The Glycolytic Enzyme Aldolase Mediates Assembly, Expression, and Activity of Vacuolar H+-ATPase
2004; Elsevier BV; Volume: 279; Issue: 10 Linguagem: Inglês
10.1074/jbc.m303871200
ISSN1083-351X
AutoresMing Lu, Yuri Y. Sautin, L. Shannon Holliday, Stephen L. Gluck,
Tópico(s)Biochemical and Molecular Research
ResumoVacuolar H+-ATPases (V-ATPases) are a family of highly conserved proton pumps that couple hydrolysis of cytosolic ATP to proton transport out of the cytosol. How ATP is supplied for V-ATPase-mediated hydrolysis and for coupling of proton transport is poorly understood. We have reported that the glycolytic enzyme aldolase physically associates with V-ATPase (Lu, M., Holliday, L. S., Zhang, L., Dunn, W. A., and Gluck, S. L. (2001) J. Biol. Chem. 276, 30407–30413). Here we show that aldolase interacts with three different subunits of V-ATPase (subunits a, B, and E). The binding sites for the V-ATPase subunits on aldolase appear to be on distinct interfaces of the glycolytic enzyme. Aldolase deletion mutant cells were able to grow in medium buffered at pH 5.5 but not at pH 7.5, displaying a growth phenotype similar to that observed in V-ATPase subunit deletion mutants. Abnormalities in V-ATPase assembly and protein expression observed in aldolase deletion mutant cells could be fully rescued by aldolase complementation. The interaction between aldolase and V-ATPase increased dramatically in the presence of glucose, suggesting that aldolase may act as a glucose sensor for V-ATPase regulation. Taken together, these findings provide functional evidence that the ATP-generating glycolytic pathway is directly coupled to the ATP-hydrolyzing proton pump through physical interaction between aldolase and V-ATPase. Vacuolar H+-ATPases (V-ATPases) are a family of highly conserved proton pumps that couple hydrolysis of cytosolic ATP to proton transport out of the cytosol. How ATP is supplied for V-ATPase-mediated hydrolysis and for coupling of proton transport is poorly understood. We have reported that the glycolytic enzyme aldolase physically associates with V-ATPase (Lu, M., Holliday, L. S., Zhang, L., Dunn, W. A., and Gluck, S. L. (2001) J. Biol. Chem. 276, 30407–30413). Here we show that aldolase interacts with three different subunits of V-ATPase (subunits a, B, and E). The binding sites for the V-ATPase subunits on aldolase appear to be on distinct interfaces of the glycolytic enzyme. Aldolase deletion mutant cells were able to grow in medium buffered at pH 5.5 but not at pH 7.5, displaying a growth phenotype similar to that observed in V-ATPase subunit deletion mutants. Abnormalities in V-ATPase assembly and protein expression observed in aldolase deletion mutant cells could be fully rescued by aldolase complementation. The interaction between aldolase and V-ATPase increased dramatically in the presence of glucose, suggesting that aldolase may act as a glucose sensor for V-ATPase regulation. Taken together, these findings provide functional evidence that the ATP-generating glycolytic pathway is directly coupled to the ATP-hydrolyzing proton pump through physical interaction between aldolase and V-ATPase. Vacuolar H+-ATPases (V-ATPases) 1The abbreviations used are: V-ATPase; vacuolar H+-ATPase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; aa, amino acid(s); GST, glutathione S-transferase; MBP, maltose-binding protein. are a family of highly conserved proton pumps that couple hydrolysis of cytosolic ATP to proton transport out of the cytosol (1Forgac M. J. Biol. Chem. 1999; 274: 12951-12954Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar, 2Gluck S.L. Underhill D.M. Iyori M. Holliday L.S. Kostrominova T.Y. Lee B.S. Annu. Rev. Physiol. 1996; 58: 427-445Crossref PubMed Scopus (124) Google Scholar, 3Stevens T.H. Forgac M. Annu. Rev. Cell. Dev. Biol. 1997; 13: 779-808Crossref PubMed Scopus (523) Google Scholar). They reside on intracellular membranes of all eukaryotic cells and are essential for the normal functions of secretory vesicles, the trans-Golgi network, endosomes, lysosomes, and the yeast vacuole (1Forgac M. J. Biol. Chem. 1999; 274: 12951-12954Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar, 3Stevens T.H. Forgac M. Annu. Rev. Cell. Dev. Biol. 1997; 13: 779-808Crossref PubMed Scopus (523) Google Scholar). In some specialized cells such as kidney epithelial cells and osteoclasts, V-ATPases reside at high levels on the plasma membrane where they are responsible for transepithelial or cellular proton transport required for normal acid-base homeostasis or bone remodeling (2Gluck S.L. Underhill D.M. Iyori M. Holliday L.S. Kostrominova T.Y. Lee B.S. Annu. Rev. Physiol. 1996; 58: 427-445Crossref PubMed Scopus (124) Google Scholar). Although V-ATPases consume ATP in these cellular processes, the mechanism for coupling of activity to metabolism is poorly understood. V-ATPase consists of two macrodomains, V1, a catalytic sector, composed of peripheral membrane proteins, and V0, a transmembrane sector, composed of intrinsic membrane proteins, that transmits protons through the lipid bilayer (4Kane P. FEBS Lett. 2000; 469: 137-141Crossref PubMed Scopus (64) Google Scholar). The V1 sector attaches at the cytoplasmic face of the membrane to the V0 sector. Dissociated V1 and V0 sectors of V-ATPase in yeast are present in a dynamic equilibrium with the fully assembled proton pump (4Kane P. FEBS Lett. 2000; 469: 137-141Crossref PubMed Scopus (64) Google Scholar). ATP-driven proton transport requires structural and functional coupling of the V1 and V0 sectors. The dissociated V1 sector lacks ATP hydrolytic activity, and the free V0 sector does not form an open proton pore (5Hirata 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). In yeast cells, the coupling of the V1 and V0 sectors is governed by glucose availability. In cells grown in glucose, the V1 and V0 sectors are assembled into V-ATPase (6Kane P. J. Biol. Chem. 1995; 270: 17025-17032Abstract Full Text Full Text PDF PubMed Google Scholar). Upon glucose deprivation, V1 dissociates from V0. A panel of yeast mutants with deficiencies in the Ras-cAMP pathway, the main glucose repression/derepression pathway, and the respiratory pathway has been investigated (7Parra K.J. Kane P.M. Mol. Cell. Biol. 1998; 18: 7064-7074Crossref PubMed Google Scholar). V-ATPase was found to assemble normally in all the yeast mutant strains tested, suggesting that glucose-induced assembly of V-ATPase is independent of these pathways (7Parra K.J. Kane P.M. Mol. Cell. Biol. 1998; 18: 7064-7074Crossref PubMed Google Scholar). The glycolytic pathway comprises nine enzymatic steps that convert one glucose molecule with six carbon atoms into two molecules of pyruvate, each with three carbon atoms. At the end of glycolysis, the ATP balance sheet shows a net profit of two ATP molecules/glucose molecule. For most animal cells, glycolysis is only a prelude to the citric acid cycle since the pyruvic acid quickly enters the mitochondria to be completely oxidized to CO2 and H2O. However, in yeast such as Saccharomyces cerevisiae, glycolysis is the preferred metabolic pathway for ATP generation (8Alberts B. Bray D. Lewis J. Raff M. Roberts K. Watson J.D. Molecular Biology of the Cell. Garland Publishing, Inc., New York1989: 41-86Google Scholar). Instead of being degraded in mitochondria, the pyruvate molecules generated in yeast cells stay in the cytosol and are converted into ethanol and CO2, which is then excreted. Although yeast cells can utilize a variety of carbon sources, they use up the available glucose before turning to alternative fuels (9Rolland F. Winderickx J. Thevelein J.M. Trends Biochem. Sci. 2001; 26: 310-317Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar). Addition of glucose to carbon-starved yeast cells or cells growing on a nonfermentable carbon source results in two major changes in enzymatic activities and gene expression. First, glucose represses expression of genes encoding proteins in the respiratory pathway and enzymes for utilization of alternative carbon sources. Second, glucose induces expression of genes required for glucose utilization, including genes encoding glycolytic enzymes (9Rolland F. Winderickx J. Thevelein J.M. Trends Biochem. Sci. 2001; 26: 310-317Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar). In a search for protein partners that interact with V-ATPase, we discovered that the glycolytic enzyme aldolase binds directly to V-ATPase (10Lu M. Holliday L.S. Zhang L. Dunn W.A. Gluck S.L. J. Biol. Chem. 2001; 276: 30407-30413Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). In yeast cells deficient in aldolase, we observed complete disassembly and a dramatic reduction in the steady-state level of V-ATPase (10Lu M. Holliday L.S. Zhang L. Dunn W.A. Gluck S.L. J. Biol. Chem. 2001; 276: 30407-30413Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). In this report, we show that yeast mutant cells deficient in aldolase display a growth phenotype similar to that observed in V-ATPase subunit deletion mutants. V-ATPase abnormalities observed in aldolase deletion mutant cells can be rescued to normal levels by aldolase complementation. These data provide functional evidence that the glycolytic enzyme aldolase regulates V-ATPase assembly, protein expression, and activity by physical association with the proton pump. Materials—The high fidelity Expand Long enzyme system was purchased from Roche Applied Science. Restriction endonucleases and T4 DNA ligase were obtained from New England Biolabs (Beverly, MA). The yeast expression vector YC2/CT and polyclonal rabbit antiserum against aldolase were from Invitrogen. Monoclonal antibody against the B subunit of V-ATPase (13D11) was purchased from Molecular Probes (Eugene, OR). The monoclonal antibody against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was from Biogenesis (Kingston, NH). Zymolyase 100T was purchased from ICN (Costa Mesa, CA). Bafilomycin A1, 3-aminotriazole, and isopropylthiogalactoside were from Sigma. [35S]Methionine was obtained from Amersham Biosciences. Yeast Two-hybrid Assays—High fidelity PCR was performed to amplify a cDNA fragment coding for 389 aa at the amino terminus that represents the cytoplasmic portion of the human a4 subunit of V-ATPase using the forward primer 5′-ATGGCGTCTGTGTTTCGAAG-3′ and the reverse primer 5′-GTAGCTGCCGACACCATAG-3′. The cDNA fragment that corresponds to the 100 aa at the amino terminus of the human B1 subunit of V-ATPase was amplified by PCR using the forward primer 5′-ATGGAGATAGACAGCAGGCC-3′ and the reverse primer 5′-CATCGATCCCTGATGTCC-3′. The cDNA fragment that corresponds to the 100 aa at the carboxyl terminus of the human B1 subunit of V-ATPase was amplified by PCR using the forward primer 5′-ACCAGCTGTACGCCTGCTATG-3′ and the reverse primer 5′-TAGAGCGCAGTGTCAG-3′. The amplified cDNA fragments were cloned in-frame with the GAL-4 DNA binding domain on the yeast expression vector pAS2-1 (Clontech). PJ69 yeast cells harboring three reporter genes for adenine, histidine, and β-galactosidase were transformed with the cDNA constructs and spread onto 100-mm plates of minimum synthetic medium lacking tryptophan (10Lu M. Holliday L.S. Zhang L. Dunn W.A. Gluck S.L. J. Biol. Chem. 2001; 276: 30407-30413Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). Colonies of yeast cells were allowed to grow for 4 days and harvested for subsequent transformation with a human kidney cDNA expression library fused in-frame with the GAL-4 activation domain in the vector pACT2 (Clontech). After selection for growth on adenine–/Leu–/Trp– triple dropout plates, positive clones were streaked on His–/Leu–/Trp– dropout plates in the presence of 1 mm 3-aminotriazole and subsequently assayed for β-galactosidase activity. Analysis of the positive colonies was carried out as described previously (10Lu M. Holliday L.S. Zhang L. Dunn W.A. Gluck S.L. J. Biol. Chem. 2001; 276: 30407-30413Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). Yeast cells were grown overnight to stationary phase. DNA was isolated and introduced into bacteria by electroporation. DNA sequencing was carried out by incubation with BigDye Terminators and read by an automatic DNA sequencer (PE Applied Biosystems, Foster City, CA). The derived DNA sequences were analyzed by searching the GenBank™ data base for homology to known DNA and peptide sequences. Glutathione S-Transferase (GST) Precipitation Assays—The cytoplasmic portion of the human a4 subunit of V-ATPase was amplified by high fidelity PCR as described above using the forward primer 5′-AATTGGATCCATGGCTCTCAGCGATGCTGACGTGC-3′ and the reverse primer 5′-GAGAGAATTCGTCCAAAAACTTCCTGTTGGCATTTGC-3′. A 150-aa cDNA fragment from the amino terminus of the human B1 subunit of V-ATPase was amplified using the forward primer 5′-ATGGAGATAGACAGCAGGCC-3′ and the reverse primer 5′-GCTGGCCATTGATATCCAG-3′. The amplified PCR products were purified with QIAEX II beads (Qiagen), digested with restriction enzymes, purified again with QIAEX II beads, and cloned in-frame into the GST expression vector pGEX-2TK (Amersham Biosciences). Clones containing the fusion constructs were identified by DNA minipreparation and restriction enzyme digestion and verified by DNA sequencing. Purification of the fusion proteins was performed as described previously (10Lu M. Holliday L.S. Zhang L. Dunn W.A. Gluck S.L. J. Biol. Chem. 2001; 276: 30407-30413Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). Briefly bacterial colonies were inoculated into LB medium containing ampicillin and grown for 16 h at 37 °C in a shaking incubator. The cultures were diluted 1:10 and grown for 3 h, isopropylthiogalactoside was added to a final concentration of 0.2 mm, and incubation was continued for 1 h. Bacteria were pelleted by centrifugation at 10,000 × g for 2 min and resuspended in ice-cold phosphate-buffered saline. Cell lysis was carried out by sonication (Sonic Dismembrater, Fisher Scientific) for 2 × 30 s. Triton X-100 was added to a final concentration of 1% to minimize aggregation of the fusion protein with bacterial proteins. Samples were centrifuged at 10,000 × g for 5 min, and the supernatants were collected, mixed with a 50% slurry of glutathione-agarose beads (Sigma) in phosphate-buffered saline, and incubated for 5 min at room temperature. The beads were collected by centrifugation and incubated with 35S-labeled aldolase in Nonidet P-40 buffer (150 mm NaCl, 1% Nonidet P-40, 50 mm Tris, pH 8) at 4 °C for 3 h with shaking. The beads were recovered by centrifugation, washed five times in Nonidet P-40 buffer, resuspended in Laemmli sample loading buffer (2% SDS, 10% glycerol, 100 mm dithiothreitol, 60 mm Tris, pH 6.8, and 0.001% bromphenol blue) and boiled for 3 min. After centrifugation, the supernatants were collected and applied to SDS-polyacrylamide gels. The gels were dried for autoradiography analysis. Immunoprecipitation and Immunoblotting—Yeast cells were grown in supplemented minimal medium lacking methionine, harvested by centrifugation at 1000 × g for 5 min, resuspended in pretreatment buffer (0.1 m Tris-HCl, pH 9.0, 10 mm dithiothreitol), and incubated for 5 min. Spheroplasts were generated by treatment with zymolyase 100T (ICN) for 20 min in SPC buffer (1% glucose, 1 m sorbitol, 50 mm K2HPO4, 16 mm citric acid, pH 5.8), labeled with [35S]methionine for 60 min, lysed in solubilization buffer (10 mm Tris-HCl, pH 7.5, 10% glycerol, 1 mm EDTA, 2 mm dithiothreitol, 1% polyoxyethylene-9-lauryl ether) for 15 min and incubated with the anti-B subunit antibody 13D11 (Molecular Probes) for 60 min. After another 60-min incubation with protein A-agarose beads, the immunoprecipitates were collected by centrifugation, washed three times, and incubated for 5 min at 70 °C in 50 μl of cracking buffer (50 mm Tris-HCl, pH 7.0, 8 m urea, 5% SDS, 5% β-mercaptoethanol) for SDS-polyacrylamide gel electrophoresis and autoradiography analysis. Quantitation of the V0 and V1 subunits was performed using a densitometer. Vacuolar membrane vesicles were prepared from midlog phase yeast cells as described previously (11Lu M. Vergara S. Zhang L. Holliday L.S. Aris J. Gluck S.L. J. Biol. Chem. 2002; 277: 38409-38415Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). Western blotting was carried out by separating V-ATPase immunoprecipitates on 10% polyacrylamide gels as described previously (10Lu M. Holliday L.S. Zhang L. Dunn W.A. Gluck S.L. J. Biol. Chem. 2001; 276: 30407-30413Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar) using a Bio-Rad minigel apparatus and transferring electrophoretically to Hybond nitrocellulose membranes (Amersham Biosciences) using a Trans-Blot apparatus (Bio-Rad). The membranes were incubated to block nonspecific binding in 5% nonfat dry milk in TTBS (10 mm Tris, pH 8.0, 500 mm NaCl, and 0.05% Tween 20) with gentle agitation for 1 h at room temperature. Polyclonal rabbit antiserum against aldolase was diluted 1:1000 in TTBS and applied to the membranes for 1 h. After washing with TTBS, the membranes were incubated with peroxidase-conjugated goat anti-rabbit IgG in TTBS for 1 h, washed five times in TTBS, and incubated with SuperSignal West Dura Substrate working solution (Pierce) according to the manufacturer’s instructions. ATPase and Proton Translocating Activities—Bafilomycin A1-sensitive ATP hydrolysis of vacuolar membranes was assayed by measuring the production of inorganic phosphate as described previously (11Lu M. Vergara S. Zhang L. Holliday L.S. Aris J. Gluck S.L. J. Biol. Chem. 2002; 277: 38409-38415Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). Briefly vacuolar membrane vesicles in ATPase buffer (150 mm NaCl, 2 mm MgCl2, 1 mm vanadate, 1 mm azide, pH 6.75) were preincubated for 15 min at room temperature in the presence and absence of bafilomycin A1. The reaction was initiated by addition of ATP in a final concentration of 3 mm and stopped by addition of trichloroacetic acid. The samples were extracted with chloroform to remove lipid and detergent. After centrifugation, the upper aqueous phase was transferred to clean test tubes and incubated with buffers containing ascorbic acid and ammonium molybdate. The concentration of inorganic phosphate was measured by a spectrophotometer at 700 nm and converted to rate of ATP hydrolysis. Proton transport activity was measured by ATP-dependent quenching of acridine orange using a PerkinElmer Life Sciences fluorescence spectrometer with excitation at 493 nm and emission at 545 nm (11Lu M. Vergara S. Zhang L. Holliday L.S. Aris J. Gluck S.L. J. Biol. Chem. 2002; 277: 38409-38415Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). Purified vacuolar membrane vesicles were resuspended in 25 mm Tris, pH 7.2, 25 mm KCl, 5 mm acridine orange, and 5 mm MgCl2 in the presence and absence of bafilomycin A1. ATP was added at a final concentration of 5 mm to initiate transport. The Glycolytic Enzyme Aldolase Interacts with Three Distinct Subunits of V-ATPase—We previously detected interaction between aldolase and the E subunit of V-ATPase (10Lu M. Holliday L.S. Zhang L. Dunn W.A. Gluck S.L. J. Biol. Chem. 2001; 276: 30407-30413Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). To search for protein molecules that interact with other subunits of V-ATPase, we carried out yeast two-hybrid assays using the a and B subunits of V-ATPase as baits, respectively. Subunit a is an integral membrane protein possessing an amino-terminal hydrophilic domain and a carboxyl-terminal hydrophobic domain containing multiple membrane-spanning segments (12Nishi T. Forgac M. J. Biol. Chem. 2000; 275: 6824-6830Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 13Toyomura T. Oka T. Yamaguchi C. Wada Y. Futai M. J. Biol. Chem. 2000; 275: 8760-8765Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar, 14Oka T. Murata Y. Namba M. Yoshimizu T. Toyomura T. Yamamoto A. Sun-Wada G.-H. Hamadaki N. Wada Y. Futai M. J. Biol. Chem. 2001; 276: 40050-40054Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 15Smith A.N. Finberg K.E. Wagner C.A. Lifton R.P. Devonald M. Su Y. Karet F.E. J. Biol. Chem. 2001; 276: 42382-42388Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). The 389 aa at the amino terminus that represent the cytoplasmic portion of the a4 subunit was cloned as the bait. The catalytic “head” of V-ATPase consists of three pairs of A and B subunits arrayed as a hexagon. The A subunit is the site of ATP hydrolysis, and the B subunit may have a regulatory role (16Adachi I. Arai H. Pimental R. Forgac M. J. Biol. Chem. 1990; 265: 960-966Abstract Full Text PDF PubMed Google Scholar, 17Wang Z.Q. Gluck S.L. J. Biol. Chem. 1990; 265: 21957-21965Abstract Full Text PDF PubMed Google Scholar). The central portion of the B subunit is believed to be required for binding to the A subunit. To maximize chances for identification of protein molecules that may regulate V-ATPase but are not elements of the core structure of the enzyme, we cloned two regions, 100 aa each, from the amino and carboxyl termini of the B subunit as baits. Screening a human kidney cDNA expression library identified the glycolytic enzyme aldolase to interact with the a subunit and the B amino-terminal fragment but not with the B carboxyl-terminal fragment (Table I). Taken together, aldolase interacts with the a, B, and E subunits of V-ATPase. These interactions were confirmed by GST precipitation assays (Fig. 1A).Table IAldolase interacts with the a, B, and E subunits of V-ATPase on distinct interfacesAldolase deletion mutantspAS2-apAS2-B-NpAS2-B-CpAS2-EAldolase-(1-364)++-+Aldolase-(108-364)++--Aldolase-(121-364)+---Aldolase-(126-364)+---Aldolase-(163-364)+---Aldolase-(188-364)+--- Open table in a new tab Furthermore our data indicate that distinct domains exist on aldolase for interactions with the a, B, and E subunits of V-ATPase (Table I). When the cytoplasmic portion of the a subunit was used as the bait for two-hybrid assays, clones representing a wide spectrum of mutations in aldolase were identified, ranging from the full-length cDNA of 364 aa to cDNAs with deletions up to the 187 aa from the amino terminus. Thus, the 177 aa at the carboxyl terminus of aldolase is sufficient for interaction with the a subunit. As for the domain in aldolase required for interaction with the B subunit of V-ATPase, our two-hybrid assay identified the full-length aldolase cDNA and clones with deletions up to 107 aa from the amino terminus of aldolase. Clones with deletions beyond the 107 aa from the amino terminus that interact with the a subunit failed to bind to the B subunit. In contrast, aldolase deletion mutants over a wide spectrum of amino acids failed to show any interaction with the E subunit, suggesting that the entire aldolase protein was required to fold in a particular configuration for interaction with the E subunit (Table I). These observations were further confirmed by GST precipitation assays (Fig. 1, B–D). Simultaneous interactions of aldolase with the a, B, and E subunits of V-ATPase were further confirmed by in vitro binding assays (Fig. 1, E–H). In the absence of aldolase, no binding was detected among V-ATPase subunits (Fig. 1, F–H), suggesting that the interactions occur directly through aldolase. Our findings that aldolase binds to both the V1 and V0 subunits of V-ATPase support the notion that aldolase may act as a nucleus in assembly of V1 and V0 sectors into the V-ATPase holoenzyme. Aldolase Deletion Mutant Cells Display a Growth Phenotype Similar to That Observed in the V-ATPase Subunit Deletion Mutants—Previous studies showed that deletion of individual c, B, or E subunits of V-ATPase disrupted V-ATPase assembly and resulted in undetectable levels of ATP hydrolytic and proton transport activities (18Foury F. J. Biol. Chem. 1990; 265: 18554-18560Abstract Full Text PDF PubMed Google Scholar, 19Nelson H. Nelson N. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 3503-3507Crossref PubMed Scopus (245) Google Scholar). Yeast cells lacking the c, B, or E subunit of V-ATPase, however, were able to grow on culture plates buffered at pH 5.5 but not at pH 7.5, most likely due to abnormalities in cytosolic calcium homeostasis resulting in a lethal phenotype at high extracellular pH (20Ohya Y. Umemoto N. Tanida I. Ohta A. Iida H. Anraku Y. J. Biol. Chem. 1991; 266: 13971-13977Abstract Full Text PDF PubMed Google Scholar, 21Forster C. Kane P.M. J. Biol. Chem. 2000; 275: 38245-38253Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). To investigate the relationship between aldolase and V-ATPase activity, we examined the growth pattern of a yeast mutant strain deficient in aldolase (22Lobo Z J. Bacteriol. 1984; 160: 222-226Crossref PubMed Google Scholar). The aldolase deletion mutant strain, fba1, expressed undetectable levels of aldolase protein (Fig. 2A) and displayed aldolase enzymatic activity below 2% of that observed in wild-type cells (22Lobo Z J. Bacteriol. 1984; 160: 222-226Crossref PubMed Google Scholar). The aldolase mutant cells were able to grow on culture plates buffered at pH 5.5 but not at pH 7.5 (Fig. 2B), displaying a growth phenotype similar to that observed in the V-ATPase subunit deletion mutants. In contrast, mutant cells deficient in the glycolytic enzyme glucose-6-phosphate isomerase were found to grow on culture plates buffered at both pH 5.5 and pH 7.5 (Fig. 2B), consistent with our previous observation that the assembly and expression of V-ATPase were much less affected in these mutant cells (10Lu M. Holliday L.S. Zhang L. Dunn W.A. Gluck S.L. J. Biol. Chem. 2001; 276: 30407-30413Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). These results support the notion that aldolase deficiency results in V-ATPase malfunction by disrupting the physical association between aldolase and V-ATPase. Restoration of V-ATPase Assembly, Protein Expression, and Activity to Normal Levels by Aldolase Complementation—Since aldolase physically interacts with V-ATPase in vivo and deletion of the aldolase gene results in complete disassembly and a 3-fold reduction in the steady-state level of V-ATPase (10Lu M. Holliday L.S. Zhang L. Dunn W.A. Gluck S.L. J. Biol. Chem. 2001; 276: 30407-30413Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar), we examined whether the V-ATPase abnormalities observed in aldolase deletion mutant cells could be restored to normal levels by aldolase complementation. The open reading frame coding for the yeast aldolase gene was amplified by high fidelity PCR. The amplified cDNA fragment was verified by DNA sequencing and subsequently cloned into the expression vector pYC2/CT (Invitrogen), which is designed for inducible expression of recombinant proteins in S. cerevisiae. The vector contains the galactose-inducible GAL-1 promoter and harbors the CEN6 origin for non-integrative centromeric maintenance and low copy replication of the plasmid (generally one to two copies per cell). Aldolase deletion mutant cells were transformed with the aldolase-containing expression vector. The transformed cells were spread onto 100-mm culture plates containing 0.2% glucose and 2% galactose. Since the presence of glucose inhibits the growth of aldolase deletion mutant cells (22Lobo Z J. Bacteriol. 1984; 160: 222-226Crossref PubMed Google Scholar), aldolase-expressing colonies were selected by growth on glucose-containing plates and confirmed by Western blotting. The aldolase-expressing cells were subsequently examined for V-ATPase assembly and expression. Normal levels of assembly and protein expression of V-ATPase were observed as measured by immunoprecipitation using an antibody against the B subunit of V-ATPase, 13D11. Our data, therefore, demonstrate restoration of V-ATPase assembly and protein expression to normal levels in aldolase deletion mutant cells by aldolase complementation. To examine whether V-ATPase activity was also restored, we prepared vacuolar membrane vesicles as described previously (11Lu M. Vergara S. Zhang L. Holliday L.S. Aris J. Gluck S.L. J. Biol. Chem. 2002; 277: 38409-38415Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar) and measured ATP hydrolytic activity that is sensitive to the specific inhibitor of V-ATPase, bafilomycin A1. ATP hydrolysis of vacuolar membrane vesicles was assayed by measuring the production of inorganic phosphate (11Lu M. Vergara S. Zhang L. Holliday L.S. Aris J. Gluck S.L. J. Biol. Chem. 2002; 277: 38409-38415Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). As shown in Fig. 3A, aldolase complementation restored ATP hydrolysis to normal levels in the aldolase deletion mutant cells. The proton transport activity of V-ATPase was also measured by tracking the ATP-dependent quenching of acridine orange using a fluorescence spectrometer with excitation at 493 nm and emission at 545 nm (11Lu M. Vergara S. Zhang L. Holliday L.S. Aris J. Gluck S.L. J. Biol. Chem. 2002; 277: 38409-38415Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). As expected, aldolase complementation restored V-ATPase proton transport activity to normal levels (Fig. 3B). Up-regulation of Aldolase-V-ATPase Interaction in the Presence of Glucose—Based on its interaction with the V1 domain of V-ATPase and genetic manipulation analysis, the RAVE complex was proposed to play a role in glucose-dependent assembly of V-ATPase (23Seol J.H. Shevchenko A. Shevchenko A. Deshaies R.J. Nat. Cell Biol. 2001; 3: 384-391Crossref PubMed Scopus (207) Google Scholar). Further studies, however, demonstrated that the interaction between RAVE and V1 was not glucose-sensitive, consistent with a constitutive rather than a regulatory role for the RAVE complex in V-ATPase assembly (24Smardon A.M. Tarso M. Kane P J. Biol. Chem. 2002; 277: 13831-13839Abstract Full Text Full Text P
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