphate medium (NPM; 2% glucose, 0.5% casamino acids, 1× complete amino acid mix, 1% proline, 2 mm NaCl, 4 mmMgCl2). Restriction endonucleases and DNA modifying enzymes were from New England Biolabs (Beverly, MA). Proteinase K and complete EDTA-free protease inhibitor mixture were from Roche Molecular Biochemicals. Antiserum to proteinase A (PrA), ALP, and Vac8p were described previously (12Wang Y.X. Catlett N.L. Weisman L.S. J. Cell Biol. 1998; 140: 1063-1074Crossref PubMed Scopus (145) Google Scholar, 17Klionsky D.J. Banta L.M. Emr S.D. Mol. Cell. Biol. 1988; 8: 2105-2116Crossref PubMed Scopus (190) Google Scholar, 18Klionsky D.J. Emr S.D. EMBO J. 1989; 8: 2241-2250Crossref PubMed Scopus (235) Google Scholar). Antiserum to Apg13p and Cvt9p were generated as described.2,3 Antiserum to phosphoglycerate kinase (PGK) was generously provided by Dr. Jeremy Thorner (19Baum P. Thorner J. Honig L. Proc. Natl. Acad. Sci. U. S. A. 1978; 75: 4962-4966Crossref PubMed Scopus (50) Google Scholar). Monoclonal antibodies to the hemagglutinin epitope were from Covance Research Products (Richmond, CA). Horseradish peroxidase-conjugated anti-rabbit goat antibody was from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA). ECL reagents were from Amersham Pharmacia Biotech. EXPRE35S35S protein-labeling mix and [33P]orthophosphate were from NEN Life Science Products. Oxalyticase was from Enzogenetics (Corvallis, OR) and Zymolyase 100T from Seikagaku Kogyo (Tokyo, Japan). Immobilon-P (polyvinylidene fluoride) was from Millipore Corp. (Bedford, MA). Plasmid pTrcHisB was from Invitrogen (Carlsbad, CA). Antiserum to Apg1p and additional antiserum to Apg13p were generated as follows: Synthetic peptides corresponding to amino acids 105–123 and 643–664 of Apg1p and amino acids 613–633 and 715–726 of Apg13p were conjugated to keyhole limpet hemocyanin (Multiple Peptide Systems, San Diego, CA) and injected into New Zealand White rabbits. All other reagents were from Sigma. The plasmid pCuCvt9 expressing the CVT9 gene under control of the regulableCUP1 promoter will be described elsewhere.3Plasmid pYK128 contains the APG1 gene with an N-terminal 3xHA epitope tag cloned into the vector pRS423, pYW10 containsVAC8 cloned into pRS426 (12Wang Y.X. Catlett N.L. Weisman L.S. J. Cell Biol. 1998; 140: 1063-1074Crossref PubMed Scopus (145) Google Scholar) and YEp351(APG13) contains theAPG13 gene cloned into the YEp351 plasmid.2 The following plasmids encode VAC8 bearing deletions in the indicated armadillo repeat domains: pLT4, ΔArm1; pLT5, ΔArm2; pLT6, ΔArm3; pYWT1, ΔArm4–7 4F. Tang, J. Nau, E. Kauffman, and L. Weisman, manuscript in preparation.; and pYW51, ΔArm1–3 (12Wang Y.X. Catlett N.L. Weisman L.S. J. Cell Biol. 1998; 140: 1063-1074Crossref PubMed Scopus (145) Google Scholar). APG13 was amplified from strain SEY6210 genomic DNA into two overlapping fragments by two PCR reactions. For reaction one, the sense primer was 5′-CGCGTCAGAGCAAGAGGTGAAAGGGTTGCCACGTA-3′ and the antisense primer was 5′-GTGGCCCGTAGTTGGAGCTCATACTTGGCC-3′. For reaction two, the sense primer was 5′-GTCTATCCAATATCGAGACCTGTTCAACCA-3′ and the antisense primer was 5′-GGAAACGCAGTCAGCGGGTGACAAATAAGC-3′. Utilizing the overlapping fragments as a template, the APG13 coding region was PCR-amplified with the primers 5′-GTTGAATAGCTGCAGTCCTGGTTGCCGAAG-3′, which adds a PstI site, and 5′-TAAAATAAAAGCTTACCATTTTTA-3′, which adds aHindIII site. The amplified fragment was inserted into thePstI and HindIII sites of pTrcHisB to generate pTrcHisBAPG13. TheVAC8coding region was PCR-amplified with the primers V22 (5′-CATGCCATGGTGGGTTCATGTTGTAGTTGC-3′), which adds aNcoI site and V23 (5′-ACGCGTCGACTCAATGTAAAAATTGTAAAATCTGTTG-3′), which adds aSalI site. NcoI-SalI-digestedVAC8was cloned intoNcoI-SalI-digested pAS2 (20Harper J.W. Adami G.R. Wei N. Keyomarsi K. Elledge S.J. Cell. 1993; 75: 805-816Abstract Full Text PDF PubMed Scopus (5250) Google Scholar) to create theVAC8two-hybrid bait plasmid pYW31. The same strategy was used to clone the mutated vac8 gene from pLT4, pLT5, pLT6, pYWT1, and pYW51 into the two-hybrid vector. The bait plasmid pYW31 was transformed into the yeast strain PJ69-4A (21James P. Halladay J. Craig E.A. Genetics. 1996; 144: 1425-1436Crossref PubMed Google Scholar) and checked for activation of the reporter genes. The resulting strain PJ69-4A + pYW31 was transformed with the two-hybrid yeast library pool Y2HL-C1 (21James P. Halladay J. Craig E.A. Genetics. 1996; 144: 1425-1436Crossref PubMed Google Scholar). Approximately 280,000 transformants were screened. The transformants were plated onto SC-His-Leu-Trp + 3 mm3-amino-1,2,4-triazole and incubated at 24 °C for 14 days. These plates were replica plated onto SC-Ade-Leu-Trp and incubated at 24 °C for 5 days. Colonies were picked and tested for β-galactosidase activity. Library plasmids were isolated from the yeast strain and retransformed into PJ69-4A + pYW31. Plasmids that showed positive responses after retransformation were sequenced and identified by comparison with the Saccharomyces Genome Data base. Two identical clones of APG13 that coded for amino acids 567–738 were isolated. Plasmid pTrcHisBAPG13 was digested with PstI and HindIII. The 2.2-kb APG13 fragment was cloned intoPstI-HindIII-cut pBlueScript SK to create pJN21. The following plasmids were generated by digesting pJN21 with the indicated enzymes, isolating DNA of the size noted, and ligating into pGAD-C (1Scott S.V. Klionsky D.J. Curr. Opin. Cell Biol. 1998; 10: 523-529Crossref PubMed Scopus (80) Google Scholar, 2Kim J. Scott S.V. Oda M.N. Klionsky D.J. J. Cell Biol. 1997; 137: 609-618Crossref PubMed Scopus (117) Google Scholar, 3Baba M. Osumi M. Scott S.V. Klionsky D.J. Ohsumi Y. J. Cell Biol. 1997; 139: 1687-1695Crossref PubMed Scopus (280) Google Scholar) that had been digested with the indicated enzymes to construct prey plasmids encoding the indicated number of amino acids of Apg13p: pJN22 (pADAPG13), SalI and partially digested withBamHI (2.2 kb), into BamHI-SalI sites of C2, full-length Apg13p; pJN23 (pAD1–520APG13), BamHI (1.6 kb), into BamHI sites of C2, amino acids 1–520; pJN24 (pAD1–279APG13), BamHI-NsiI (0.8 kb), intoBamHI-PstI sites of C2, amino acids 1–279; pJN25 (pAD280–520APG13), NsiI-BamHI (0.8 kb), intoPstI-BglII sites of C2, amino acids 280–520; pJN26 (pAD280–714APG13), NsiI (1.3 kb), intoPstI sites of C2, amino acids 280–714; pJN27 (pAD521–738APG13), BamHI-SalI (0.7 kb), intoBamHI-SalI sites of C3, amino acids 521–738; pJN28 (pAD521–692APG13), BamHI-ClaI (0.5 kb), into BamHI-ClaI sites of C3, amino acids 521–692; pJN29 (pAD693–738APG13), ClaI-SalI (0.2 kb), into ClaI-SalI sites of C1, amino acids 692–738. Control plasmids for the two-hybrid analysis were pSE1111 containingSNF4 fused to the GAL4 activation domain in pACT and pSE1112 containing SNF1 fused to the GAL4binding domain in pAS1 (22Bai C. Elledge S. Methods Enzymol. 1997; 283: 141-156Crossref PubMed Scopus (76) Google Scholar). Yeast strains were grown to anA 600 = 0.8–1, and converted to spheroplasts as described (23Scott S.V. Klionsky D.J. J. Cell Biol. 1995; 131: 1727-1735Crossref PubMed Scopus (30) Google Scholar). vac8Δ cells were grown in SMD, andapg13Δ cells were grown in YPD. Spheroplasts were washed in PS1000 (20 mm K-PIPES, pH 6.8, 1 msorbitol), and subjected to osmotic lysis (23Scott S.V. Klionsky D.J. J. Cell Biol. 1995; 131: 1727-1735Crossref PubMed Scopus (30) Google Scholar) by pipetting up and down 10 times in PS200 with MgCl2 (20 mm K-PIPES, pH 6.8, 200 mm sorbitol, 5 mm MgCl2) at a cell concentration of 20 A 600/ml. The supernatant and pellet fractions were collected after centrifugation at 5000 × g for 5 min. A flotation analysis was carried out as described previously (23Scott S.V. Klionsky D.J. J. Cell Biol. 1995; 131: 1727-1735Crossref PubMed Scopus (30) Google Scholar). The pellet fraction was resuspended in 100 μl of 15% Ficoll, and then overlaid with 1 ml of 13% Ficoll and 300 μl of 2% Ficoll. The resulting step gradients were subjected to centrifugation in a microcentrifuge at 12,000 × gfor 10 min. The float fraction was collected from the 2% Ficoll/13% Ficoll interface. Proteinase K treatment was with 50 μg of proteinase K for 15 min on ice with a concentration of osmotically lysed spheroplasts equivalent to 20 A 600/ml. All fractions were precipitated with trichloroacetic acid, resolved by SDS-polyacrylamide gel electrophoresis, and subjected to immunoblotting as described (4Harding T.M. Morano K.A. Scott S.V. Klionsky D.J. J. Cell Biol. 1995; 131: 591-602Crossref PubMed Scopus (401) Google Scholar). Fractionation of Apg13p and Apg1p was as above except that the isolated spheroplasts were washed with PS1000 with complete EDTA-free protease inhibitor mixture before lysis and then lysed in PS200 with 5 mm MgCl2 and protease inhibitors. The pellet fraction was collected by centrifugation at 13,000 × gfor 5 min at 4 °C. Pulse labeling experiments were as described (5Scott 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 (216) Google Scholar). Yeast strains were grown in SMD medium to anA 600 = 0.8–1, pelleted, and then pulse-labeled at 30 °C in fresh SMD with 11 μCi/A 600Expre35S35S label. Chase reactions were initiated by addition of 10 μm cysteine and 20 μm methionine. Cells were then pelleted and resuspended at a concentration of 1 A 600/ml in either SMD or SD-N for the duration of the chase reaction. API was immunoprecipitated as described (24Klionsky D.J. Cueva R. Yaver D.S. J. Cell Biol. 1992; 119: 287-299Crossref PubMed Scopus (307) Google Scholar). Indicated yeast strains were grown in YPD to an A 600 = 1, harvested, washed, incubated in SD-N for 4 h, and fixed with 1.5% KMnO4 as described (25Erdmann R. Veenhuis M. Mertens D. Kunau W.H. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 5419-5423Crossref PubMed Scopus (264) Google Scholar). Cells were grown to anA 600 of 0.5–1 in NPM. 4A 600 units of cells were harvested and labeled for 30 min at 30 °C in 100 μl of fresh NPM with 25 μCi of [33P]orthophosphate. Cells were recovered by centrifugation, and proteins were precipitated with 10% trichloroacetic acid and subjected to immunoprecipitation as described previously (24Klionsky D.J. Cueva R. Yaver D.S. J. Cell Biol. 1992; 119: 287-299Crossref PubMed Scopus (307) Google Scholar), except that single precipitation reactions were performed. BJ2168 cells harboring pYW10 and YEp351(APG13) were grown in 100 ml of YPD, collected by centrifugation, washed with water, and resuspended in 50 ml of Z buffer (50 mm Tris-HCl pH7.5, 1 m sorbitol, 1% yeast extract, 2% peptone, 1% glucose) containing 0.5 mg/ml Zymolyase 100T. The suspension was incubated for 40 min at 30 °C with gentle shaking to generate spheroplasts. The resultant spheroplasts were washed once with Z buffer, resuspended in 50 ml of Z buffer, and divided into two 25-ml aliquots. Rapamycin (0.4 μg/ml, prepared in 90% ethanol, 10% Triton X-100) was added to one aliquot, and the spheroplasts were incubated for 1 h at 30 °C with gentle shaking. The spheroplasts were collected and washed with 50 mm Tris-HCl, pH 7.5, 1 m sorbitol, and lysed by suspending in 1 ml of lysis buffer (phosphate-buffered saline, pH 7.4, 1 mm EDTA, 1 mm EGTA, 2 mm Na3VO4, 50 mm KF, 15 mm sodium pyrophosphate, 15 mm p-nitrophenylphosphate, 20 μg/ml leupeptin, 20 μg/ml benzamidine, 10 μg/ml pepstatin A, 40 μg/ml aprotinin, 1 mm phenylmethylsulfonyl fluoride, and 0.5% Tween 20). The cell lysate was further incubated on ice for 5 min, and spun down at 6,500 rpm for 10 min at 4 °C to remove cell debris. The resultant supernatant was collected and incubated with 20 μl of protein G-Sepharose (50% suspension) for 30 min at 4 °C with gentle agitation. The cell lysate was spun down, and the supernatant (two 500-μl aliquots) was incubated with or without 2 μl of anti-Vac8p serum for 1.5 h at 4 °C with gentle agitation. 20 μl of protein G-Sepharose (50% suspension) was added to the lysate/antiserum mixture, followed by further incubation for 1.5 h at 4 °C with gentle agitation. The immune complex was washed three times with lysis buffer, and the resulting immunocomplex was subjected to immunoblotting using anti-Apg13p antibody (1:5,000 dilution) or anti-Vac8p antibody (1:5,000 dilution). For secondary antibody, horseradish peroxidase-conjugated anti-rabbit goat antibody (1:10,000) was used. Immunodetection was carried out with the ECL system. The vac8 mutant was identified in a screen for strains defective in vacuolar inheritance (13Wang Y.X. Zhao H. Harding T.M. Gomes de Mesquita D.S. Woldringh C.L. Klionsky D.J. Munn A.L. Weisman L.S. Mol. Biol. Cell. 1996; 7: 1375-1389Crossref PubMed Scopus (75) Google Scholar). Vac8p is unusual among those proteins involved in organelle segregation in that it was also found to be required for the import of the resident vacuolar hydrolase aminopeptidase I through the cytoplasm to vacuole targeting pathway. To gain further information on the role of Vac8p in the Cvt pathway, a two-hybrid screen was used to identify proteins that interact with Vac8p. The VAC8 gene was cloned into a two-hybrid vector containing a DNA-binding domain. A his3 ade2 strain was transformed with the resulting pBD-VAC8 plasmid and a plasmid library that had been cloned into the transcriptional activating domain vector. Transformants were screened for growth on His-free plates, and then growth on Ade-free plates and finally for β-galactosidase activity. Plasmids were recovered from cells that were positive by all three criteria and were subjected to DNA sequencing. One of the genes that was identified from the screen was found to contain part of the open reading frame corresponding toAPG13 (Fig. 1 A). The APG13 gene was identified as being required for macroautophagy (16Funakoshi T. Matsuura A. Noda T. Ohsumi Y. Gene (Amst.). 1997; 192: 207-213Crossref PubMed Scopus (140) Google Scholar) and was subsequently shown to be needed for import of prAPI (5Scott 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 (216) Google Scholar). To determine the domain of Apg13p that interacts with Vac8p, a series of deletions was constructed in the APG13 gene that was used as the prey plasmid for the two-hybrid intera" @default.
• W2131799247 created "2016-06-24" @default.
• W2131799247 creator A5000851275 @default.
• W2131799247 creator A5010689898 @default.
• W2131799247 creator A5034310619 @default.
• W2131799247 creator A5041858353 @default.
• W2131799247 creator A5056765809 @default.
• W2131799247 creator A5064819609 @default.
• W2131799247 creator A5065223412 @default.
• W2131799247 creator A5066515392 @default.
• W2131799247 creator A5081220025 @default.
• W2131799247 creator A5088939968 @default.
• W2131799247 date "2000-08-01" @default.
• W2131799247 modified "2023-10-11" @default.
• W2131799247 title "Apg13p and Vac8p Are Part of a Complex of Phosphoproteins That Are Required for Cytoplasm to Vacuole Targeting" @default.
• W2131799247 cites W1589290041 @default.
• W2131799247 cites W1631725776 @default.
• W2131799247 cites W1668735973 @default.
• W2131799247 cites W1750072934 @default.
• W2131799247 cites W1903792504 @default.
• W2131799247 cites W1973130575 @default.
• W2131799247 cites W1985482115 @default.
• W2131799247 cites W1991313744 @default.
• W2131799247 cites W1991369070 @default.
• W2131799247 cites W2011580247 @default.
• W2131799247 cites W2016204905 @default.
• W2131799247 cites W2020710502 @default.
• W2131799247 cites W2022456133 @default.
• W2131799247 cites W2026244176 @default.
• W2131799247 cites W2036999913 @default.
• W2131799247 cites W2045200845 @default.
• W2131799247 cites W2045856345 @default.
• W2131799247 cites W2057764483 @default.
• W2131799247 cites W2059520792 @default.
• W2131799247 cites W2065304353 @default.
• W2131799247 cites W2072544389 @default.
• W2131799247 cites W2078294331 @default.
• W2131799247 cites W2089218510 @default.
• W2131799247 cites W2093521208 @default.
• W2131799247 cites W2095263179 @default.
• W2131799247 cites W2101011430 @default.
• W2131799247 cites W2105137727 @default.
• W2131799247 cites W2109402440 @default.
• W2131799247 cites W2111362374 @default.
• W2131799247 cites W2111792196 @default.
• W2131799247 cites W2126801593 @default.
• W2131799247 cites W2129109987 @default.
• W2131799247 cites W2134348354 @default.
• W2131799247 cites W2137690550 @default.
• W2131799247 cites W2163508965 @default.
• W2131799247 cites W2165191318 @default.
• W2131799247 cites W2168427881 @default.
• W2131799247 cites W2268036223 @default.
• W2131799247 cites W2413057282 @default.
• W2131799247 cites W2416689305 @default.
• W2131799247 cites W4211037514 @default.
• W2131799247 doi "https://doi.org/10.1074/jbc.m002813200" @default.
• W2131799247 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/10837477" @default.
• W2131799247 hasPublicationYear "2000" @default.
• W2131799247 type Work @default.
• W2131799247 sameAs 2131799247 @default.
• W2131799247 citedByCount "207" @default.
• W2131799247 countsByYear W21317992472012 @default.
• W2131799247 countsByYear W21317992472013 @default.
• W2131799247 countsByYear W21317992472014 @default.
• W2131799247 countsByYear W21317992472015 @default.
• W2131799247 countsByYear W21317992472016 @default.
• W2131799247 countsByYear W21317992472017 @default.
• W2131799247 countsByYear W21317992472018 @default.
• W2131799247 countsByYear W21317992472019 @default.
• W2131799247 countsByYear W21317992472020 @default.
• W2131799247 countsByYear W21317992472021 @default.
• W2131799247 countsByYear W21317992472022 @default.
• W2131799247 countsByYear W21317992472023 @default.
• W2131799247 crossrefType "journal-article" @default.
• W2131799247 hasAuthorship W2131799247A5000851275 @default.
• W2131799247 hasAuthorship W2131799247A5010689898 @default.
• W2131799247 hasAuthorship W2131799247A5034310619 @default.
• W2131799247 hasAuthorship W2131799247A5041858353 @default.
• W2131799247 hasAuthorship W2131799247A5056765809 @default.
• W2131799247 hasAuthorship W2131799247A5064819609 @default.
• W2131799247 hasAuthorship W2131799247A5065223412 @default.
• W2131799247 hasAuthorship W2131799247A5066515392 @default.
• W2131799247 hasAuthorship W2131799247A5081220025 @default.
• W2131799247 hasAuthorship W2131799247A5088939968 @default.
• W2131799247 hasBestOaLocation W21317992471 @default.
• W2131799247 hasConcept C102568950 @default.
• W2131799247 hasConcept C185592680 @default.
• W2131799247 hasConcept C190062978 @default.
• W2131799247 hasConcept C55493867 @default.
• W2131799247 hasConcept C70721500 @default.
• W2131799247 hasConcept C86803240 @default.
• W2131799247 hasConcept C95444343 @default.
• W2131799247 hasConceptScore W2131799247C102568950 @default.
• W2131799247 hasConceptScore W2131799247C185592680 @default.
• W2131799247 hasConceptScore W2131799247C190062978 @default.
• W2131799247 hasConceptScore W2131799247C55493867 @default.
• W2131799247 hasConceptScore W2131799247C70721500 @default.

• next