The RAVE Complex Is Essential for Stable Assembly of the Yeast V-ATPase
2002; Elsevier BV; Volume: 277; Issue: 16 Linguagem: Inglês
10.1074/jbc.m200682200
ISSN1083-351X
AutoresAnne M. Smardon, Maureen Tarsio, Patricia M. Kane,
Tópico(s)Photosynthetic Processes and Mechanisms
ResumoVacuolar proton-translocating ATPases are composed of a peripheral complex, V1, attached to an integral membrane complex, Vo. Association of the two complexes is essential for ATP-driven proton transport and is regulated post-translationally in response to glucose concentration. A new complex, RAVE, was recently isolated and implicated in glucose-dependent reassembly of V-ATPase complexes that had disassembled in response to glucose deprivation (Seol, J. H., Shevchenko, A., and Deshaies, R. J. (2001) Nat. Cell Biol. 3, 384–391). Here, we provide evidence supporting a role for RAVE in reassembly of the V-ATPase but also demonstrate an essential role in V-ATPase assembly under other conditions. The RAVE complex associates reversibly with V1 complexes released from the membrane by glucose deprivation but binds constitutively to cytosolic V1 sectors in a mutant lacking Vosectors. V-ATPase complexes from cells lacking RAVE subunits show serious structural and functional defects even in glucose-grown cells or in combination with a mutation that blocks disassembly of the V-ATPase. RAVE·V1 interactions are specifically disrupted in cells lacking V1 subunits E or G, suggesting a direct involvement for these subunits in interaction of the two complexes. Skp1p, a RAVE subunit involved in many different signal transduction pathways, binds stably to other RAVE subunits under conditions that alter RAVE·V1 binding; thus, Skp1p recruitment to the RAVE complex does not appear to provide a signal for V-ATPase assembly. Vacuolar proton-translocating ATPases are composed of a peripheral complex, V1, attached to an integral membrane complex, Vo. Association of the two complexes is essential for ATP-driven proton transport and is regulated post-translationally in response to glucose concentration. A new complex, RAVE, was recently isolated and implicated in glucose-dependent reassembly of V-ATPase complexes that had disassembled in response to glucose deprivation (Seol, J. H., Shevchenko, A., and Deshaies, R. J. (2001) Nat. Cell Biol. 3, 384–391). Here, we provide evidence supporting a role for RAVE in reassembly of the V-ATPase but also demonstrate an essential role in V-ATPase assembly under other conditions. The RAVE complex associates reversibly with V1 complexes released from the membrane by glucose deprivation but binds constitutively to cytosolic V1 sectors in a mutant lacking Vosectors. V-ATPase complexes from cells lacking RAVE subunits show serious structural and functional defects even in glucose-grown cells or in combination with a mutation that blocks disassembly of the V-ATPase. RAVE·V1 interactions are specifically disrupted in cells lacking V1 subunits E or G, suggesting a direct involvement for these subunits in interaction of the two complexes. Skp1p, a RAVE subunit involved in many different signal transduction pathways, binds stably to other RAVE subunits under conditions that alter RAVE·V1 binding; thus, Skp1p recruitment to the RAVE complex does not appear to provide a signal for V-ATPase assembly. Vacuolar proton-translocating ATPases (V-ATPases) 1The abbreviations used are: V-ATPasevacuolar proton-translocating ATPaseRAVEregulator of the ATPase of vacuolar and endosomal membranesYEPDyeast extract-peptone-2% dextrose mediumSDfully supplemented minimal mediumSCFSkp1-cullin-F-boxE2ubiquitin conjugating enzymeE3ubiquitin-protein isopeptide ligaseMes4-morpholineethanesulfonic acidMOPS4-morpholinepropanesulfonic acidare highly conserved proton pumps responsible for acidification of organelles such as the lysosome/vacuole, Golgi apparatus, and endosomes in all eukaryotic cells (1.Forgac M. J. Bioenerg. Biomembr. 1999; 31: 57-65Crossref PubMed Scopus (38) Google Scholar, 2.Nelson N. Harvey W.R. Physiol. Rev. 1999; 79: 361-385Crossref PubMed Scopus (371) Google Scholar, 3.Stevens T.H. Forgac M. Annu. Rev. Cell Dev. Biol. 1997; 13: 779-808Crossref PubMed Scopus (523) Google Scholar). In some cells, VATPases are also present at high levels at the plasma membrane, where they pump protons from the cytosol out of the cell (2.Nelson N. Harvey W.R. Physiol. Rev. 1999; 79: 361-385Crossref PubMed Scopus (371) Google Scholar, 4.Wieczorek H. Brown D. Grinstein S. Ehrenfeld J. Harvey W.R. Bioessays. 1999; 21: 637-648Crossref PubMed Scopus (226) Google Scholar). In all of these locations and in organisms ranging from yeast to humans, V-ATPases have a very similar structure and subunit composition. They are comprised of 13 or 14 subunits arranged as a complex of cytosolic peripheral membrane subunits containing the sites of ATP hydrolysis, the V1sector, attached to a membrane complex containing the proton pore, the Vo sector. ATP-driven proton transport occurs only when the two sectors are structurally and functionally coupled. Free V1 sectors do not catalyze hydrolysis of MgATP, the physiological substrate, and free Vo sectors do not appear to form open proton pores (5.Graf R. Harvey W.R. Wieczorek H. J. Biol. Chem. 1996; 271: 20908-20913Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 6.Parra K.J. Keenan K.L. Kane P.M. J. Biol. Chem. 2000; 275: 21761-21767Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 7.Zhang J. Feng Y. Forgac M. J. Biol. Chem. 1994; 269: 23518-23523Abstract Full Text PDF PubMed Google Scholar). vacuolar proton-translocating ATPase regulator of the ATPase of vacuolar and endosomal membranes yeast extract-peptone-2% dextrose medium fully supplemented minimal medium Skp1-cullin-F-box ubiquitin conjugating enzyme ubiquitin-protein isopeptide ligase 4-morpholineethanesulfonic acid 4-morpholinepropanesulfonic acid The assembly pathways for V-ATPases are complicated and incompletely understood. Both mammalian cells and yeast contain free V1and Vo sectors in vivo (8.Myers M. Forgac M. J. Cell. Physiol. 1993; 156: 35-42Crossref PubMed Scopus (48) Google Scholar, 9.Peng S.B. Li X. Crider B.P. Zhou Z. Andersen P. Tsai S.J. Xie X.S. Stone D.K. J. Biol. Chem. 1999; 274: 2549-2555Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 10.Doherty R.D. Kane P.M. J. Biol. Chem. 1993; 268: 16845-16851Abstract Full Text PDF PubMed Google Scholar), and yeast mutants lacking one subunit of either sector are able to assemble the other sector (10.Doherty R.D. Kane P.M. J. Biol. Chem. 1993; 268: 16845-16851Abstract Full Text PDF PubMed Google Scholar). Yet there is evidence that the major pathway for biosynthetic assembly of V-ATPases does not involve independent assembly of free V1 and Vo sectors followed by attachment of the two sectors. Instead, pulse-chase studies indicate very early association of V1 and Vo sector subunits followed by addition of subunits from both sectors (11.Kane P.M. Tarsio M. Liu J. J. Biol. Chem. 1999; 274: 17275-17283Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Definition of assembly pathways was further complicated by the observation that fully assembled V-ATPases could rapidly and reversibly disassemble into free V1 and Vo sectors (12.Kane P.M. J. Biol. Chem. 1995; 270: 17025-17032Abstract Full Text Full Text PDF PubMed Google Scholar,13.Sumner J.P. Dow J.A. Earley F.G. Klein U. Jager D. Wieczorek H. J. Biol. Chem. 1995; 270: 5649-5653Abstract Full Text Full Text PDF PubMed Scopus (287) Google Scholar). Disassembly of V-ATPases in yeast and insects, the two systems in which the process is best characterized, occurs in response to low extracellular glucose concentrations. Reversible disassembly is believed to be an important regulatory mechanism; disassembly of V-ATPases conserves ATP under conditions of nutrient limitation by silencing the ATPase activity of the enzyme and reassembly rapidly reactivates the pump with no need for new protein synthesis (14.Wieczorek H. Grber G. Harvey W.R. Huss M. Merzendorfer H. Zeiske W. J. Exp. Biol. 2000; 203: 127-135Crossref PubMed Google Scholar,15.Kane P.M. Parra K.J. J. Exp. Biol. 2000; 203: 81-87Crossref PubMed Google Scholar). The signaling pathways connecting V-ATPase assembly with extracellular glucose concentration have proven to be somewhat elusive. Many of the pathways that signal glucose availability in yeast cells do not appear to be involved in modulating the assembly state of the V-ATPase (16.Parra K.J. Kane P.M. Mol. Cell. Biol. 1998; 18: 7064-7074Crossref PubMed Google Scholar). Recently, a new player in V-ATPase assembly was identified. Seolet al. (17.Seol J.H. Shevchenko A. Deshaies R.J. Nat. Cell Biol. 2001; 3: 384-391Crossref PubMed Scopus (210) Google Scholar) identified a Skp1p-containing complex they called RAVE, regulator of the ATPase of vacuolar and endosomal membranes, through affinity chromatography to detect Skp1p binding partners, and subsequently showed that RAVE also bound at least four of the V1 subunits of the yeast V-ATPase. The RAVE complex contains three members, Rav1p, Rav2p, and Skp1p. RAV1 andRAV2 were previously uncharacterized yeast open reading frames, but RAV1 has homologues in all eukaryotes. Deletion of the RAV1 and RAV2 genes resulted in a temperature-dependent Vma− phenotype, characterized by sensitivity to elevated pH and poor growth on glycerol-containing medium. The V-ATPase also showed assembly defects in the rav mutants, and the V1 subunits that were assembled on the vacuolar membrane in a rav1Δ mutant disassembled rapidly in response to glucose deprivation but reassembled much more slowly upon glucose re-addition than in wild-type cells. These results implicated the RAVE complex in glucose-triggered reassembly of the V-ATPase, but it was not clear whether this aspect of RAVE function accounted for the V-ATPase assembly defects of rav1Δ mutants observed even in the presence of glucose. Skp1p is a very highly conserved protein primarily known as a component of SCF (Skp1-cullin-F-box) ubiquitin ligases, which are involved in signal-induced protein degradation (18.Deshaies R.J. Annu. Rev. Cell Dev. Biol. 1999; 15: 435-467Crossref PubMed Scopus (1083) Google Scholar, 19.Patton E.E. Willems A.R. Tyers M. Trends Genet. 1998; 14: 236-243Abstract Full Text Full Text PDF PubMed Scopus (446) Google Scholar, 20.Willems A.R. Goh T. Taylor L. Chernushevich I. Shevchenko A. Tyers M. Philos. Trans. R Soc. Lond. B Biol. Sci. 1999; 354: 1533-1550Crossref PubMed Scopus (107) Google Scholar). SCF complexes regulate a wide range of fundamental cell processes ranging from cell cycle progression to nutrient utilization to transcription. Skp1p mediates this wide range of functions by binding a variety of proteins with little structural or sequence similarity beyond an F-box motif, a degenerate, 40-amino acid sequence motif originally identified by sequence alignment of several Skp1p-binding proteins and subsequently shown to be directly involved in binding to Skp1p (19.Patton E.E. Willems A.R. Tyers M. Trends Genet. 1998; 14: 236-243Abstract Full Text Full Text PDF PubMed Scopus (446) Google Scholar, 21.Bai C. Sen P. Hofmann K. Ma L. Goebl M. Harper J.W. Elledge S.J. Cell. 1996; 86: 263-274Abstract Full Text Full Text PDF PubMed Scopus (991) Google Scholar). In SCF complexes Skp1p is seen primarily as an adaptor capable of bridging F-box proteins with specificity for phosphorylated substrates to be degraded and E2/E3 ubiquitin ligase components bound to the cullin (19.Patton E.E. Willems A.R. Tyers M. Trends Genet. 1998; 14: 236-243Abstract Full Text Full Text PDF PubMed Scopus (446) Google Scholar,21.Bai C. Sen P. Hofmann K. Ma L. Goebl M. Harper J.W. Elledge S.J. Cell. 1996; 86: 263-274Abstract Full Text Full Text PDF PubMed Scopus (991) Google Scholar). The RAVE complex diverges from the SCF model in several important ways. Although Seol et al. (17.Seol J.H. Shevchenko A. Deshaies R.J. Nat. Cell Biol. 2001; 3: 384-391Crossref PubMed Scopus (210) Google Scholar) determined that Rav1p binds to Skp1p, RAV1 contains no identifiable F-box sequence or resemblance to the cullin proteins. More importantly, the RAVE complex does not contain the yeast cullin, Cdc53p (17.Seol J.H. Shevchenko A. Deshaies R.J. Nat. Cell Biol. 2001; 3: 384-391Crossref PubMed Scopus (210) Google Scholar), and there is no apparent role for protein degradation in disassembly or reassembly of the V-ATPase (12.Kane P.M. J. Biol. Chem. 1995; 270: 17025-17032Abstract Full Text Full Text PDF PubMed Google Scholar). Taken together, these data indicate that RAVE is a non-SCF Skp1p-containing complex. The role of Skp1p in the RAVE complex is mysterious, but the presence of Skp1p invokes rich possibilities for cross-talk between V-ATPase assembly and the wide variety of signaling pathways in yeast that ultimately act through other Skp1-containing complexes. In this work we have taken a closer look at the role of RAVE in both biosynthetic assembly and glucose-dependent disassembly and reassembly of the V-ATPase in yeast. Our results support those of Seolet al. (17.Seol J.H. Shevchenko A. Deshaies R.J. Nat. Cell Biol. 2001; 3: 384-391Crossref PubMed Scopus (210) Google Scholar) implicating RAVE in glucose-induced reassembly of the V-ATPase but also indicate that the RAVE complex must play a more general role in V-ATPase assembly. We provide evidence that the RAVE complex appears to bind V1 whenever it is in the cytosol and that RAVE is required for stable assembly even in a mutant V-ATPase incapable of disassembly in response to glucose deprivation. We also find that the association of Skp1p with the RAVE complex does not change with changes in carbon source or under conditions where the RAVE complex cannot bind to V1. These results suggest that Skp1p is a stable component of the RAVE complex and that the RAVE complex is critical for stable assembly of the V-ATPase under many different conditions. Oligonucleotides were purchased from MWG Biotech. LA-Taq polymerase was from Panvera and native Pfu DNA polymerase was from Stratagene. Other molecular biology reagents were purchased from New England BioLabs. Tran35S-label and zymolyase 100T were purchased from ICN. Concanamycin A was from Wako Biochemicals. Monoclonal antibody 9E10 against the myc epitope was obtained from Roche Molecular Biotechnology or Zymed Laboratories Inc., and alkaline phosphatase second antibodies were from Promega. Yeast and Escherichia coli media were from Difco or Fisher. All other reagents were purchased from Sigma Chemical Co. Yeast cells were grown in yeast extract-peptone-2% dextrose (YEPD) medium or fully supplemented minimal medium (SD) lacking individual nutrients as described previously (22.Sherman F. Fink G.R. Hicks J.B. Methods in Yeast Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1982Google Scholar). For growth of vma mutant strains, YEPD was buffered to pH 5.0 with 50 mm sodium phosphate/50 mm sodium succinate (YEPD, pH 5). For analysis of the Vma− growth phenotype, YEPD was buffered to pH 7.5 with 50 mm Mes, 50 mm MOPS buffer, and 60 mm CaCl2 was added (YEPD, pH 7.5, + Ca2+). The strains used in this work are listed in Table I. Therav1Δ::LEU2 andrav2Δ::URA3 alleles were constructed by fusion PCR. Sequences immediately upstream and downstream of theRAV1 and RAV2 open reading frames were amplified using oligonucleotides YJR1-7 and YJR2-8 to amplify the 5′-end ofRAV1, oligonucleotides YJR5-11 and YJR6-12 to amplify the 3′-end of RAV1, oligonucleotides YDR1-1 and YDR2-2 to amplify the 5′-end of RAV2, and oligonucleotides YDR5-5 and YDR6-6 to amplify the 3′-end of RAV2. Oligonucleotide sequences are shown in Table II. TheURA3 and LEU2 genes were amplified using oligonucleotides YDR3-3 and YDR4-4 with pRS316 as a template and YJR3-9 and YJR4-10 with pRS315 as a template, respectively (23.Sikorski R.S. Hieter P. Genetics. 1989; 122: 19-27Crossref PubMed Google Scholar). The deletion alleles were constructed by combining the RAV2 5′ and 3′-ends and URA3 fragment into a single fusion PCR reaction containing oligonucleotides YDR1-1 and YDR6-6. The RAV1 5′- and 3′-ends and LEU2 fragment were combined in a fusion PCR reaction containing oligonucleotides YJR1-7 and YJR6-12. The PCR reactions employed a mixture of LA-Taq and native Pfuthermostable DNA polymerases in LA-Taq buffer provided by the manufacturer. The products of the fusion PCR reactions were gel-purified and used to transform wild-type strain SF838-5A in a one-step gene replacement (24.Rothstein R.J. Methods Enzymol. 1983; 101: 202-211Crossref PubMed Scopus (2033) Google Scholar). Replacement of the wild-type with the mutant alleles was confirmed by PCR. To create therav1Δrav2Δ double mutant, therav2Δ::URA2 fusion product was transformed into the rav1Δ strain, and transformants were selected on SD-uracil.Table IGenotypes of strains used in this studyStrainGenotypeSourceSF838–5AMATαura3–52 leu2–3,112 his4–519 ade6(54.Stevens T.H. Rothman J.H. Payne G.S. Schekman T.H. J. Cell Biol. 1986; 102: 1551-1557Crossref PubMed Scopus (148) Google Scholar)SF838–5A rav1ΔMATα ura3–52 leu2–3,112 his4–519 ade6 rav1Δ::LEU2This studySF838–5A rav2ΔMATα ura3–52 leu2–3,112 his4–519 ade6 rav2Δ::URA3This studySF838–5A rav1Δ rav2ΔMATαura3–52 leu2–3,112 his4–519 ade6 rav1Δ::LEU2 rav2Δ::URA3This studySF838–5Arav1Δ vma11E145LMATα ura3–52 leu2–3,112 his4–519 ade6 rav1Δ::LEU2kanMX6-vma11E145LRDY1512RAV1-Myc9MATαade2–1 trp1–1 can1–100 leu2–3,112 his3–11,15 ura3–1 ssd1 pep4::TRP1 bar1::LEU2 RAV1-Myc9-HIS5(17.Seol J.H. Shevchenko A. Deshaies R.J. Nat. Cell Biol. 2001; 3: 384-391Crossref PubMed Scopus (210) Google Scholar)RDY1513RAV2-Myc9MATa ade2–1 trp1–1 can1–100 leu2–3,112 his3–11,15 ura3–1 ssd1 pep4::TRP1 bar1::LEU2 RAV2-Myc9-HIS5(17.Seol J.H. Shevchenko A. Deshaies R.J. Nat. Cell Biol. 2001; 3: 384-391Crossref PubMed Scopus (210) Google Scholar)RDY1512RAV1-Myc9 vma1ΔMATa ade2–1 trp1–1 can1–100 leu2–3,112 his3–11,15 ura3–1 ssd1 pep4::TRP1 bar1::LEU2 RAV1-Myc9-HIS5 vma1Δ::URA3This studyRDY1512RAV1-Myc9 vma2ΔMATa ade2–1 trp1–1 can1–100 leu2–3,112 his3–11,15 ura3–1 ssd1 pep4::TRP1 bar1::LEU2 RAV1-Myc9-HIS5 vma2Δ::URA3This studyRDY1512RAV1-Myc9 vma4ΔMATa ade2–1 trp1–1 can1–100 leu2–3,112 his3–11,15 ura3–1 ssd1 pep4::TRP1 bar1::LEU2 RAV1-Myc9-HIS5 vma4Δ::URA3This studyRDY1512RAV1-Myc9 9 vma7ΔMATa ade2–1 trp1–1 can1–100 leu2–3,112 his3–11,15 ura3–1 ssd1 pep4::TRP1 bar1 LEU2 RAV1-Myc9-HIS5 vma7Δ::URA3This studyRDY1512RAV1-Myc9 vma10ΔMATa ade2–1 trp1–1 can1–100 leu2–3,112 his3–11,15 ura3–1 ssd1 bar1::LEU2 RAV1-Myc9-HIS5 vma10Δ::URA3This studyRDY1512RAV1-Myc9 vma3ΔMATa ade2–1 trp1–1 can1–100 leu2–3,112 his3–11,15 ura3–1 ssd1 bar1::LEU2 RAV1-Myc9-HIS5 vma3Δ::URA3This studyRDY1513RAV2-Myc9 vma3ΔMATa ade2–1 trp1–1 can1–100 leu2–3,112 his3–11,15 ura3–1 ssd1 pep4::TRP1 bar1::LEU2 RAV2-Myc9-HIS5 vma3Δ::URAThis studySF838–5A RAV1-Myc13Matα ura3–52 leu2–3,112 his4–519 ade6 RAV1-Myc13-kanMX6This studySF838–5A RAV1-Myc13 vma5ΔMatα ura3–52 leu2–3,112 his4–519 ade6 RAV1-Myc13-kanMX6 vma5Δ::LEU2This studySF838–5ARAV1-Myc13 vma8ΔMatα ura3–52 leu2–3,112 his4–519 ade6 RAV1-Myc13-kanMX6 vma8Δ::LEU2This studySF838–5ARAV1-Myc13 vma13ΔMatα ura3–52 leu2–3,112 his4–519 ade6 RAV1-Myc13-kanMX6 vma13Δ::LEU2This studySF838–5ARAV1-Myc13 rav2ΔMatα ura3–52 leu2–3,112 his4–519 ade6 RAV1-Myc13-kanMX6 rav2Δ::LEU2This study Open table in a new tab Table IIOligonucleotides used in this studyOligonucleotide nameOligonucleotide sequence 5′ → 3′YJR1–7TGTTGATCGTACTTGCCAGTCYJR2–8GTCGATATGCGGTGTGAAACTCAATTGTTTGCGGTGTTCYJR3–9GAACACCGCAAACAATTGAGTTTCACACCGCATATCGACYJR4–10TTTCACCCACAGAGTCGTTTCGGCATCAGAGCAGATTGTAYJR5–11TACAATCTGCTCTGATGCCGAAACGACTCTGTGGGTGAAAYJR6–12CGTTCCTAACAGAGATCAAGCYDR1–1GTTGTACCGAGGTTCCATTGYDR2–2GCTGCATGTGTCAGAGGTTTCCACTGTGTGCTTGATCAGAYDR3–3TCTGATCAAGCACACAGTGGAAACCTCTGACACATGCAGCYDR4–4GTTCGCCATCGAAGCTTTGTATTTCACACCGCAGGGTAAYDR5–5TTACCCTGCGGTGTGAAATACAAAGCTTCGATGGCGAACYDR6–6CCGTTTAGTTCCCGAATATGLONGTINE F2CGGATCCCCGGGTTAATTAALONGTINE R1GAATTCGAGCTCGTTTAAACRAV1M2TCACCGTTAATTAACCCGGGGATCCGTACAAAGTCATCTAGTAAGTTCTTGGTAATAGCRAV1M3CATCCAGTTTAAACGAGCTCGAATTCCAGCCTTACAAGTAACTAATTAGGAG Open table in a new tab The strain containing the integrated vma11-E145L allele was constructed by first cloning the SmaI-EcoRV fragment containing kanMX6 gene from plasmid pFA6A-KanMX6 into the SnaBI site 445 bp upstream from the start codon of a plasmid-borne copy of the vma11-E145L allele in pRHA176 (25.Longtine M.S. McKenzie 3rd, A. Demarini D.J. Shah N.G. Wach A. Brachat A. Philippsen P. Pringle J.R. Yeast. 1998; 14: 953-961Crossref PubMed Scopus (4193) Google Scholar, 26.Hirata R. Graham L.A. Takatsuki A. Stevens T.H. Anraku Y. J. Biol. Chem. 1997; 272: 4795-4803Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar). The 3.3-kb kanMX-containing vma11-E145L was excised from the plasmid with SacII and KpnI and used to transform the SF838-5A wild-type strain. Transformants were selected on YEPD medium containing 200 μg/ml G418, and integration of the vma11-E145L allele at the VMA11 locus was confirmed by sequencing. Therav1Δ::LEU2 mutation was then introduced into this strain as described above. To combine a myc-tagged Rav1p with the various vma deletion alleles, two different strategies were used. The vma mutant strains containing myc9-tagged RAV1 (Table I) were obtained by amplifying vmaΔ::URA3 alleles from genomic DNA of existing vma1Δ (27.Shih C.K. Wagner R. Feinstein S. Kanik-Ennulat C. Neff N. Mol. Cell. Biol. 1988; 8: 3094-3103Crossref PubMed Scopus (52) Google Scholar), vma2Δ (28.Yamashiro C.T. Kane P.M. Wolczyk D.F. Preston R.A. Stevens T.H. Mol. Cell. Biol. 1990; 10: 3737-3749Crossref PubMed Scopus (146) Google Scholar), vma3Δ (29.Kane P.M. Kuehn M.C. Howald-Stevenson I. Stevens T.H. J. Biol. Chem. 1992; 267: 447-454Abstract Full Text PDF PubMed Google Scholar), vma4Δ (30.Foury F. J. Biol. Chem. 1990; 265: 18554-18560Abstract Full Text PDF PubMed Google Scholar), andvma7Δ and vma10Δ (11.Kane P.M. Tarsio M. Liu J. J. Biol. Chem. 1999; 274: 17275-17283Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar) strains by PCR. In each case, oligonucleotides recognizing sequences 200–500 bp 5′ and 3′ of the URA3 insertion into each gene were used, and the PCR fragment containing the disrupted allele was gel-purified. Wild-type strain RDY1512 (17.Seol J.H. Shevchenko A. Deshaies R.J. Nat. Cell Biol. 2001; 3: 384-391Crossref PubMed Scopus (210) Google Scholar) was then transformed with the mutant alleles, and transformants were selected on SD-uracil medium. Correct integration was confirmed by either PCR or immunoblot. The vma mutant strains containing myc13-tagged RAV1 (Table I) were obtained by introducing a RAV1-myc13-kanMX allele, which placed the myc13 epitope immediately before the stop codon of RAV1, into existing vma deletion strains. Themyc13-kanMX cassette was amplified from plasmid pFA6a-myc13-kanMX (25.Longtine M.S. McKenzie 3rd, A. Demarini D.J. Shah N.G. Wach A. Brachat A. Philippsen P. Pringle J.R. Yeast. 1998; 14: 953-961Crossref PubMed Scopus (4193) Google Scholar), and RAV1fragments upstream and downstream of the stop codon were amplified with oligonucleotides YJR1-7 and RAV1M2 and RAV1M3 and YJR6-12, respectively. The final myc13-tagged RAV1 was generated by fusion PCR as described previously (31.Wach A. Yeast. 1996; 12: 259-265Crossref PubMed Scopus (705) Google Scholar). Previously characterized vma5Δ (32.Ho M.N. Hill K.J. Lindorfer M.A. Stevens T.H. J. Biol. Chem. 1993; 268: 221-227Abstract Full Text PDF PubMed Google Scholar), vma13Δ (33.Ho M.N. Hirata R. Umemoto N. Ohya Y. Takatsuki A. Stevens T.H. Anraku Y. J. Biol. Chem. 1993; 268: 18286-18292Abstract Full Text PDF PubMed Google Scholar), andvma8Δ (11.Kane P.M. Tarsio M. Liu J. J. Biol. Chem. 1999; 274: 17275-17283Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar) strains were transformed with the fusion PCR fragment. Transformants were selected on YEPD, pH 5, medium containing 200 μg/ml G418 and confirmed by PCR and/or immunoblotting. Immunoprecipitation of V-ATPase complexes from biosynthetically labeled cells was performed as described previously (12.Kane P.M. J. Biol. Chem. 1995; 270: 17025-17032Abstract Full Text Full Text PDF PubMed Google Scholar). For immunoprecipitation of cytosolic RAVE·V1 complexes, cytosolic fractions were obtained from the indicated strains (150 A600 of each) as described previously (6.Parra K.J. Keenan K.L. Kane P.M. J. Biol. Chem. 2000; 275: 21761-21767Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). Protein from 10% of the cytosolic fraction was directly precipitated with 10% trichloroacetic acid, and the remaining 90% was combined with 100 μl of anti-V1monoclonal antibody (8B1 or 13D11 (29.Kane P.M. Kuehn M.C. Howald-Stevenson I. Stevens T.H. J. Biol. Chem. 1992; 267: 447-454Abstract Full Text PDF PubMed Google Scholar)) or 5 μg of anti-myc antibody (9E10, Roche Molecular Biochemicals) followed by 60 μl of a 50% (v/v) suspension of Protein A-Sepharose CL4B. The immunoprecipitated proteins were solubilized at 75 °C in cracking buffer (50 mm Tris-HCl, pH 6.8, 8 m urea, 5% SDS, 5% β-mercaptoethanol) for analysis by SDS-PAGE and immunoblotting. For comparison of immunoprecipitations from the variousvmaΔ strains (Fig. 6), cells (100A600 of each strain) were lysed by agitation with glass beads as described (34.Seol J.H. Feldman R.M. Zachariae W. Shevchenko A. Correll C.C. Lyapina S. Chi Y. Galova M. Claypool J. Sandmeyer S. Nasmyth K. Deshaies R.J. Genes Dev. 1999; 13: 1614-1626Crossref PubMed Scopus (360) Google Scholar) and cytosol was obtained by centrifugation for 30 min at 100,000 × g in a Beckman TLA-100 Ultracentrifuge. Protein concentration in the cytosolic fractions from the various strains was measured by Lowry assay (35.Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar); 0.4 mg of protein was directly trichloroacetic acid-precipitated, and 4 mg was immunoprecipitated and analyzed by immunoblotting as described below. Vacuolar vesicles were isolated as described (36.Roberts C.J. Raymond C.K. Yamashiro C.T. Stevens T.H. Methods Enzymol. 1991; 194: 644-661Crossref PubMed Scopus (287) Google Scholar). ATP hydrolysis activity was determined at 37 °C using a coupled enzyme assay (37.Lotscher H.R. deJong C. Capaldi R.A. Biochemistry. 1984; 23: 4140-4143Crossref PubMed Scopus (99) Google Scholar), in the presence and absence of 100 nm concanamycin A. For immunoblotting, vacuolar vesicles were pelleted by centrifugation and solubilized in cracking buffer. For all immunoblots, samples were separated by SDS-PAGE then transferred to nitrocellulose. Blots were probed with mouse monoclonal antibodies 8B1, 13D11, 7A2, and 10D7 against V1 subunits A, B, and C, and Vo subunit a, respectively. Myc-tagged RAVE subunits were detected with mouse monoclonal antibody 9E10. Rabbit polyclonal antisera against yeast Skp1 was a generous gift from Ray Deshaies, California Institute of Technology. Primary antibodies were bound by alkaline phosphatase-conjugated goat anti-mouse or goat anti-rabbit secondary antibody and detected by colorimetric assay as described (38.Kane P.M. Stevens T.H. J. Bioenerg. Biomembr. 1992; 24: 383-393Crossref PubMed Scopus (56) Google Scholar). Seol et al. (17.Seol J.H. Shevchenko A. Deshaies R.J. Nat. Cell Biol. 2001; 3: 384-391Crossref PubMed Scopus (210) Google Scholar) reported that the reduced levels of V1 sectors that were present at the vacuolar membrane in a rav1Δ mutant in vivodissociated rapidly from the membrane upon glucose deprivation but were slow to re-associate upon glucose re-addition. This result implicated the RAVE complex in the glucose-dependent reassembly of the V-ATPase. To probe this aspect of RAVE activity further, we examined the effects of extracellular glucose on the RAVE·V1interaction by using a monoclonal antibody against the V1 B subunit to immunoprecipitate free V1 complexes from cytosol along with bound RAVE subunits. Immunoblots of the cytosolic fractions probed with antibody against another V1 subunit, subunit A, or against Rav1p and Rav2p proteins tagged with a myc9epitope are shown in Fig. 1A. These immunoblots demonstrate that there are detectable levels of V1 subunits in the cytosol even in the presence of glucose (+), but the level of V1 subunits in the cytosol increases markedly when the cells are deprived of glucose for 15 min (−) and then falls to the original level when glucose is restored to the cells (−/+). (Although this immunoblot was probed only with the anti-A subunit antibody, previous results indicate that all of the V1 subunits except subunit C are present in the cytosolic V1 complexes (6.Parra K.J. Keenan K.L. Kane P.M. J. Biol. Chem. 2000; 275: 21761-21767Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 12.Kane P.M. J. Biol. Chem. 1995; 270: 17025-17032Abstract Full Text Full Text PDF PubMed Google Scholar).) In contrast, the levels of Rav1p and Rav2p in the cytosol remain the same regardless of the level of extracellular glucose. In the right panels of Fig. 1A, complexes immunoprecipitated under non-denaturing conditions with an anti-B subunit antibody were probed for the presence of V1 subunit A, Rav1p, and Rav2p on immunoblots. The levels of co-precipitated A subunit reflect the levels present in the cytosolic fractions, as expected; they increase in the absence of glucose and decrease in the presence of glucose. It is striking, however, that the levels of coprecipitated Rav1p and Rav2p also rise and fall with the changes in glucose concentration. This indicates that a part of the population of cytosolic RAVE complexes is recruited to interact with V1 sectors released from the membrane by glucose deprivation and that these RAVE complexes are able to rapidly release the bound V1 when extracellular glucose is restored. Disassembly and reassembly of the yeast V-ATPase has been shown to be entirely post-tran
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