Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 7-Day Trial for You or Your Team.

Learn More →

Site-directed Mutagenesis of the 100-kDa Subunit (Vph1p) of the Yeast Vacuolar (H+)-ATPase

Site-directed Mutagenesis of the 100-kDa Subunit (Vph1p) of the Yeast Vacuolar (H+)-ATPase THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 271, No. 37, Issue of September 13, pp. 22487–22493, 1996 © 1996 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Site-directed Mutagenesis of the 100-kDa Subunit (Vph1p) of the Yeast Vacuolar (H )-ATPase* (Received for publication, April 29, 1996, and in revised form, June 21, 1996) Xing-Hong Leng, Morris F. Manolson‡, Qing Liu, and Michael Forgac§ From the Department of Cellular and Molecular Physiology, Tufts University School of Medicine, Boston, Massachusetts 02111 and the ‡Division of Cell Biology, Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada as synaptic vesicles and chromaffin granules, and the central Vacuolar (H )-ATPases (V-ATPases) are multisubunit complexes responsible for acidification of intracellular vacuoles of plants, Neurospora and yeast. Acidification of these compartments in eukaryotic cells. V-ATPases possess a compartments, in turn, plays an important role in such proc- subunit of approximate molecular mass 100 kDa of un- esses as receptor-mediated endocytosis, intracellular mem- known function that is composed of an amino-terminal brane traffic, protein processing and degradation, and coupled hydrophilic domain and a carboxyl-terminal hydropho- transport of small molecules. In yeast, acidification of the cen- bic domain. To test whether the 100-kDa subunit plays a tral vacuole is important both to maintain the activity of deg- role in proton transport, site-directed mutagenesis of 21 radative enzymes and to drive uptake of solutes such as Ca the VPH1 gene, which is one of two genes that encodes and amino acids (5). this subunit in yeast, has been carried out in a strain The V-ATPases are multisubunit complexes composed of two lacking both endogenous genes. Ten charged and twelve structural domains. The peripheral V domain is a 500-kDa polar residues located in the seven putative transmem- complex responsible for ATP hydrolysis, whereas the integral brane helices in the COOH-terminal domain of the mol- V domain is a 250-kDa complex responsible for proton trans- ecule were individually changed, and the effects on pro- location (10). In Saccharomyces cerevisiae, the V domain is ton transport, ATPase activity, and assembly of the 1 composed of seven different subunits of molecular masses 69 yeast V-ATPase were measured. Two mutations (R735L (subunit A encoded by VMA1 (11, 12)), 60 (subunit B/VMA2 and Q634L) in transmembrane helix 6 and at the border of transmembrane helix 5, respectively, showed greatly (13)), 54 (VMA13 (14)), 42 (subunit C/VMA5 (15, 16)), 32 (sub- reduced levels of the 100-kDa subunit in the vacuolar unit D/VMA8 (17)), 27 (subunit E/VMA4 (15, 18)), and 14 kDa membrane, suggesting that these mutations affected sta- (subunit F/VMA7 (19, 20)). The V domain is composed of at bility of the 100-kDa subunit. Two mutations, D425N and least four subunits of molecular masses approximately 100 K538A, in transmembrane helix 1 and at the border of (encoded by VPH1 and STV1 (21, 22)), 36 (VMA6 (23)), 13 transmembrane helix 3, respectively, showed reduced (VMA10 (24)), and 17 kDa (subunit c/VMA3 and VMA11 (25, assembly of the V-ATPase, with the D425N mutation also 26)). By analogy with the bovine coated vesicle V-ATPase (27), reducing the activity of V-ATPase complexes that did the V domain has the structure A B C D E F , whereas V 1 3 3 1 1 1 1 0 assemble. Two mutations, H743A and K593A, in trans- domain has the structure 100 36 c . No mammalian homolog 1 1 6 membrane helix 6 and at the border of transmembrane to Vma10p has yet been identified, and the yeast counterpart to helix 4, respectively, have significantly greater effects the bovine 19-kDa V subunit is also uncertain. on activity than on assembly, with proton transport and Although the V-ATPases are homologous to the F-ATPases ATPase activity inhibited 40–60%. One mutation, E789Q, (28–31), both in overall structure (10, 27) and in sequence in transmembrane helix 7, virtually completely abol- homology of several of the subunits (11–13, 25, 32–38), no ished proton transport and ATPase activity while hav- ing no effect on assembly. These results suggest that the obvious structural homolog exists for the 100-kDa subunit in 100-kDa subunit may be required for activity as well as the F-ATPases. The 100-kDa subunit of the V-ATPase in yeast assembly of the V-ATPase complex and that several is encoded by two homologous genes, VPH1 (21) and STV1 (22). charged residues in the last four putative transmem- VPH1 encodes a 95-kDa protein, which possesses a hydrophilic brane helices of this subunit may play a role in proton amino-terminal domain of approximately 45 kDa and a carbox- transport. yl-terminal hydrophobic domain of approximately 50 kDa con- taining 6–7 putative transmembrane helices (21). STV1 en- codes a 102-kDa protein that shares the same domain 1 1 The vacuolar (H )-ATPases (V-ATPases) are a family of arrangement and is 54% identical in amino acid sequence with proton pumps responsible for acidification of intracellular com- the product of the VPH1 gene (Vph1p) (22). Disruption of the partments in eukaryotic cells (for reviews see Refs. 1–9). VPH1 gene leads to somewhat reduced growth at neutral pH Among the compartments acidified by V-ATPases are clathrin- relative to acidic pH (21) and disruption of STV1 has no obvious coated vesicles, endosomes, lysosomes, secretory vesicles, such phenotypic consequences (22). By contrast, disruption of both VPH1 and STV1 leads to the typical Vma phenotype, includ- * This work was supported by National Institutes of Health Grant ing the inability to grow at neutral pH and hypersensitivity to GM 34478 (to M. F.) and a Medical Research Council of Canada grant 21 Ca (22). These results suggest that the VPH1 and STV1 gene (to M. F. M.). Fluorescence facilities were provided by National Insti- products are at least partially able to substitute for each other. tutes of Health Grant DK 34928. The costs of publication of this article were defrayed in part by the payment of page charges. This article must The reason that yeast possess two genes encoding the 100-kDa therefore be hereby marked “advertisement” in accordance with 18 subunit is uncertain, but the results available thus far suggests U.S.C. Section 1734 solely to indicate this fact. that V-ATPases possessing Vph1p and Stv1p may be targeted § To whom correspondence should be addressed. to different intracellular membranes (22). In order to deter- The abbreviations used are: V-ATPase, vacuolar proton-translocat- ing adenosine triphosphatase; Mes, 4-morpholineethanesulfonic acid. mine whether the 100-kDa subunit plays a direct role in proton This is an open access article under the CC BY license. 22487 22488 Vph1p Mutagenesis a coupled spectrophotometric assay (45) with the modification of using transport by the V-ATPase complex, site-directed mutagenesis 0.35 mM of NADH instead of 0.5 mM NADH. ATP-dependent proton of the VPH1 gene product has been carried out in a strain transport was measured in transport buffer (25 mM Mes/Tris, pH 7.2, 5 lacking endogenous Vph1p and Stv1p. mM MgCl , and 25 mM KCl) using the fluorescence probe amino-6- chloro-2-methoxyacridine as described previously (46) in the presence EXPERIMENTAL PROCEDURES or the absence of 10 nM bafilomycin A . Protein concentration was Materials and Strains—Zymolyase 100T was obtained from Seika- measured using the Lowry method (47). SDS-polyacrylamide gel elec- gaku America, Inc. [ S]Trans-label was purchased from ICN. Bafilo- trophoresis was carried out as described by Laemmli (48). Silver stain- mycin A was a kind gift from Dr. Karlheinz Altendorf (University of ing was performed using the method of Oakley et al. (49). Osnabruck). Leupeptin was from Boehringer Mannheim. 9-Amino-6- Western blots were probed with mouse monoclonal antibodies chloro-2-methoxyacridine was from Molecular Probes, Inc. ATP, phen- 8B1-F3 against the 69-kDa subunit, (from Molecular Probes, Inc.) or ylmethylsulfonylfluoride, and most other chemicals were purchased 10D7 against the 100-kDa subunit, (a generous gift from Dr. P.Kane), from Sigma. followed by horseradish peroxidase-conjugated secondary antibody MM322 (VPH1 in pRS316) and yeast strain MM112 (MATa (Bio-Rad). Blots were developed using a chemiluminescent detection Dvph1::LEU2 Dstv1::LYS2 his3-D200 leu2 lys2 ura3–52) (22) were used method obtained from KPL. Quantitations were done using an IS-1000 to generate and study VPH1 mutants. Yeast cells were grown in YPD Digital imaging system (Alpha Innotech Corporation). medium (yeast extract-peptone-dextrose) or synthetic dropout synthetic Immunoprecipitations were carried out as described (50), with the medium (39). following modifications. Cells were grown overnight in supplemented Mutagenesis—Mutagenesis was performed on SalI-EcoRI or EcoRI- minimal medium lacking methionine and then converted to sphero- BamHI fragments of the wild type VPH1 gene in the vector pALTER-1 plasts by incubation for 20 min with 0.5 unit of zymolase/10 cells in (Promega) following the manufacturer’s protocol. The mutagenesis SD-Met, 1.2 M sorbitol, 50 mM Tris-Mes, pH 7.5. Aliquots containing 5 3 6 35 oligonucleotides were as follows with substitution sites underlined: 10 spheroplasts were then incubated with [ S]Trans-label (50 mCi) for T411A, 59-GTGACAATTGCGGGTAAACCAG-39; T414A, 59-ACATGAA- 60 min at 30 °C. Spheroplasts were then pelleted, lysed in phosphate- AGGGAATGCGACAATTGTGGG-39; D425N, 59-GACCCATATTACC buffered saline with C E and immunoprecipitated (50) using 7.5 mgof 12 9 AAACATG-39; H428, 59-GTCATTAAGAACCCGGCACCCATATCACC- purified 8B1-F3 antibody and protein A-Sepharose followed by SDS- A-39; R462V, 59-AACAAAATAATGTATACACCAGTGAAGGCCA-39; polyacrylamide gel electrophoresis on 12% acrylamide gels and autora- Y463F, 59-ACAAAATAATGAATCTACCAG-39; S472A, 59-GTGTACAT- diography as described (50). GGCAAAGACACCCA-39; Y474F, 59-GAAACCTGTGAACATGGAAAA- RESULTS G-39; T475A, 59-GAAACCTGCGTACATGG-39; K538A, 59-ATTAAAAT- TGATAGTGCCATTTTGTAAGAATTA-39; S540A, 59-GAACCCCATTA- It has previously been shown that deletion of genes encoding AAATGGCTAGTTTCATTTTGTA-39; H547A, 59-ATAAGAATAGGTCA- subunits of the yeast V-ATPase leads to a conditional lethal TCGCGATGAACCCCAT-TAA-39; Y585F, 59-AACGGAAAGAAAACCA- phenotype such that strains carrying such deletions are unable AAGA-39; Y592F, 59-ATCAACAGCCCATTTAAAAACAATACAAACG-39; to grow at neutral pH but are able to grow at acidic pH (51). We K593A, 59-AATCAACAGCCCACGCGTAAACAATACAAACG-39; Q634L, 59-ACAAAAACACTAGGACC TTTG-39; C644A,59-GCAACCAAGGAATG- have employed a strain in which both the VPH1 and STV1 GCAACCAAGGCCATCA-39; K652A, 59-GAAATGTAATGGCGCCACCA- genes encoding the 100-kDa subunit of the V-ATPase have ATAGCAACCA-39; R735L, 59-CCTATTTACTTTTATGGGC-39; H743A, 2 been disrupted, leading to the typical Vma phenotype (22). 59-CTTATCATTGGCAGCTGCTCAATTGTCTA-39; T781V, 59-GTGGT- Expression of the VPH1 gene on a CEN plasmid in this strain TCGCACTAGTATGTGCAGTTCTTG-39; E789Q, 59-GTTTTGATGCAA- leads to growth at pH 7.5. Twenty-two individual site-directed GGT ACA-39. Fragments containing the indicated mutations were sequ- mutations were introduced into the VPH1 gene and the mutant enced using the diedoxy method (40) to confirm that no other mutations had been introduced and were then substituted back into the vector proteins expressed in the double knock out strain. The residues containing the wild type VPH1 gene. selected for mutation all correspond to polar or charged resi- Transformation—Yeast cells were transformed with the wild type dues located within the seven putative transmembrane helices plasmid (pRS316-VPH1), mutants or the vector pRS316 alone (as a in the COOH-terminal half of the 100-kDa subunit. These negative control) using the lithium acetate procedure (41) and were 2 residues were selected in an effort to identify buried polar or selected on Ura plates as described previously (42). The mutants were charged amino acids that might directly contribute to the pro- tested for growth on pH 7.5 or pH 5.5 YPD plates buffered with 50 mM KH PO and 50 mM succinic acid (43). ton conduction pathway in the V-ATPase complex. Polar resi- 2 4 Isolation of Vacuolar Membrane Vesicles—Vacuolar membrane ves- dues were changed to nonpolar residues of similar size (for icles were isolated using a modification of the protocol described by Kim example substituting alanine for serine or phenylalanine for et al. (44). Yeast were grown overnight at 30 °C to 1 3 10 cells/ml in 1 tyrosine), whereas charged residues were replaced either by liter of selective medium. Cells were pelleted, washed once with water, similar polar amino acids (i.e. glutamine for glutamic acid) or and resuspended in 50 ml of 10 mM dithiothreitol, 100 mM Tris-HCl, pH by nonpolar amino acids (usually alanine). It is anticipated 9.4. After incubation at 30 °C for 15 min, cells were pelleted again, resuspended in 50 ml of YEPD medium containing 0.7 M sorbitol, 2 mM that replacement of a polar or charged amino acid that directly dithiothreitol, and 100 mM Tris-Mes, pH 7.5, and 5 mg of Zymolyase participates in proton movement across the membrane will 100T and incubated at 30 °C with gentle shaking for 90 min. The disrupt ATP-dependent proton transport. Of these 22 muta- resulting spheroplasts were washed twice with ice-cold 1.2 M sorbitol, tions, only three showed greatly reduced growth at neutral pH and pelleted at 3500 3 g for 10 min at 4 °C. The pellet was resuspended (E789Q, D425N, and R735L) (data not shown), with the latter in 40 ml of homogenization buffer (10% glycerol, 1.5% polyvinylpyrro- two having an identical growth phenotype to the double knock lidone (M 40,000), 0.25 mM MgCl , 2 mg/ml bovine serum albumin, 50 r 2 mM Tris-ascorbate, pH 7.5, 1 mM phenylmethylsulfonyl fluoride, 1 out strain. Because as little as 20% of the wild type V-ATPase mg/ml leupeptin), transferred to a Dounce glass homogenizer and sub- activity is sufficient to rescue the growth phenotype of a Vma jected to 20 strokes with a tight fitting pestle. The homogenate was 2 strain, it was necessary to measure the vacuolar proton pump- centrifuged at 3500 3 g for 15 min at 4 °C, and the supernatant was ing and V-ATPase activity in each mutant strain to assess the transferred to a Ti 45 centrifuge tube and spun for 35 min at 100,000 3 effect of the mutation on V-ATPase activity. g at 4 °C. The pellets were resuspend in 8 ml of overlay medium, which Fig. 1 shows the ATP-dependent proton transport and the contained 1.1 M glycerol, 2 mM dithiothreitol, 0.25 mM MgCl , 2 mg/ml bovine serum albumin, and 5 mM Tris-Mes, pH 7.6, and homogenized by bafilomycin-sensitive ATPase activity for isolated vacuoles pu- 10 strokes in a Dounce glass homogenizer using a tight fitting pestle. rified from a Dvph1Dstv1 strain expressing the pRS316 plasmid The homogenate was overlaid onto a one step 30-ml 10–30% sucrose alone, pRS316 containing the wild type VPH1 gene, or the gradient and centrifuged for2hat 100,000 3 g in an SW-28 rotor. VPH1 gene bearing the indicated point mutations. Bafilomycin Material at the 10–30% interface was collected, diluted 10-fold with A has been shown to be a specific inhibitor of the V-ATPases overlay medium, and centrifuged at 100,000 3 g for 35 min at 4 °C. The pellets were resuspend in 0.5–1 ml of overlay medium, quick frozen with liquid nitrogen, and stored at 280 °C until used. Biochemical Characterization—ATPase activity was measured using P. Kane, personal communication. Vph1p Mutagenesis 22489 FIG.1. Effect of Vph1p mutations on bafilomycin-sensitive ATPase activity and ATP-dependent proton transport in purified vacuolar membrane vesicles. ATPase activity and ATP-dependent proton transport were measured on aliquots of purified vacuolar membrane vesicles containing 5 mg of protein as described under “Experimental Procedures.” Activities are expressed relative to the Dvph1Dstv1 strain expressing the pRS316 plasmid containing the wild type VPH1 gene (defined as 100%). The specific ATPase activity of these vacuolar membrane vesicles was 3.3 mmol ATP/min/mg protein at saturating ATP and 37° C, with approximately 80% of the ATPase activity inhibitable by 10 nM bafilomycin. All of the ATP-dependent proton transport in vacuolar membrane vesicles isolated from cells expressing the wild type Vph1p was inhibitable by 10 nM bafilomycin. No ATP-dependent proton transport or bafilomycin sensitive-ATPase activity was observed in vacuolar membrane vesicles isolated form cells transformed with the vector alone. Each bar represents the average of two or three determinations made on two or three independent vacuolar membrane preparations, with the error corresponding to the standard deviation. WT, wild type. (52). As expected on the basis of the growth phenotype, three ence of the 100-kDa and A subunits on the vacuolar membrane mutations (D425N, R735L, and E789Q) showed 20% or less of provides a reasonable measure of V-ATPase assembly. the wild type proton transport and V-ATPase activity, with Fig. 2 shows a Western blot carried out on vacuolar mem- both D425N and R735L having virtually no detectable activity. branes from the wild type, deletion, and mutant strains using Four additional mutations (K538A, K593A, Q634L, and antibodies directed against either Vph1p or the A subunit, H743A) showed between 20 and 70% of wild type activity, with whereas Fig. 3 shows the results of quantitative analysis of the Q634L having the lowest activity of this group. The remaining Western blot as well as the activity data on each mutant for mutants all displayed 70% or greater of wild type activity. comparison. As can be seen, two mutants (R735L and Q634L) None of the mutations resulted in significant uncoupling of showed greatly reduced levels of the 100-kDa subunit in the proton transport from ATP hydrolysis (i.e. a greater loss of vacuolar membrane as well as greatly reduced levels of A proton transport than ATPase activity), although one mutation subunit. These mutations thus appear to affect stability of the (Q634L) did decrease ATPase activity to a somewhat greater 100-kDa subunit. Two other mutations (D425N and K538A) extent than proton transport, suggesting a possible increase in show significantly reduced levels of A subunit associated with coupling efficiency. the vacuolar membrane with only slightly reduced levels of the The most obvious ways in which mutations in the 100-kDa 100-kDa subunit. These mutations thus appear to have an subunit might lead to a decrease in V-ATPase activity is if effect on assembly of the V-ATPase complex. It should be noted these mutations resulted in a 100-kDa subunit that was un- from Fig. 3 that the D425N mutant, while still having detect- stable and rapidly degraded or that was unable to correctly able levels of assembly as assessed by the presence of the A assemble with the remaining subunits of the V-ATPase com- subunit, is completely devoid of proton transport and ATPase plex. To assess these possibilities, Western blot analysis was activity, suggesting that any V-ATPase that does assemble is performed on isolated vacuoles using antibodies directed inactive. This result suggests that Asp may play a role in against Vph1p and the A subunit of the V-ATPase. It has both assembly and activity of the V-ATPase. previously been shown that disruption of the VPH1 gene leads The remaining mutants have near normal levels of the 100- to the inability of the V domain (including the A subunit) to kDa and A subunits. In particular, the E789Q mutant shows assemble onto the vacuolar membrane (21). Moreover, the ab- wild type levels of both subunits while possessing less than sence of any of the V subunits (with the exception of the 20% of the wild type levels of proton transport and ATPase 54-kDa subunit (14)) leads to loss of assembly of the entire V activity. Similarly, examination of Fig. 3 reveals that both the domain onto the vacuolar membrane (53, 54). Thus the pres- K593A and H743A mutants have approximately half as much 22490 Vph1p Mutagenesis DISCUSSION The V-ATPases resemble the F-ATPases of mitochondria, chloroplasts, and bacteria (28–31) both in overall structure (10, 27) and in sequence homology between several of the subunits, including the nucleotide binding A and B subunits (32–37, 11–13) and the dicyclohexylcarbodiimide-reactive c subunit (25, 38). As with the F-ATPases, the V-ATPases possess a peripheral domain (V ) responsible for ATP hydrolysis and an integral domain (V ) responsible for proton translocation. Un- like the corresponding F-ATPase domains, however, the V and V domains do not retain their respective activities when sep- arated from each other (55–57). An important question with regard to the V-ATPases is the mechanism by which they translocate protons. For the bovine coated vesicle V-ATPase, the V domain, which is responsible for proton translocation (58), is composed of four subunits of approximate molecular masses 100, 38, 19, and 17 kDa (sub- unit c) (57) that are present in a stoichiometry of 100 38 19 c 1 1 1 6 FIG.2. Effect of Vph1p mutations on stability of the 100-kDa (27). Of these subunits, only the 17-kDa c subunit has been subunit and association of the 69-kDa A subunit with the vacu- shown to directly participate in proton translocation by virtue olar membrane. Vacuolar membrane vesicles (5 mg) isolated from the of its reaction with dicyclohexylcarbodiimide (59). By analogy Dvph1Dstv1 strain expressing the wild type VPH1 gene, the VPH1 gene with the F-ATPase c subunit (60), dicyclohexylcarbodiimide bearing the indicated mutations, or the vector alone were subjected to reacts with a buried carboxyl group located near the middle of SDS-polyacrylamide gel electrophoresis on a 12% acrylamide gel fol- lowed by transfer to nitrocellulose and Western blot analysis using the the last of four putative transmembrane helices. Although a monoclonal antibody 10D7 against Vph1p or 8B1-F3 against the 69-kDa low level of proton translocation has been reported for the A subunit as described under “Experimental Procedures.” WT, wild isolated, reconstituted c subunit (61), optimal levels of dicyclo- type. hexylcarbodiimide-inhibitable proton transport are only ob- served when the complete complement of V subunits are re- proton transport and ATPase activity as predicted on the basis assembled prior to reconstitution (58). The basis for difference of the amount of assembled V-ATPase. To test whether, for in proton conduction properties of the native and reassembled these mutations, the observed decrease in activity might be due V domains remains uncertain. to loss of one of the other V-ATPase subunits from the complex, In the case of the F-ATPases, the F domain of Escherichia the following experiment was performed. Cells were converted 35 coli is composed of three subunits of molecular masses 30 (a), to spheroplasts and metabolically labeled with [ S]Trans-label 17 (b), and 8 kDa (c) that are present in a stoichiometry of for 60 min at 30 °C followed by cell lysis, detergent solubiliza- a b c (62). Reassembly studies indicate that for F , all 1 2 10–12 0 tion, and immunoprecipitation using the anti-A subunit anti- three subunits are required to form a functional proton channel body 8B1-F3 and protein A-Sepharose. As can be seen in Fig. 4, (63, 64). Mutational analysis has demonstrated that the buried the complete complement of V and V subunits are immuno- 1 0 carboxyl group located in the second of two transmembrane precipitated from cells expressing wild type Vph1p, whereas helices of the c subunit is critical for proton translocation (60). only the V subunits are immunoprecipitated in the vector Moreover, genetic analysis of the a subunit indicates the pres- control. Of the three mutants listed above, H743A and E789Q ence of several buried polar and charged residues, particularly showed wild type patterns of immunoprecipitation, whereas in the last two transmembrane helices, which also play a crit- K593A showed the normal pattern of subunits but with some- ical role in proton transport (65, 66). Thus, substitutions at what reduced levels of the V subunits, as predicted on the 206 210 219 Ser , Arg , and Glu in the fifth putative transmembrane basis of the data shown in Fig. 3. Thus, all three of these helix or His in the sixth putative transmembrane helix of the mutations appear to impair activity rather than assembly of a subunit significantly impair proton translocation through F . the V-ATPase complex. It should be noted, however, that the 0 These studies have led to a model in which the proton conduc- absence of one of the smaller V-ATPase subunits (i.e. the VMA7 tion pathway is composed of several residues of the a subunit, or VMA10 gene products) might not be detectable by this possibly arranged as an amphipathic helix, with the buried method and that no test for “correct” assembly of the V-ATPase carboxylate of the c subunit providing a gate in the conduction complex has been performed. pathway (60, 65). To further characterize these mutants, we have determined By analogy with the F-ATPases, we would predict that the the K for ATP and V values as well as the pH optima for m max V-ATPases should have some homolog to the a subunit, which the wild type and for each of the mutants affecting activity as would serve a comparably important role in proton transloca- well as for Q634L. As can be seen from the data in Table I, no tion. Of the V subunits besides the c subunit (which contains change greater than 40% was observed in K values for any of 0 no more buried polar or charged residues than the F c subunit the mutations tested, although the V values are in good max (38, 25)), the 36-kDa subunit (Vma6p) contains no putative agreement with the activity data shown in Fig. 1. These data transmembrane helices (23, 67). The sequence of the highly indicate that the observed decrease in activity in these mutants hydrophobic mammalian 19-kDa subunit has not been ob- is not due to a greatly diminished affinity for ATP. Interest- tained; however, no yeast counterpart to this subunit has yet ingly, two of the mutations did result in a very significant shift been identified. The remaining yeast V subunit, the 13-kDa in the pH optima of the enzyme. Thus, whereas the optimum pH for the wild type V-ATPase was approximately 7.2, that for product of the VMA10 gene (24), resembles the product of the VMA6 gene in possessing no putative transmembrane helices. H743A was 8.2, whereas that for E789Q was 9.7. In addition, the activity at the pH optimum of these mutants was approx- Thus the only yeast V subunit that has been identified that imately 75% (for H743A) and 30% (for E789Q) of the activity at might serve the role of the a subunit in the V-ATPase complex the optimum pH of the wild type. is the 100-kDa subunit. Vph1p Mutagenesis 22491 FIG.3. Comparison of effects of mutations in Vph1p on bafilomycin-sensitive ATPase activity, ATP-dependent proton transport, and the presence of A subunit on isolated vacuolar membrane vesicles. Bafilomycin-sensitive ATPase activity and ATP-dependent proton transport were measured on vacuolar membrane vesicles (5 mg of protein) as described in the legend to Fig. 1. Each bar represents the average of two determinations made on a single vacuolar preparation. An aliquot of the same preparation containing 5 mg of protein was analyzed by Western blot using the antibody 8B1-F3 against the 69-kDa A subunit as described in the legend to Fig. 2, and the resultant blot was quantitated using an IS-1000 Digital Imaging System from Alpha Innotech Corporation. WT, wild type. The 100-kDa subunit is composed of an NH -terminal hydro- plasmic side of the vacuole. Further work is thus necessary to philic domain of 45 kDa and a COOH-terminal hydrophobic resolve the actual orientation of the 100-kDa subunit in the domain of 55 kDa containing 6–7 putative transmembrane membrane. helices (21, 22, 68). In addition to the two yeast 100-kDa sub- Like the F a subunit, the 100-kDa subunit possesses mul- unit genes (VPH1 and STV1), three additional cDNAs from tiple polar and charged residues located within the putative mouse, rat, and bovine have been cloned (68–70), with 40–95% transmembrane helices in the hydrophobic COOH-terminal do- amino acid identity observed between pairs of these sequences main. To test whether the 100-kDa subunit might be playing (22). Based upon hydropathy analysis (21), a tentative model an analogous role to the a subunit in proton translocation, we for the folding of the 100-kDa subunit in the membrane is carried out site-directed mutagenesis of 22 such polar and shown in Fig. 5. The amino-terminal hydrophilic domain has charged residues in this domain. The residues selected for been placed on the lumenal side of the membrane based upon mutagenesis (shown circled in Fig. 5) are all conserved between two observations. First, labeling of the coated vesicle 100-kDa the available 100-kDa sequences from yeast, mouse, rat, and subunit by membrane impermeant reagents is only observed bovine. after detergent permeabilization of the membrane (27), sug- Interestingly, most of the mutations tested did not show any gesting that the large hydrophilic domain is sequestered within impairment of proton translocation. These residues are pre- the lumen of the coated vesicle. Second, proteolysis of the sumably not individually critical for proton translocation by 100-kDa subunit by trypsin in intact coated vesicles results in the V-ATPase, although it is possible that replacement of sev- cleavage at a site between transmembrane helices five and six. eral of these residues together might impair proton transport. 735 634 Because coated vesicles are oriented with the cytoplasmic sur- Mutations at Arg and Gln in H6 and the border of H5, face exposed, this places the loop between H5 and H6 on the respectively, led to the nearly complete absence of the 100-kDa cytoplasmic side of the membrane. Tracing of the polypeptide subunit in the vacuolar membrane, suggesting that the pro- back to the amino terminus places the amino-terminal domain teins containing these mutations were either unstable and on the lumenal side of the membrane. There are other data, rapidly degraded or else mistargeted to some other intracellu- however, that make this assignment tentative. Thus protease lar membrane. Given the results that suggest that the 100-kDa treatment of intact yeast vacuoles leads to disappearance on subunit possesses targeting information in yeast (22), the lat- Western blots of any band recognized by a polyclonal antiserum ter possibility should not be ruled out. However, the fact that raised against a peptide located in the amino-terminal domain for most integral membrane proteins in yeast targeting to the of Vph1p, suggesting that this epitope is exposed on the cyto- vacuole represents the default pathway (71) makes it more 3 4 I. Adachi and M. Forgac, unpublished data. M. Manolson, unpublished observations. 22492 Vph1p Mutagenesis likely that these mutations have affected stability of the 100- on the vacuolar membrane. Because these four mutations pre- 425 538 kDa subunit. Mutations at Asp and Lys in H1 and at the vented the appearance of a V-ATPase complex in the vacuolar border of H3, on the other hand, did not prevent folding and membrane, it is not possible to determine the role of the cor- targeting of the 100-kDa subunit to the vacuolar membrane but responding residues in proton transport. did interfere with proper assembly of the V-ATPase complex as The mutations of greatest interest are those that inhibited demonstrated by the reduced level of the peripheral A subunit proton transport and ATPase activity but did not have obvious effects on assembly or stability of the V-ATPase complex. The three residues that fell into this last category are Lys at the 743 789 border of H4, His in H6, and Glu in H7. For both K593A and H743A, proton transport and ATPase activity were only 50% of that predicted on the basis of the amount of assembly observed, whereas for E789Q, the V-ATPase complex was vir- TABLE I Comparison of K for ATP, V and pH optima for wild type, m max K593A, Q634L, H743A, and E789Q pH optima were determined by measurement of bafilomycin-sensitive ATPase activity in purified vacuolar membranes (5 mg of protein) over the pH range of 5.5–10.5 in the assay mixture. K for ATP and V m max values were determined for bafilomycin-sensitive ATPase activity on purified vacuolar membranes (5 mg of protein) over a range of ATP concentrations of 0.5–10 mM. MgCl was varied such that the total Mg concentration was in all cases 1.0 mM higher than the ATP concentration. This ensured that most of the ATP was present as the MgATP complex but avoided the possibility of inhibition of activity due to high concentrations of free Mg . The V values are reported max relative to the wild type, which had a specific activity of 3.3 mmol ATP/min/mg protein. The values shown are the average of two inde- FIG.4. Effect of Vph1p mutations on V-ATPase assembly. Yeast pendent determinations (at five ATP concentrations each) on a single cells (the Dvph1Dstv1 strain) expressing the wild type VPH1 gene, the vacuolar membrane preparation for each mutant, with the errors cor- indicated mutations or the vector alone were grown overnight in me- responding to the average deviation from the mean. thionine-free medium followed by conversion to spheroplasts and incu- 35 6 bation with [ S]Trans-label (50 mCi/5 3 10 spheroplasts) for 60 min at ATP rel Mutation pH optimum K V m max 30 °C. Spheroplasts were then pelleted and lysed in phosphate-buffered saline with C E , and the V-ATPase immunoprecipitated using the mM 12 9 monoclonal antibody 8B1-F3 directed against the 69-kDa A subunit and Wild type 7.2 0.70 6 0.08 1.00 6 0.07 protein A-Sepharose followed by SDS-polyacrylamide gel electrophore- K593A 7.0 0.61 6 0.17 0.33 6 0.03 sis on a 12% acrylamide gel and autoradiography as described under Q634L 7.0 0.43 6 0.11 0.18 6 0.02 “Experimental Procedures.” The positions of the V-ATPase subunits are H743A 8.2 0.79 6 0.35 0.50 6 0.02 indicated and were confirmed by comparison with the migration of E789Q 9.7 0.41 6 0.04 0.11 6 0.02 C-labeled molecular mass standards. WT, wild type. FIG.5. Model for folding of the 100-kDa subunit (Vph1p) of the yeast V-ATPase and the location and effects of point mutations in Vph1p. The orientation of the 100-kDa subunit in the membrane is based upon labeling and proteolysis data obtained for the 100-kDa subunit of the bovine coated vesicle V-ATPase (see text). In particular, the position at which trypsin cleaves the bovine 100-kDa subunit from the cytoplasmic side of the membrane is indicated. Residues that were mutated in this study are circled. Two mutations (R735L and Q634L) affected stability of the 100-kDa subunit, two mutations (D425N and K538A) affected assembly of the V-ATPase complex, whereas three mutations (H743A, K593A, and E789Q) affected proton transport and ATPase activity of the V-ATPase complex. Vph1p Mutagenesis 22493 25974–25977 tually completely devoid of activity. In addition, both the 19. Nelson, H., Mandiyan, S., and Nelson, N. (1994) J. Biol. Chem. 269, H743A and E789Q mutations resulted in a significant change 24150–24155 in the pH optimum of the enzyme. While it is difficult to assign 20. Manolson, M. F., Proteau, D., Preston, R. A., Stenbit, A., Roberts, B. T., Hoyt, M., Preuss, D., Mulholland, J., Botstein, D., and Jones, E. W. (1992) J. Biol. a precise interpretation to these findings, they may reflect an Chem. 267, 14294–14303 alteration in the environment of residues whose protonation 21. Manolson, M. F., Wu, B., Proteau, D., Taillon, B. E., Roberts, B. T., Hoyt, M. A., and Jones, E. W. (1994) J. Biol. Chem. 269, 14064–14074 state is important for transport to occur. Interestingly, as with 22. Bauerle, C., Ho, M. N., Lindorfer, M. A., and Stevens, T. H. (1993) J. Biol. the F a subunit, positively and negatively charged residues in Chem. 268, 12749–12757 the last two putative transmembrane helices appear to be 23. Supekova, L., Supek, F., and Nelson, N. (1995) J. Biol. Chem. 270, 13726–13732 important for activity, possibly serving to line a polar channel 24. Umemoto, N., Yoshihisa, T., Hirata, R., and Anraku, Y. (1990) J. Biol. Chem. necessary to allow protons to gain access to the buried carboxyl 265, 18447–18453 25. Umemoto, N., Ohya, Y., and Anraku, Y. (1991) J. Biol. Chem. 266, group of the c subunit. These results suggest that the 100-kDa 24526–24532 subunit may serve an analogous role to the a subunit in the 26. Arai, H., Terres, G., Pink, S., and Forgac, M. (1988) J. Biol. Chem. 263, V-ATPase complex. It is important to recognize, however, that 8796–8802 27. Senior, A. E. (1990) Annu. Rev. Biophys. Biophys. Chem. 19, 7–41 further work will be required to determine whether the resi- 28. Penefsky, H. S., and Cross, R. L. (1991) Adv. Enzymol. 64, 173–214 dues identified play a direct role in proton translocation or 29. Pedersen, P. L., and Amzel, L. M. (1993) J. Biol. Chem. 268, 9937–9940 whether they serve some other function in the V-ATPase com- 30. Futai, M., Park, M. Y., Iwamoto, A., Omote, H., and Maeda, M. (1994) Biochem. Biophys. Acta 1187, 165–170 plex, such as coupling of proton transport to ATP hydrolysis. 31. Bowman, E. J., Tenney, K., and Bowman, B. (1988) J. Biol. Chem. 263, We have previously presented data that suggest that the 13994–14001 32. Bowman, B. J., Allen, R., Wechser, M. A., and Bowman, E. J. (1988) J. Biol. 100-kDa subunit may also possess the binding site for the Chem. 263, 14002–14007 specific V-ATPase inhibitor bafilomycin A (58). Thus, recon- 33. Zimniak, L., Dittrich, P., Gogarten, J. P., Kibak, H., and Taiz, L. (1988) J. Biol. stituted V or isolated 100-kDa subunit are both able to protect Chem. 263, 9102–9112 34. Manolson, M. F., Ouellette, B. F. F., Filion, M., and Poole, R. J. (1988) J. Biol. the intact V-ATPase from inhibition by bafilomycin. When the Chem. 263, 17987–17994 100-kDa mutants constructed in the present study were tested 35. Puopolo, K., Kumamoto, C., Adachi, I., and Forgac, M. (1991) J. Biol. Chem. 266, 24564–24572 for their sensitivity to 1 nM bafilomycin (a subsaturating con- 36. Puopolo, K., Kumamoto, C., Adachi, I., Magner, R., and Forgac, M. (1992) centration that inhibits 40–50% of the V-ATPase activity in J. Biol. Chem. 267, 3696–3706 yeast vacuoles), no significant differences in bafilomycin sensi- 37. Mandel, M., Moriyama, Y., Hulmes, J. D., Pan, Y. C., Nelson, H., and Nelson, N. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 5521–5524 tivity were observed. Obviously mutations that resulted in 38. Guthrie, C., and Fink, G. R. (1991) Methods Enzymol. 194, 1–932 complete loss of activity (such as D425N and R735L) could not 39. Sanger, F., Niklen, S., and Coulson, A. R. (1977) Proc. Natl. Acad. Sci. U. S. A. be tested for bafilomycin sensitivity, but none of the remaining 74, 5463–5467 40. Gietz, D., St. Jean, A., Woods, R. A., and Schiestl, R. H. (1992) Nucleic Acids residues appears to be critical for bafilomycin binding to the Res. 20, 1425 V-ATPase. It is possible that the bafilomycin binding site might 41. Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K. (eds) (1992) Short Protocols in Molecular Biology, reside on the soluble amino-terminal domain or that hydropho- Wiley, New York bic rather than hydrophilic residues in the integral domain are 42. Yamashiro, C. T., Kane, P. M., Wolczyk, D. F., Preston, R. A., and Stevens, T. important in bafilomycin binding. Further studies will be nec- H. (1990) Mol. Cell. Biol. 10, 3737–3749 43. Kim, E. K., Zhen, R. G., and Rea, P. A. (1994) Proc. Natl. Acad. Sci. U. S. A 91, essary to resolve this question. 6128–6132 44. Roberts, C. J., Raymond, C. K., Yamashiro, C. T., and Stevens, T. H. (1991) Acknowledgments—We acknowledge Dr. Patricia Kane of the De- Methods Enzymol. 194, 644–661 partment of Biochemistry and Molecular Biology at SUNY, Syracuse, 45. Feng, Y., and Forgac, M. (1992) J. Biol. Chem. 267, 5817–5822 for the kind gift of the 10D7 antibody against the 100-kDa subunit and 46. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. for many helpful discussions and suggestions. We dedicate this paper to Chem. 193, 265–275 Dr Yasuhiro Anraku, whose many contributions over the years have 47. Laemmli, U. K. (1970) Nature 227, 680–685 48. Oakley, B. R., Kirsch, D. R., and Morris, N. R. (1980) Anal. Biochem. 105, greatly enriched the field of the vacuolar ATPases. 361–363 REFERENCES 49. Kane, P. M. (1995) J. Biol. Chem. 270, 17025–17032 50. Nelson, H., and Nelson, N. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 3503–3507 1. Forgac, M. (1989) Physiol. Rev. 69, 765–796 51. Bowman, E. J., Siebers, A., and Altendorf, K. (1988) Proc. Natl. Acad. Sci. 2. Forgac, M. (1992) J. Bioenerg. Biomembr. 24, 341–350 U. S. A. 85, 7972–7976 3. Bowman, B. J., Vazquez-Laslop, N., and Bowman, E. J. (1992) J. Bioenerg. 52. Kane, P. M., Kuehn, M. C., Howald-Stevenson, I., and Stevens, T. (1992) Biomembr. 24, 361–370 J. Biol. Chem. 267, 447–454 4. Kane, P. M., and Stevens, T. H. (1992) J. Bioenerg. Biomembr. 24, 383–394 53. Doherty, R. D., and Kane, P. M. (1993) J. Biol. Chem. 268, 16845–16851 5. Anraku, Y., Umemoto, N., Hirata, R., and Ohya, Y. (1992) J. Bioenerg. 54. Puopolo, K., and Forgac, M. (1990) J. Biol. Chem. 265, 14836–14841 Biomembr. 24, 395–406 55. Puopolo, K., Sczekan, M., Magner, R., and Forgac, M. (1992) J. Biol. Chem. 6. Gluck, S. L. (1992) J. Bioenerg. Biomembr. 24, 351–360 267, 5171–5176 7. Kibak, H., Taiz, L., Starke, T., Bernasconi, P., and Gogarten, J. P. (1992) 56. Zhang, J., Myers, M., and Forgac, M. (1992) J. Biol. Chem. 267, 9773–9778 J. Bioenerg. Biomembr. 24, 415–424 57. Zhang, J., Feng, Y., and Forgac, M. (1994) J. Biol. Chem. 269, 23518–23523 8. Nelson, N. (1992) J. Bioenerg. Biomembr. 24, 407–414 58. Arai, H., Berne, M., and Forgac, M. (1987) J. Biol. Chem. 262, 11006–11011 9. Adachi, I., Puopolo, K., Marquez-Sterling, N., Arai, H., and Forgac, M. (1990) 59. Fillingame, R. H. (1992) J. Bioenerg. Biomembr. 24, 485–491 J. Biol. Chem. 265, 967–973 60. Sun, S.-Z., Xie, X.-S., and Stone, D. K. (1987) J. Biol. Chem. 262, 14790–14794 10. Hirata, R., Ohsumi, Y., Nakano, A., Kawasaki, H., Suzuki, K., and Anraku, Y. 61. Foster, D. L., and Fillingame, R. H. (1982) J. Biol. Chem. 257, 2009–2015 (1990) J. Biol. Chem. 265, 6726–6733 62. Schneider, E., and Altendorf, K. (1985) EMBO J. 4, 515–518 11. Kane, P. M., Yamashiro, C. T., Wolczyk, D. F., Neff, N., Goebl, M., and Stevens, 63. Schneider, E., and Altendorf, K. (1987) Microbiol. Rev. 51, 477–497 T. H. (1990) Science 250, 651–657 64. Cain, B. D., and Simoni, R. D. (1986) J. Biol. Chem. 261, 10043–10050 12. Nelson, H., Mandiyan, S., and Nelson, N. (1989) J. Biol. Chem. 264, 1775–1778 65. Cain, B. D., and Simoni, R. D. (1988) J. Biol. Chem. 263, 6606–6612 13. Ho, M. N., Hirata, R., Umemoto, N., Ohya, Y., Takatsuki, A., Stevens, T. H., 66. Wang, S.-Y., Moriyama, Y., Mandel, M., Hulmes, J. D., Pan, Y.-C. E., Danho, and Anraku, Y. (1993) J. Biol. Chem. 268, 18286–18292 W., Nelson, H., and Nelson, N. (1988) J. Biol. Chem. 263, 17638–17642 14. Ho, M. N., Hill, K. J., Lindorfer, M. A., and Stevens, T. H. (1993) J. Biol. Chem. 67. Perin, M. S., Fried, V. A., Stone, D. K., Xie, X.-S., and Sudhof, T. C. (1991) 268, 221–227 15. Beltran, C., Kopecky, J., Pan, Y.-C. E., Nelson, H., and Nelson, N. (1992) J. Biol. Chem. 266, 3877–3881 68. Lee, C., Ghoshal, K., and Beaman, K. D. (1990) Mol. Immunol. 27, 1137–1144 J. Biol. Chem. 267, 774–779 16. Nelson, H., Mandiyan, S., and Nelson, N. (1995) Proc. Natl. Acad. Sci. U. S. A. 69. Peng, S.-B., Crider, B. P., Xie, X.-S., and Stone, D. K. (1994) J. Biol. Chem. 269, 17262–17266 92, 497–501 17. Foury, F. (1990) J. Biol. Chem. 265, 18554–18560 70. Roberts, C. J., Nothwehr, S. F., and Stevens, T. H. (1992) J. Cell Biol. 119, 18. Graham, L. A., Hill, K. J., and Stevens, T. H. (1994) J. Biol. Chem. 269, 69–83 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Biological Chemistry Unpaywall

Site-directed Mutagenesis of the 100-kDa Subunit (Vph1p) of the Yeast Vacuolar (H+)-ATPase

Leng, Xing-Hong; Manolson, Morris F.; Liu, Qing; Forgac, Michael
Journal of Biological Chemistry – Sep 1, 1996
Download PDF
7 pages

Loading next page...
 
/lp/unpaywall/site-directed-mutagenesis-of-the-100-kda-subunit-vph1p-of-the-yeast-pLk65XPh2a

References (71)

Publisher
Unpaywall
ISSN
0021-9258
DOI
10.1074/jbc.271.37.22487
Publisher site
See Article on Publisher Site

Abstract

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 271, No. 37, Issue of September 13, pp. 22487–22493, 1996 © 1996 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Site-directed Mutagenesis of the 100-kDa Subunit (Vph1p) of the Yeast Vacuolar (H )-ATPase* (Received for publication, April 29, 1996, and in revised form, June 21, 1996) Xing-Hong Leng, Morris F. Manolson‡, Qing Liu, and Michael Forgac§ From the Department of Cellular and Molecular Physiology, Tufts University School of Medicine, Boston, Massachusetts 02111 and the ‡Division of Cell Biology, Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada as synaptic vesicles and chromaffin granules, and the central Vacuolar (H )-ATPases (V-ATPases) are multisubunit complexes responsible for acidification of intracellular vacuoles of plants, Neurospora and yeast. Acidification of these compartments in eukaryotic cells. V-ATPases possess a compartments, in turn, plays an important role in such proc- subunit of approximate molecular mass 100 kDa of un- esses as receptor-mediated endocytosis, intracellular mem- known function that is composed of an amino-terminal brane traffic, protein processing and degradation, and coupled hydrophilic domain and a carboxyl-terminal hydropho- transport of small molecules. In yeast, acidification of the cen- bic domain. To test whether the 100-kDa subunit plays a tral vacuole is important both to maintain the activity of deg- role in proton transport, site-directed mutagenesis of 21 radative enzymes and to drive uptake of solutes such as Ca the VPH1 gene, which is one of two genes that encodes and amino acids (5). this subunit in yeast, has been carried out in a strain The V-ATPases are multisubunit complexes composed of two lacking both endogenous genes. Ten charged and twelve structural domains. The peripheral V domain is a 500-kDa polar residues located in the seven putative transmem- complex responsible for ATP hydrolysis, whereas the integral brane helices in the COOH-terminal domain of the mol- V domain is a 250-kDa complex responsible for proton trans- ecule were individually changed, and the effects on pro- location (10). In Saccharomyces cerevisiae, the V domain is ton transport, ATPase activity, and assembly of the 1 composed of seven different subunits of molecular masses 69 yeast V-ATPase were measured. Two mutations (R735L (subunit A encoded by VMA1 (11, 12)), 60 (subunit B/VMA2 and Q634L) in transmembrane helix 6 and at the border of transmembrane helix 5, respectively, showed greatly (13)), 54 (VMA13 (14)), 42 (subunit C/VMA5 (15, 16)), 32 (sub- reduced levels of the 100-kDa subunit in the vacuolar unit D/VMA8 (17)), 27 (subunit E/VMA4 (15, 18)), and 14 kDa membrane, suggesting that these mutations affected sta- (subunit F/VMA7 (19, 20)). The V domain is composed of at bility of the 100-kDa subunit. Two mutations, D425N and least four subunits of molecular masses approximately 100 K538A, in transmembrane helix 1 and at the border of (encoded by VPH1 and STV1 (21, 22)), 36 (VMA6 (23)), 13 transmembrane helix 3, respectively, showed reduced (VMA10 (24)), and 17 kDa (subunit c/VMA3 and VMA11 (25, assembly of the V-ATPase, with the D425N mutation also 26)). By analogy with the bovine coated vesicle V-ATPase (27), reducing the activity of V-ATPase complexes that did the V domain has the structure A B C D E F , whereas V 1 3 3 1 1 1 1 0 assemble. Two mutations, H743A and K593A, in trans- domain has the structure 100 36 c . No mammalian homolog 1 1 6 membrane helix 6 and at the border of transmembrane to Vma10p has yet been identified, and the yeast counterpart to helix 4, respectively, have significantly greater effects the bovine 19-kDa V subunit is also uncertain. on activity than on assembly, with proton transport and Although the V-ATPases are homologous to the F-ATPases ATPase activity inhibited 40–60%. One mutation, E789Q, (28–31), both in overall structure (10, 27) and in sequence in transmembrane helix 7, virtually completely abol- homology of several of the subunits (11–13, 25, 32–38), no ished proton transport and ATPase activity while hav- ing no effect on assembly. These results suggest that the obvious structural homolog exists for the 100-kDa subunit in 100-kDa subunit may be required for activity as well as the F-ATPases. The 100-kDa subunit of the V-ATPase in yeast assembly of the V-ATPase complex and that several is encoded by two homologous genes, VPH1 (21) and STV1 (22). charged residues in the last four putative transmem- VPH1 encodes a 95-kDa protein, which possesses a hydrophilic brane helices of this subunit may play a role in proton amino-terminal domain of approximately 45 kDa and a carbox- transport. yl-terminal hydrophobic domain of approximately 50 kDa con- taining 6–7 putative transmembrane helices (21). STV1 en- codes a 102-kDa protein that shares the same domain 1 1 The vacuolar (H )-ATPases (V-ATPases) are a family of arrangement and is 54% identical in amino acid sequence with proton pumps responsible for acidification of intracellular com- the product of the VPH1 gene (Vph1p) (22). Disruption of the partments in eukaryotic cells (for reviews see Refs. 1–9). VPH1 gene leads to somewhat reduced growth at neutral pH Among the compartments acidified by V-ATPases are clathrin- relative to acidic pH (21) and disruption of STV1 has no obvious coated vesicles, endosomes, lysosomes, secretory vesicles, such phenotypic consequences (22). By contrast, disruption of both VPH1 and STV1 leads to the typical Vma phenotype, includ- * This work was supported by National Institutes of Health Grant ing the inability to grow at neutral pH and hypersensitivity to GM 34478 (to M. F.) and a Medical Research Council of Canada grant 21 Ca (22). These results suggest that the VPH1 and STV1 gene (to M. F. M.). Fluorescence facilities were provided by National Insti- products are at least partially able to substitute for each other. tutes of Health Grant DK 34928. The costs of publication of this article were defrayed in part by the payment of page charges. This article must The reason that yeast possess two genes encoding the 100-kDa therefore be hereby marked “advertisement” in accordance with 18 subunit is uncertain, but the results available thus far suggests U.S.C. Section 1734 solely to indicate this fact. that V-ATPases possessing Vph1p and Stv1p may be targeted § To whom correspondence should be addressed. to different intracellular membranes (22). In order to deter- The abbreviations used are: V-ATPase, vacuolar proton-translocat- ing adenosine triphosphatase; Mes, 4-morpholineethanesulfonic acid. mine whether the 100-kDa subunit plays a direct role in proton This is an open access article under the CC BY license. 22487 22488 Vph1p Mutagenesis a coupled spectrophotometric assay (45) with the modification of using transport by the V-ATPase complex, site-directed mutagenesis 0.35 mM of NADH instead of 0.5 mM NADH. ATP-dependent proton of the VPH1 gene product has been carried out in a strain transport was measured in transport buffer (25 mM Mes/Tris, pH 7.2, 5 lacking endogenous Vph1p and Stv1p. mM MgCl , and 25 mM KCl) using the fluorescence probe amino-6- chloro-2-methoxyacridine as described previously (46) in the presence EXPERIMENTAL PROCEDURES or the absence of 10 nM bafilomycin A . Protein concentration was Materials and Strains—Zymolyase 100T was obtained from Seika- measured using the Lowry method (47). SDS-polyacrylamide gel elec- gaku America, Inc. [ S]Trans-label was purchased from ICN. Bafilo- trophoresis was carried out as described by Laemmli (48). Silver stain- mycin A was a kind gift from Dr. Karlheinz Altendorf (University of ing was performed using the method of Oakley et al. (49). Osnabruck). Leupeptin was from Boehringer Mannheim. 9-Amino-6- Western blots were probed with mouse monoclonal antibodies chloro-2-methoxyacridine was from Molecular Probes, Inc. ATP, phen- 8B1-F3 against the 69-kDa subunit, (from Molecular Probes, Inc.) or ylmethylsulfonylfluoride, and most other chemicals were purchased 10D7 against the 100-kDa subunit, (a generous gift from Dr. P.Kane), from Sigma. followed by horseradish peroxidase-conjugated secondary antibody MM322 (VPH1 in pRS316) and yeast strain MM112 (MATa (Bio-Rad). Blots were developed using a chemiluminescent detection Dvph1::LEU2 Dstv1::LYS2 his3-D200 leu2 lys2 ura3–52) (22) were used method obtained from KPL. Quantitations were done using an IS-1000 to generate and study VPH1 mutants. Yeast cells were grown in YPD Digital imaging system (Alpha Innotech Corporation). medium (yeast extract-peptone-dextrose) or synthetic dropout synthetic Immunoprecipitations were carried out as described (50), with the medium (39). following modifications. Cells were grown overnight in supplemented Mutagenesis—Mutagenesis was performed on SalI-EcoRI or EcoRI- minimal medium lacking methionine and then converted to sphero- BamHI fragments of the wild type VPH1 gene in the vector pALTER-1 plasts by incubation for 20 min with 0.5 unit of zymolase/10 cells in (Promega) following the manufacturer’s protocol. The mutagenesis SD-Met, 1.2 M sorbitol, 50 mM Tris-Mes, pH 7.5. Aliquots containing 5 3 6 35 oligonucleotides were as follows with substitution sites underlined: 10 spheroplasts were then incubated with [ S]Trans-label (50 mCi) for T411A, 59-GTGACAATTGCGGGTAAACCAG-39; T414A, 59-ACATGAA- 60 min at 30 °C. Spheroplasts were then pelleted, lysed in phosphate- AGGGAATGCGACAATTGTGGG-39; D425N, 59-GACCCATATTACC buffered saline with C E and immunoprecipitated (50) using 7.5 mgof 12 9 AAACATG-39; H428, 59-GTCATTAAGAACCCGGCACCCATATCACC- purified 8B1-F3 antibody and protein A-Sepharose followed by SDS- A-39; R462V, 59-AACAAAATAATGTATACACCAGTGAAGGCCA-39; polyacrylamide gel electrophoresis on 12% acrylamide gels and autora- Y463F, 59-ACAAAATAATGAATCTACCAG-39; S472A, 59-GTGTACAT- diography as described (50). GGCAAAGACACCCA-39; Y474F, 59-GAAACCTGTGAACATGGAAAA- RESULTS G-39; T475A, 59-GAAACCTGCGTACATGG-39; K538A, 59-ATTAAAAT- TGATAGTGCCATTTTGTAAGAATTA-39; S540A, 59-GAACCCCATTA- It has previously been shown that deletion of genes encoding AAATGGCTAGTTTCATTTTGTA-39; H547A, 59-ATAAGAATAGGTCA- subunits of the yeast V-ATPase leads to a conditional lethal TCGCGATGAACCCCAT-TAA-39; Y585F, 59-AACGGAAAGAAAACCA- phenotype such that strains carrying such deletions are unable AAGA-39; Y592F, 59-ATCAACAGCCCATTTAAAAACAATACAAACG-39; to grow at neutral pH but are able to grow at acidic pH (51). We K593A, 59-AATCAACAGCCCACGCGTAAACAATACAAACG-39; Q634L, 59-ACAAAAACACTAGGACC TTTG-39; C644A,59-GCAACCAAGGAATG- have employed a strain in which both the VPH1 and STV1 GCAACCAAGGCCATCA-39; K652A, 59-GAAATGTAATGGCGCCACCA- genes encoding the 100-kDa subunit of the V-ATPase have ATAGCAACCA-39; R735L, 59-CCTATTTACTTTTATGGGC-39; H743A, 2 been disrupted, leading to the typical Vma phenotype (22). 59-CTTATCATTGGCAGCTGCTCAATTGTCTA-39; T781V, 59-GTGGT- Expression of the VPH1 gene on a CEN plasmid in this strain TCGCACTAGTATGTGCAGTTCTTG-39; E789Q, 59-GTTTTGATGCAA- leads to growth at pH 7.5. Twenty-two individual site-directed GGT ACA-39. Fragments containing the indicated mutations were sequ- mutations were introduced into the VPH1 gene and the mutant enced using the diedoxy method (40) to confirm that no other mutations had been introduced and were then substituted back into the vector proteins expressed in the double knock out strain. The residues containing the wild type VPH1 gene. selected for mutation all correspond to polar or charged resi- Transformation—Yeast cells were transformed with the wild type dues located within the seven putative transmembrane helices plasmid (pRS316-VPH1), mutants or the vector pRS316 alone (as a in the COOH-terminal half of the 100-kDa subunit. These negative control) using the lithium acetate procedure (41) and were 2 residues were selected in an effort to identify buried polar or selected on Ura plates as described previously (42). The mutants were charged amino acids that might directly contribute to the pro- tested for growth on pH 7.5 or pH 5.5 YPD plates buffered with 50 mM KH PO and 50 mM succinic acid (43). ton conduction pathway in the V-ATPase complex. Polar resi- 2 4 Isolation of Vacuolar Membrane Vesicles—Vacuolar membrane ves- dues were changed to nonpolar residues of similar size (for icles were isolated using a modification of the protocol described by Kim example substituting alanine for serine or phenylalanine for et al. (44). Yeast were grown overnight at 30 °C to 1 3 10 cells/ml in 1 tyrosine), whereas charged residues were replaced either by liter of selective medium. Cells were pelleted, washed once with water, similar polar amino acids (i.e. glutamine for glutamic acid) or and resuspended in 50 ml of 10 mM dithiothreitol, 100 mM Tris-HCl, pH by nonpolar amino acids (usually alanine). It is anticipated 9.4. After incubation at 30 °C for 15 min, cells were pelleted again, resuspended in 50 ml of YEPD medium containing 0.7 M sorbitol, 2 mM that replacement of a polar or charged amino acid that directly dithiothreitol, and 100 mM Tris-Mes, pH 7.5, and 5 mg of Zymolyase participates in proton movement across the membrane will 100T and incubated at 30 °C with gentle shaking for 90 min. The disrupt ATP-dependent proton transport. Of these 22 muta- resulting spheroplasts were washed twice with ice-cold 1.2 M sorbitol, tions, only three showed greatly reduced growth at neutral pH and pelleted at 3500 3 g for 10 min at 4 °C. The pellet was resuspended (E789Q, D425N, and R735L) (data not shown), with the latter in 40 ml of homogenization buffer (10% glycerol, 1.5% polyvinylpyrro- two having an identical growth phenotype to the double knock lidone (M 40,000), 0.25 mM MgCl , 2 mg/ml bovine serum albumin, 50 r 2 mM Tris-ascorbate, pH 7.5, 1 mM phenylmethylsulfonyl fluoride, 1 out strain. Because as little as 20% of the wild type V-ATPase mg/ml leupeptin), transferred to a Dounce glass homogenizer and sub- activity is sufficient to rescue the growth phenotype of a Vma jected to 20 strokes with a tight fitting pestle. The homogenate was 2 strain, it was necessary to measure the vacuolar proton pump- centrifuged at 3500 3 g for 15 min at 4 °C, and the supernatant was ing and V-ATPase activity in each mutant strain to assess the transferred to a Ti 45 centrifuge tube and spun for 35 min at 100,000 3 effect of the mutation on V-ATPase activity. g at 4 °C. The pellets were resuspend in 8 ml of overlay medium, which Fig. 1 shows the ATP-dependent proton transport and the contained 1.1 M glycerol, 2 mM dithiothreitol, 0.25 mM MgCl , 2 mg/ml bovine serum albumin, and 5 mM Tris-Mes, pH 7.6, and homogenized by bafilomycin-sensitive ATPase activity for isolated vacuoles pu- 10 strokes in a Dounce glass homogenizer using a tight fitting pestle. rified from a Dvph1Dstv1 strain expressing the pRS316 plasmid The homogenate was overlaid onto a one step 30-ml 10–30% sucrose alone, pRS316 containing the wild type VPH1 gene, or the gradient and centrifuged for2hat 100,000 3 g in an SW-28 rotor. VPH1 gene bearing the indicated point mutations. Bafilomycin Material at the 10–30% interface was collected, diluted 10-fold with A has been shown to be a specific inhibitor of the V-ATPases overlay medium, and centrifuged at 100,000 3 g for 35 min at 4 °C. The pellets were resuspend in 0.5–1 ml of overlay medium, quick frozen with liquid nitrogen, and stored at 280 °C until used. Biochemical Characterization—ATPase activity was measured using P. Kane, personal communication. Vph1p Mutagenesis 22489 FIG.1. Effect of Vph1p mutations on bafilomycin-sensitive ATPase activity and ATP-dependent proton transport in purified vacuolar membrane vesicles. ATPase activity and ATP-dependent proton transport were measured on aliquots of purified vacuolar membrane vesicles containing 5 mg of protein as described under “Experimental Procedures.” Activities are expressed relative to the Dvph1Dstv1 strain expressing the pRS316 plasmid containing the wild type VPH1 gene (defined as 100%). The specific ATPase activity of these vacuolar membrane vesicles was 3.3 mmol ATP/min/mg protein at saturating ATP and 37° C, with approximately 80% of the ATPase activity inhibitable by 10 nM bafilomycin. All of the ATP-dependent proton transport in vacuolar membrane vesicles isolated from cells expressing the wild type Vph1p was inhibitable by 10 nM bafilomycin. No ATP-dependent proton transport or bafilomycin sensitive-ATPase activity was observed in vacuolar membrane vesicles isolated form cells transformed with the vector alone. Each bar represents the average of two or three determinations made on two or three independent vacuolar membrane preparations, with the error corresponding to the standard deviation. WT, wild type. (52). As expected on the basis of the growth phenotype, three ence of the 100-kDa and A subunits on the vacuolar membrane mutations (D425N, R735L, and E789Q) showed 20% or less of provides a reasonable measure of V-ATPase assembly. the wild type proton transport and V-ATPase activity, with Fig. 2 shows a Western blot carried out on vacuolar mem- both D425N and R735L having virtually no detectable activity. branes from the wild type, deletion, and mutant strains using Four additional mutations (K538A, K593A, Q634L, and antibodies directed against either Vph1p or the A subunit, H743A) showed between 20 and 70% of wild type activity, with whereas Fig. 3 shows the results of quantitative analysis of the Q634L having the lowest activity of this group. The remaining Western blot as well as the activity data on each mutant for mutants all displayed 70% or greater of wild type activity. comparison. As can be seen, two mutants (R735L and Q634L) None of the mutations resulted in significant uncoupling of showed greatly reduced levels of the 100-kDa subunit in the proton transport from ATP hydrolysis (i.e. a greater loss of vacuolar membrane as well as greatly reduced levels of A proton transport than ATPase activity), although one mutation subunit. These mutations thus appear to affect stability of the (Q634L) did decrease ATPase activity to a somewhat greater 100-kDa subunit. Two other mutations (D425N and K538A) extent than proton transport, suggesting a possible increase in show significantly reduced levels of A subunit associated with coupling efficiency. the vacuolar membrane with only slightly reduced levels of the The most obvious ways in which mutations in the 100-kDa 100-kDa subunit. These mutations thus appear to have an subunit might lead to a decrease in V-ATPase activity is if effect on assembly of the V-ATPase complex. It should be noted these mutations resulted in a 100-kDa subunit that was un- from Fig. 3 that the D425N mutant, while still having detect- stable and rapidly degraded or that was unable to correctly able levels of assembly as assessed by the presence of the A assemble with the remaining subunits of the V-ATPase com- subunit, is completely devoid of proton transport and ATPase plex. To assess these possibilities, Western blot analysis was activity, suggesting that any V-ATPase that does assemble is performed on isolated vacuoles using antibodies directed inactive. This result suggests that Asp may play a role in against Vph1p and the A subunit of the V-ATPase. It has both assembly and activity of the V-ATPase. previously been shown that disruption of the VPH1 gene leads The remaining mutants have near normal levels of the 100- to the inability of the V domain (including the A subunit) to kDa and A subunits. In particular, the E789Q mutant shows assemble onto the vacuolar membrane (21). Moreover, the ab- wild type levels of both subunits while possessing less than sence of any of the V subunits (with the exception of the 20% of the wild type levels of proton transport and ATPase 54-kDa subunit (14)) leads to loss of assembly of the entire V activity. Similarly, examination of Fig. 3 reveals that both the domain onto the vacuolar membrane (53, 54). Thus the pres- K593A and H743A mutants have approximately half as much 22490 Vph1p Mutagenesis DISCUSSION The V-ATPases resemble the F-ATPases of mitochondria, chloroplasts, and bacteria (28–31) both in overall structure (10, 27) and in sequence homology between several of the subunits, including the nucleotide binding A and B subunits (32–37, 11–13) and the dicyclohexylcarbodiimide-reactive c subunit (25, 38). As with the F-ATPases, the V-ATPases possess a peripheral domain (V ) responsible for ATP hydrolysis and an integral domain (V ) responsible for proton translocation. Un- like the corresponding F-ATPase domains, however, the V and V domains do not retain their respective activities when sep- arated from each other (55–57). An important question with regard to the V-ATPases is the mechanism by which they translocate protons. For the bovine coated vesicle V-ATPase, the V domain, which is responsible for proton translocation (58), is composed of four subunits of approximate molecular masses 100, 38, 19, and 17 kDa (sub- unit c) (57) that are present in a stoichiometry of 100 38 19 c 1 1 1 6 FIG.2. Effect of Vph1p mutations on stability of the 100-kDa (27). Of these subunits, only the 17-kDa c subunit has been subunit and association of the 69-kDa A subunit with the vacu- shown to directly participate in proton translocation by virtue olar membrane. Vacuolar membrane vesicles (5 mg) isolated from the of its reaction with dicyclohexylcarbodiimide (59). By analogy Dvph1Dstv1 strain expressing the wild type VPH1 gene, the VPH1 gene with the F-ATPase c subunit (60), dicyclohexylcarbodiimide bearing the indicated mutations, or the vector alone were subjected to reacts with a buried carboxyl group located near the middle of SDS-polyacrylamide gel electrophoresis on a 12% acrylamide gel fol- lowed by transfer to nitrocellulose and Western blot analysis using the the last of four putative transmembrane helices. Although a monoclonal antibody 10D7 against Vph1p or 8B1-F3 against the 69-kDa low level of proton translocation has been reported for the A subunit as described under “Experimental Procedures.” WT, wild isolated, reconstituted c subunit (61), optimal levels of dicyclo- type. hexylcarbodiimide-inhibitable proton transport are only ob- served when the complete complement of V subunits are re- proton transport and ATPase activity as predicted on the basis assembled prior to reconstitution (58). The basis for difference of the amount of assembled V-ATPase. To test whether, for in proton conduction properties of the native and reassembled these mutations, the observed decrease in activity might be due V domains remains uncertain. to loss of one of the other V-ATPase subunits from the complex, In the case of the F-ATPases, the F domain of Escherichia the following experiment was performed. Cells were converted 35 coli is composed of three subunits of molecular masses 30 (a), to spheroplasts and metabolically labeled with [ S]Trans-label 17 (b), and 8 kDa (c) that are present in a stoichiometry of for 60 min at 30 °C followed by cell lysis, detergent solubiliza- a b c (62). Reassembly studies indicate that for F , all 1 2 10–12 0 tion, and immunoprecipitation using the anti-A subunit anti- three subunits are required to form a functional proton channel body 8B1-F3 and protein A-Sepharose. As can be seen in Fig. 4, (63, 64). Mutational analysis has demonstrated that the buried the complete complement of V and V subunits are immuno- 1 0 carboxyl group located in the second of two transmembrane precipitated from cells expressing wild type Vph1p, whereas helices of the c subunit is critical for proton translocation (60). only the V subunits are immunoprecipitated in the vector Moreover, genetic analysis of the a subunit indicates the pres- control. Of the three mutants listed above, H743A and E789Q ence of several buried polar and charged residues, particularly showed wild type patterns of immunoprecipitation, whereas in the last two transmembrane helices, which also play a crit- K593A showed the normal pattern of subunits but with some- ical role in proton transport (65, 66). Thus, substitutions at what reduced levels of the V subunits, as predicted on the 206 210 219 Ser , Arg , and Glu in the fifth putative transmembrane basis of the data shown in Fig. 3. Thus, all three of these helix or His in the sixth putative transmembrane helix of the mutations appear to impair activity rather than assembly of a subunit significantly impair proton translocation through F . the V-ATPase complex. It should be noted, however, that the 0 These studies have led to a model in which the proton conduc- absence of one of the smaller V-ATPase subunits (i.e. the VMA7 tion pathway is composed of several residues of the a subunit, or VMA10 gene products) might not be detectable by this possibly arranged as an amphipathic helix, with the buried method and that no test for “correct” assembly of the V-ATPase carboxylate of the c subunit providing a gate in the conduction complex has been performed. pathway (60, 65). To further characterize these mutants, we have determined By analogy with the F-ATPases, we would predict that the the K for ATP and V values as well as the pH optima for m max V-ATPases should have some homolog to the a subunit, which the wild type and for each of the mutants affecting activity as would serve a comparably important role in proton transloca- well as for Q634L. As can be seen from the data in Table I, no tion. Of the V subunits besides the c subunit (which contains change greater than 40% was observed in K values for any of 0 no more buried polar or charged residues than the F c subunit the mutations tested, although the V values are in good max (38, 25)), the 36-kDa subunit (Vma6p) contains no putative agreement with the activity data shown in Fig. 1. These data transmembrane helices (23, 67). The sequence of the highly indicate that the observed decrease in activity in these mutants hydrophobic mammalian 19-kDa subunit has not been ob- is not due to a greatly diminished affinity for ATP. Interest- tained; however, no yeast counterpart to this subunit has yet ingly, two of the mutations did result in a very significant shift been identified. The remaining yeast V subunit, the 13-kDa in the pH optima of the enzyme. Thus, whereas the optimum pH for the wild type V-ATPase was approximately 7.2, that for product of the VMA10 gene (24), resembles the product of the VMA6 gene in possessing no putative transmembrane helices. H743A was 8.2, whereas that for E789Q was 9.7. In addition, the activity at the pH optimum of these mutants was approx- Thus the only yeast V subunit that has been identified that imately 75% (for H743A) and 30% (for E789Q) of the activity at might serve the role of the a subunit in the V-ATPase complex the optimum pH of the wild type. is the 100-kDa subunit. Vph1p Mutagenesis 22491 FIG.3. Comparison of effects of mutations in Vph1p on bafilomycin-sensitive ATPase activity, ATP-dependent proton transport, and the presence of A subunit on isolated vacuolar membrane vesicles. Bafilomycin-sensitive ATPase activity and ATP-dependent proton transport were measured on vacuolar membrane vesicles (5 mg of protein) as described in the legend to Fig. 1. Each bar represents the average of two determinations made on a single vacuolar preparation. An aliquot of the same preparation containing 5 mg of protein was analyzed by Western blot using the antibody 8B1-F3 against the 69-kDa A subunit as described in the legend to Fig. 2, and the resultant blot was quantitated using an IS-1000 Digital Imaging System from Alpha Innotech Corporation. WT, wild type. The 100-kDa subunit is composed of an NH -terminal hydro- plasmic side of the vacuole. Further work is thus necessary to philic domain of 45 kDa and a COOH-terminal hydrophobic resolve the actual orientation of the 100-kDa subunit in the domain of 55 kDa containing 6–7 putative transmembrane membrane. helices (21, 22, 68). In addition to the two yeast 100-kDa sub- Like the F a subunit, the 100-kDa subunit possesses mul- unit genes (VPH1 and STV1), three additional cDNAs from tiple polar and charged residues located within the putative mouse, rat, and bovine have been cloned (68–70), with 40–95% transmembrane helices in the hydrophobic COOH-terminal do- amino acid identity observed between pairs of these sequences main. To test whether the 100-kDa subunit might be playing (22). Based upon hydropathy analysis (21), a tentative model an analogous role to the a subunit in proton translocation, we for the folding of the 100-kDa subunit in the membrane is carried out site-directed mutagenesis of 22 such polar and shown in Fig. 5. The amino-terminal hydrophilic domain has charged residues in this domain. The residues selected for been placed on the lumenal side of the membrane based upon mutagenesis (shown circled in Fig. 5) are all conserved between two observations. First, labeling of the coated vesicle 100-kDa the available 100-kDa sequences from yeast, mouse, rat, and subunit by membrane impermeant reagents is only observed bovine. after detergent permeabilization of the membrane (27), sug- Interestingly, most of the mutations tested did not show any gesting that the large hydrophilic domain is sequestered within impairment of proton translocation. These residues are pre- the lumen of the coated vesicle. Second, proteolysis of the sumably not individually critical for proton translocation by 100-kDa subunit by trypsin in intact coated vesicles results in the V-ATPase, although it is possible that replacement of sev- cleavage at a site between transmembrane helices five and six. eral of these residues together might impair proton transport. 735 634 Because coated vesicles are oriented with the cytoplasmic sur- Mutations at Arg and Gln in H6 and the border of H5, face exposed, this places the loop between H5 and H6 on the respectively, led to the nearly complete absence of the 100-kDa cytoplasmic side of the membrane. Tracing of the polypeptide subunit in the vacuolar membrane, suggesting that the pro- back to the amino terminus places the amino-terminal domain teins containing these mutations were either unstable and on the lumenal side of the membrane. There are other data, rapidly degraded or else mistargeted to some other intracellu- however, that make this assignment tentative. Thus protease lar membrane. Given the results that suggest that the 100-kDa treatment of intact yeast vacuoles leads to disappearance on subunit possesses targeting information in yeast (22), the lat- Western blots of any band recognized by a polyclonal antiserum ter possibility should not be ruled out. However, the fact that raised against a peptide located in the amino-terminal domain for most integral membrane proteins in yeast targeting to the of Vph1p, suggesting that this epitope is exposed on the cyto- vacuole represents the default pathway (71) makes it more 3 4 I. Adachi and M. Forgac, unpublished data. M. Manolson, unpublished observations. 22492 Vph1p Mutagenesis likely that these mutations have affected stability of the 100- on the vacuolar membrane. Because these four mutations pre- 425 538 kDa subunit. Mutations at Asp and Lys in H1 and at the vented the appearance of a V-ATPase complex in the vacuolar border of H3, on the other hand, did not prevent folding and membrane, it is not possible to determine the role of the cor- targeting of the 100-kDa subunit to the vacuolar membrane but responding residues in proton transport. did interfere with proper assembly of the V-ATPase complex as The mutations of greatest interest are those that inhibited demonstrated by the reduced level of the peripheral A subunit proton transport and ATPase activity but did not have obvious effects on assembly or stability of the V-ATPase complex. The three residues that fell into this last category are Lys at the 743 789 border of H4, His in H6, and Glu in H7. For both K593A and H743A, proton transport and ATPase activity were only 50% of that predicted on the basis of the amount of assembly observed, whereas for E789Q, the V-ATPase complex was vir- TABLE I Comparison of K for ATP, V and pH optima for wild type, m max K593A, Q634L, H743A, and E789Q pH optima were determined by measurement of bafilomycin-sensitive ATPase activity in purified vacuolar membranes (5 mg of protein) over the pH range of 5.5–10.5 in the assay mixture. K for ATP and V m max values were determined for bafilomycin-sensitive ATPase activity on purified vacuolar membranes (5 mg of protein) over a range of ATP concentrations of 0.5–10 mM. MgCl was varied such that the total Mg concentration was in all cases 1.0 mM higher than the ATP concentration. This ensured that most of the ATP was present as the MgATP complex but avoided the possibility of inhibition of activity due to high concentrations of free Mg . The V values are reported max relative to the wild type, which had a specific activity of 3.3 mmol ATP/min/mg protein. The values shown are the average of two inde- FIG.4. Effect of Vph1p mutations on V-ATPase assembly. Yeast pendent determinations (at five ATP concentrations each) on a single cells (the Dvph1Dstv1 strain) expressing the wild type VPH1 gene, the vacuolar membrane preparation for each mutant, with the errors cor- indicated mutations or the vector alone were grown overnight in me- responding to the average deviation from the mean. thionine-free medium followed by conversion to spheroplasts and incu- 35 6 bation with [ S]Trans-label (50 mCi/5 3 10 spheroplasts) for 60 min at ATP rel Mutation pH optimum K V m max 30 °C. Spheroplasts were then pelleted and lysed in phosphate-buffered saline with C E , and the V-ATPase immunoprecipitated using the mM 12 9 monoclonal antibody 8B1-F3 directed against the 69-kDa A subunit and Wild type 7.2 0.70 6 0.08 1.00 6 0.07 protein A-Sepharose followed by SDS-polyacrylamide gel electrophore- K593A 7.0 0.61 6 0.17 0.33 6 0.03 sis on a 12% acrylamide gel and autoradiography as described under Q634L 7.0 0.43 6 0.11 0.18 6 0.02 “Experimental Procedures.” The positions of the V-ATPase subunits are H743A 8.2 0.79 6 0.35 0.50 6 0.02 indicated and were confirmed by comparison with the migration of E789Q 9.7 0.41 6 0.04 0.11 6 0.02 C-labeled molecular mass standards. WT, wild type. FIG.5. Model for folding of the 100-kDa subunit (Vph1p) of the yeast V-ATPase and the location and effects of point mutations in Vph1p. The orientation of the 100-kDa subunit in the membrane is based upon labeling and proteolysis data obtained for the 100-kDa subunit of the bovine coated vesicle V-ATPase (see text). In particular, the position at which trypsin cleaves the bovine 100-kDa subunit from the cytoplasmic side of the membrane is indicated. Residues that were mutated in this study are circled. Two mutations (R735L and Q634L) affected stability of the 100-kDa subunit, two mutations (D425N and K538A) affected assembly of the V-ATPase complex, whereas three mutations (H743A, K593A, and E789Q) affected proton transport and ATPase activity of the V-ATPase complex. Vph1p Mutagenesis 22493 25974–25977 tually completely devoid of activity. In addition, both the 19. Nelson, H., Mandiyan, S., and Nelson, N. (1994) J. Biol. Chem. 269, H743A and E789Q mutations resulted in a significant change 24150–24155 in the pH optimum of the enzyme. While it is difficult to assign 20. Manolson, M. F., Proteau, D., Preston, R. A., Stenbit, A., Roberts, B. T., Hoyt, M., Preuss, D., Mulholland, J., Botstein, D., and Jones, E. W. (1992) J. Biol. a precise interpretation to these findings, they may reflect an Chem. 267, 14294–14303 alteration in the environment of residues whose protonation 21. Manolson, M. F., Wu, B., Proteau, D., Taillon, B. E., Roberts, B. T., Hoyt, M. A., and Jones, E. W. (1994) J. Biol. Chem. 269, 14064–14074 state is important for transport to occur. Interestingly, as with 22. Bauerle, C., Ho, M. N., Lindorfer, M. A., and Stevens, T. H. (1993) J. Biol. the F a subunit, positively and negatively charged residues in Chem. 268, 12749–12757 the last two putative transmembrane helices appear to be 23. Supekova, L., Supek, F., and Nelson, N. (1995) J. Biol. Chem. 270, 13726–13732 important for activity, possibly serving to line a polar channel 24. Umemoto, N., Yoshihisa, T., Hirata, R., and Anraku, Y. (1990) J. Biol. Chem. necessary to allow protons to gain access to the buried carboxyl 265, 18447–18453 25. Umemoto, N., Ohya, Y., and Anraku, Y. (1991) J. Biol. Chem. 266, group of the c subunit. These results suggest that the 100-kDa 24526–24532 subunit may serve an analogous role to the a subunit in the 26. Arai, H., Terres, G., Pink, S., and Forgac, M. (1988) J. Biol. Chem. 263, V-ATPase complex. It is important to recognize, however, that 8796–8802 27. Senior, A. E. (1990) Annu. Rev. Biophys. Biophys. Chem. 19, 7–41 further work will be required to determine whether the resi- 28. Penefsky, H. S., and Cross, R. L. (1991) Adv. Enzymol. 64, 173–214 dues identified play a direct role in proton translocation or 29. Pedersen, P. L., and Amzel, L. M. (1993) J. Biol. Chem. 268, 9937–9940 whether they serve some other function in the V-ATPase com- 30. Futai, M., Park, M. Y., Iwamoto, A., Omote, H., and Maeda, M. (1994) Biochem. Biophys. Acta 1187, 165–170 plex, such as coupling of proton transport to ATP hydrolysis. 31. Bowman, E. J., Tenney, K., and Bowman, B. (1988) J. Biol. Chem. 263, We have previously presented data that suggest that the 13994–14001 32. Bowman, B. J., Allen, R., Wechser, M. A., and Bowman, E. J. (1988) J. Biol. 100-kDa subunit may also possess the binding site for the Chem. 263, 14002–14007 specific V-ATPase inhibitor bafilomycin A (58). Thus, recon- 33. Zimniak, L., Dittrich, P., Gogarten, J. P., Kibak, H., and Taiz, L. (1988) J. Biol. stituted V or isolated 100-kDa subunit are both able to protect Chem. 263, 9102–9112 34. Manolson, M. F., Ouellette, B. F. F., Filion, M., and Poole, R. J. (1988) J. Biol. the intact V-ATPase from inhibition by bafilomycin. When the Chem. 263, 17987–17994 100-kDa mutants constructed in the present study were tested 35. Puopolo, K., Kumamoto, C., Adachi, I., and Forgac, M. (1991) J. Biol. Chem. 266, 24564–24572 for their sensitivity to 1 nM bafilomycin (a subsaturating con- 36. Puopolo, K., Kumamoto, C., Adachi, I., Magner, R., and Forgac, M. (1992) centration that inhibits 40–50% of the V-ATPase activity in J. Biol. Chem. 267, 3696–3706 yeast vacuoles), no significant differences in bafilomycin sensi- 37. Mandel, M., Moriyama, Y., Hulmes, J. D., Pan, Y. C., Nelson, H., and Nelson, N. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 5521–5524 tivity were observed. Obviously mutations that resulted in 38. Guthrie, C., and Fink, G. R. (1991) Methods Enzymol. 194, 1–932 complete loss of activity (such as D425N and R735L) could not 39. Sanger, F., Niklen, S., and Coulson, A. R. (1977) Proc. Natl. Acad. Sci. U. S. A. be tested for bafilomycin sensitivity, but none of the remaining 74, 5463–5467 40. Gietz, D., St. Jean, A., Woods, R. A., and Schiestl, R. H. (1992) Nucleic Acids residues appears to be critical for bafilomycin binding to the Res. 20, 1425 V-ATPase. It is possible that the bafilomycin binding site might 41. Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K. (eds) (1992) Short Protocols in Molecular Biology, reside on the soluble amino-terminal domain or that hydropho- Wiley, New York bic rather than hydrophilic residues in the integral domain are 42. Yamashiro, C. T., Kane, P. M., Wolczyk, D. F., Preston, R. A., and Stevens, T. important in bafilomycin binding. Further studies will be nec- H. (1990) Mol. Cell. Biol. 10, 3737–3749 43. Kim, E. K., Zhen, R. G., and Rea, P. A. (1994) Proc. Natl. Acad. Sci. U. S. A 91, essary to resolve this question. 6128–6132 44. Roberts, C. J., Raymond, C. K., Yamashiro, C. T., and Stevens, T. H. (1991) Acknowledgments—We acknowledge Dr. Patricia Kane of the De- Methods Enzymol. 194, 644–661 partment of Biochemistry and Molecular Biology at SUNY, Syracuse, 45. Feng, Y., and Forgac, M. (1992) J. Biol. Chem. 267, 5817–5822 for the kind gift of the 10D7 antibody against the 100-kDa subunit and 46. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. for many helpful discussions and suggestions. We dedicate this paper to Chem. 193, 265–275 Dr Yasuhiro Anraku, whose many contributions over the years have 47. Laemmli, U. K. (1970) Nature 227, 680–685 48. Oakley, B. R., Kirsch, D. R., and Morris, N. R. (1980) Anal. Biochem. 105, greatly enriched the field of the vacuolar ATPases. 361–363 REFERENCES 49. Kane, P. M. (1995) J. Biol. Chem. 270, 17025–17032 50. Nelson, H., and Nelson, N. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 3503–3507 1. Forgac, M. (1989) Physiol. Rev. 69, 765–796 51. Bowman, E. J., Siebers, A., and Altendorf, K. (1988) Proc. Natl. Acad. Sci. 2. Forgac, M. (1992) J. Bioenerg. Biomembr. 24, 341–350 U. S. A. 85, 7972–7976 3. Bowman, B. J., Vazquez-Laslop, N., and Bowman, E. J. (1992) J. Bioenerg. 52. Kane, P. M., Kuehn, M. C., Howald-Stevenson, I., and Stevens, T. (1992) Biomembr. 24, 361–370 J. Biol. Chem. 267, 447–454 4. Kane, P. M., and Stevens, T. H. (1992) J. Bioenerg. Biomembr. 24, 383–394 53. Doherty, R. D., and Kane, P. M. (1993) J. Biol. Chem. 268, 16845–16851 5. Anraku, Y., Umemoto, N., Hirata, R., and Ohya, Y. (1992) J. Bioenerg. 54. Puopolo, K., and Forgac, M. (1990) J. Biol. Chem. 265, 14836–14841 Biomembr. 24, 395–406 55. Puopolo, K., Sczekan, M., Magner, R., and Forgac, M. (1992) J. Biol. Chem. 6. Gluck, S. L. (1992) J. Bioenerg. Biomembr. 24, 351–360 267, 5171–5176 7. Kibak, H., Taiz, L., Starke, T., Bernasconi, P., and Gogarten, J. P. (1992) 56. Zhang, J., Myers, M., and Forgac, M. (1992) J. Biol. Chem. 267, 9773–9778 J. Bioenerg. Biomembr. 24, 415–424 57. Zhang, J., Feng, Y., and Forgac, M. (1994) J. Biol. Chem. 269, 23518–23523 8. Nelson, N. (1992) J. Bioenerg. Biomembr. 24, 407–414 58. Arai, H., Berne, M., and Forgac, M. (1987) J. Biol. Chem. 262, 11006–11011 9. Adachi, I., Puopolo, K., Marquez-Sterling, N., Arai, H., and Forgac, M. (1990) 59. Fillingame, R. H. (1992) J. Bioenerg. Biomembr. 24, 485–491 J. Biol. Chem. 265, 967–973 60. Sun, S.-Z., Xie, X.-S., and Stone, D. K. (1987) J. Biol. Chem. 262, 14790–14794 10. Hirata, R., Ohsumi, Y., Nakano, A., Kawasaki, H., Suzuki, K., and Anraku, Y. 61. Foster, D. L., and Fillingame, R. H. (1982) J. Biol. Chem. 257, 2009–2015 (1990) J. Biol. Chem. 265, 6726–6733 62. Schneider, E., and Altendorf, K. (1985) EMBO J. 4, 515–518 11. Kane, P. M., Yamashiro, C. T., Wolczyk, D. F., Neff, N., Goebl, M., and Stevens, 63. Schneider, E., and Altendorf, K. (1987) Microbiol. Rev. 51, 477–497 T. H. (1990) Science 250, 651–657 64. Cain, B. D., and Simoni, R. D. (1986) J. Biol. Chem. 261, 10043–10050 12. Nelson, H., Mandiyan, S., and Nelson, N. (1989) J. Biol. Chem. 264, 1775–1778 65. Cain, B. D., and Simoni, R. D. (1988) J. Biol. Chem. 263, 6606–6612 13. Ho, M. N., Hirata, R., Umemoto, N., Ohya, Y., Takatsuki, A., Stevens, T. H., 66. Wang, S.-Y., Moriyama, Y., Mandel, M., Hulmes, J. D., Pan, Y.-C. E., Danho, and Anraku, Y. (1993) J. Biol. Chem. 268, 18286–18292 W., Nelson, H., and Nelson, N. (1988) J. Biol. Chem. 263, 17638–17642 14. Ho, M. N., Hill, K. J., Lindorfer, M. A., and Stevens, T. H. (1993) J. Biol. Chem. 67. Perin, M. S., Fried, V. A., Stone, D. K., Xie, X.-S., and Sudhof, T. C. (1991) 268, 221–227 15. Beltran, C., Kopecky, J., Pan, Y.-C. E., Nelson, H., and Nelson, N. (1992) J. Biol. Chem. 266, 3877–3881 68. Lee, C., Ghoshal, K., and Beaman, K. D. (1990) Mol. Immunol. 27, 1137–1144 J. Biol. Chem. 267, 774–779 16. Nelson, H., Mandiyan, S., and Nelson, N. (1995) Proc. Natl. Acad. Sci. U. S. A. 69. Peng, S.-B., Crider, B. P., Xie, X.-S., and Stone, D. K. (1994) J. Biol. Chem. 269, 17262–17266 92, 497–501 17. Foury, F. (1990) J. Biol. Chem. 265, 18554–18560 70. Roberts, C. J., Nothwehr, S. F., and Stevens, T. H. (1992) J. Cell Biol. 119, 18. Graham, L. A., Hill, K. J., and Stevens, T. H. (1994) J. Biol. Chem. 269, 69–83

Journal

Journal of Biological Chemistry – Unpaywall

Published: Sep 1, 1996

There are no references for this article.