THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1093 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 268, No. 24, Iaaue of August 25, pp. 18286-18292, 1993 Printed in LISA.

VlMA13 Encodes a 54-kDa Vacuolar H+-ATPase Subunit Required for Activity but Not Assembly of the Enzyme Complex in Saccharomyces cerevisiae”

(Received for publication, February 25, 1993)

Margaret N. Ho$#, Ryogo Hiratall, Naoyuki Umemotoll , Yoshikazu Ohya**, Akira Takatsukill, Tom H. Stevens$, and Yasuhiro Anraku$$ From the Department of Biology, Faculty of Science, University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113, Japan, the $Institute of Molecular Biology, University of Oregon, Eugene, Oregon 97403, and the lllnstitute of Physical and Chemical Research (RIKEN), Hirosawa, Wako-shi, Saitama 351-01, Japan

Previous purifications and characterizations of the Saccharomyces cerevisiae vacuolar proton-translocat- ing ATPase (V-ATPase) have indicated that this en- zyme is a multisubunit complex composed of at least eight subunits of loo-, 69-, 60-, 42-, 36-, 32-, 27-, and 17-kDa (Kane, P. M., Yamashiro, C. T., and Stevens, T. H. (1989) J. Biol. Chem. 264, 19236-19244). We report the cloning and characterization of an addi- tional V-ATPase subunit, the 54-kDa subunit, which is encoded by the VMA13 gene. VMA13 was isolated by complementation of the growth phenotypes associ- ated with the una23 mutation, which was originally described as c l s l l (Ohya, Y., Umemoto, N., Tanida, I., Ohta, A., Iida, H., and Anraku, Y. (1991) J. Biol. Chem. 266, 13971-13977). The nucleotide sequence of the VMA13 gene predicted a hydrophilic polypeptide with a calculated molecular mass of 54,415 daltons. The VMA13 54-kDa gene product resides on the vac- uolar membrane and co-purified with the active V- ATPase complex. Characterization of a null umal3 mutant (Auma13) revealed that the Vmal3 polypep- tide is essential for V-ATPase activity. However, the Vmal3 polypeptide is not required for targeting of the other V-ATPase subunits (loo-, 69-, 60-, 42-, 27-, or 17-kDa subunits) to the vacuolar membrane as shown by the association of these subunits with vacuolar mem- branes isolated from Aumal3 cells. The nature of the V-ATPase “complex” in Avmal3 mutant is, neverthe- less, fundamentally different from the wild-type en- zyme. This is evidenced by the fact that the inactive V-

* This work was supported by a grant-in-aid for scientific research on priority areas of “Cellular Energy” from the Ministry of Education, Science and Culture of Japan (to Y. A.), a grant from the Japan Society for the promotion of science for Japanese junior scientists (to R. H.), a grant for “Biodesign Research Program” from the Institute of Physical and Chemical Research (RIKEN) (to R. H.), and Grant GM38006 from the National Institutes of Health (to T. H. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in thispaper has been submitted

013916. to the GenBankTM/EMBL Data Bank with accession number($

State University, Corvallis OR 97331. § Present address: Dept. of Biochemistry and Biophysics, Oregon

(1 Central Laboratory for Key Technology, Kirin Brewery Co., Ltd., Fukuura, Kanazawa-ku, Yokohama-shi, Kanagawa 236, Japan.

Stanford, CA 94305-5120. ** Dept. of Genetics, Stanford University School of Medicine,

2111 (ext. 4461); Fax: 81-3-3812-4929. $$ To whom correspondence should be addressed. Tel.: 81-3-3812-

ATPase complex from Aumal3 cells is less stable than the wild-type enzyme. Taken together, these results indicate that VMA13 encodes the 54-kDa subunit of the V-ATPase and that this subunit is essential for activity, but not assembly, of the enzyme complex.

The vacuolar proton-translocating ATPases (V-ATPase)’ are a class of multisubunit enzymes present in the membranes of eukaryotic cells (Forgac, 1989). In almost all cases, the V- ATPase function is to regulate organelle pH. The degree of acidification varies among the various organelles and between different organisms (e.g. the yeast vacuole is pH 6.1 and the mammalian lysosome pH 5 5.0) (Yamashiro et al., 1990; Kornfeld and Mellman, 1989). V-ATPases purified from mammals, plants, and fungi have been reported to have sim- ilar subunit compositions (Kane et al., 1989). For example, both the yeast vacuolar V-ATPase and the mammalian clath- rin-coated vesicle ATPase are composed of at least eight subunits ranging in size from 17- to 100-kDa. These enzymes all appear to be composed of peripheral V-ATPase subunits (VI) that constitute the catalytic sector as well as integral membrane V-ATPase subunits (V,) that constitute the mem- brane pore (Forgac, 1992; Kane and Stevens, 1992).

Despite much effort, it remains unclear exactly how many subunits comprise the V-ATPase enzyme complexes. It is also not known what allows these enzymes to maintain widely different organelle pH. The mechanisms that determine V- ATPase organelle specificity, or that direct their assembly and targeting, have also not been elucidated. It is possible that specific V-ATPase subunits are responsible for regulating the degree of organelle acidification, or allow for the targeting of the enzyme complexes to specific organelles. Characteriza- tion of the subunit composition of V-ATPases and determi- nation of the function of individual subunits within the com- plex are, therefore, necessary for a general understanding of the biological regulation and species diversification of this enzyme.

This paper reports the cloning of the VMA13 gene, which encodes a subunit of the Saccharomyces cerevisiae yacuolar - membrane ATPase ( V M A ) . vmal3 mutants were originally identified as clsll based on the sensitivity of this mutant to

The abbreviations used are: V-ATPase, vacuolar proton-translo- cating ATPase; VmalSp, VMA13 gene product; PAGE, polyacryl- amide gel electrophoresis; SSC, sodium chloride sodium citrate buffer (Ausubel et al., 1987); TBS, Tris-buffered saline; DPAP-B, dipeptidyl aminopeptidase B; kb, kilobase(s); ZW3-14, zwitterionic detergent, N-tetradecyl-N,N-dimethyl-3-ammonio-l-propanesulfonate.

18286

Vmal3p Is a 54-kDa V-ATPase Subunit 18287

elevated levels of calcium ion (Ohya et al., 1986). umal3 pNUVA450 were constructed on a single-copy plasmid, ~RS315, and mutants have also been shown to be deficient in V-ATPase tested for Integration mapping of the cloned DNA was done as follows. The

that the vMA13 gene encodes a 54-kDa polypeptide, as pre- the yeast integrative plasmid pRS305 (LEU2). The plasmid was dieted from the nucleotide sequence and confirmed through linearized by BglII digestion and integrated into the genome of the protein chemistry. This 54-kDa polypeptide co-purified with wild-type strain (YPH499). A Leu+ transformant (NUY45) was the yeast V-ATPase complex. Deletion of the VMA1.3 gene picked, and the integration of the cloned sequence into the genomic

in loss of A T P ~ ~ ~ activity from the vacuolar mem- locus of the cells was confirmed by Southern blotting analysis. NUY45 brane. These results demonstrate that the Vma13 protein was mated with a umal3-I strain, NUY32'. A diploid resulting from

(Vma13p) is a subunit Of the yeast Nucleotide sequence of the VMAl3 gene was determined for both Vmal3P is a subunit of the enzyme, this Polypeptide is not strands by the dideoxy-chain termination method (Sanger et al., required for the assembly of the remaining V-ATPase sub- 1977). Sequence similarity was searched in NBRF (Release 31) and units onto the vacuolar membrane. This is in contrast to the SWISS protein data bases (Release 20) using the FASTA algorithm

activity (Ohya et 1991). In the current work, we rep0rt 2.6-kb SpeI-XhoI fragment in pNUVA456 (see Fig. 1) was cloned into

this cross was sporulated, and tetrads were dissected.

other V-ATPase subunits examined thus far (loo-, 69-, 60-, 42-, 36-, 27-, 17-kDa),' all of which have been shown to be essential for the assembly of the remaining subunits onto the vacuolar membrane (Umemoto et al., 1990; Kane et al., 1992; Manolson et al., 1992; Ho et al., 1993; Bauerle et al., 1993). The characterization of the VMAl3 gene thus makes an important contribution toward determining the subunit com- position of the yeast V-ATPase. In addition, these data sug- gest that polypeptides can be subunits of the yeast V-ATPase without being essential for the assembly of this enzyme com- plex onto the vacuolar membrane.

EXPERIMENTAL PROCEDURES

Materials-Enzymes for recombinant DNA methods were pur- chased from Takara Shuzo (Kyoto). Modified T7-polymerase was from United States Biochemical. [cY-~'P]~CTP (-110 TBq/mmol) were from ICN. Other chemicals were as described by Uchida et al. (1988). Bafilomycin A, was a generous gift from Dr. Karlheinz Alten- dorf (University of Osnabriick).

Strains and Culture Conditions-S. cerevisiae strains used are YPH499 (MATa leu2 ura3 trpl lys2 his3 ade2), YPH5OO (MATa leu2 ura3 trpl lys2 his3 ade2), YPH5Ol (a diploid derived from a mating between YPH499 and YPH500) (Sikorski and Hieter, 1989), NUY32' (MATa vmaZ3-1 leu2 ade2 lys2 his3), NUY34 (MATa vmal3-1 leu2 his3 u r d lys2 ade2), NUY45 (VMAZS::LEU2 derivative of YPH499), and RH302 (AumaZ3:TRPI derivative of YPH500). NUY32' and NUY34 were constructed by a cross between YOC28 (MATa umaZ3- 2, leul) (Ohya et al., 1986) and YPH500. Yeast cells were grown aerobically in the following media: YPD (1% yeast extract, 2% peptone, and 2% glucose), YNBD (synthetic minimal medium; 0.67% yeast nitrogen base and 2% glucose), and YPG (1% yeast extract, 2% peptone, and 3% glycerol). When required, YNBD was supplemented with amino acids and nucleic acids as described by Sherman (1991). Ca2+ sensitivity of cells were examined on YPD medium supple- mented with 100 mM CaClz. Medium pH was adjusted by adding 50 mM succinate/phosphate buffer (Yamashiro et al., 1990). For tetrad analysis, presporulation medium (0.8% yeast extract, 0.3% polypep- tone, and 5% glucose) and sporulation medium (1% potassium ace- tate) were used. For plates, 2% agar was added.

Plasmids and Recombinant DNA Methods-Yeast single-copy plas- mid pRS315 (LEU2) (Sikorski and Hieter, 1989) was used for sub- cloning and complementation analysis of the VMAZ3 gene. pRS305 (LEU2) (Sikorski and Hieter, 1989) and pJJ281 (TRPZ) (Jones and Prakash, 1990) were used as sources of yeast selectable marker genes. Plasmid isolation, gel electrophoresis, ligation, restriction enzyme analysis, and Escherichia coli transformation were done as described by Ausubel et al. (1987). Yeast genomic DNA was isolated as described by Holm et al. (1986). Yeast transformation was carried out by the lithium acetate method of Ito et al. (1983).

Cloning, Sequencing, and Disruption of the VMA13 Gene-A umal3-1 mutant strain, NUY34 (MATa leu2) was transformed with a yeast genomic DNA library carried on the yeast multi-copy vector YEpl3 (Yoshihisa and Anraku, 1989) by the lithium acetate method. Ten plasmid-dependent transformants capable of growth on YPG medium were obtained, and all of these transformants carried the same clone (pNUVA450). Various subclones of the DNA insert in

* The names of the subunits of the enzyme were according to Kane et al. (1989). The 69-, 60-, and 17-kDa subunits are referred as subunit a, b, and c, respectively, in the papers by Anraku and his colleagues.

(Pearson and Lipman, 1988). The null umal3 mutant strain (RH302) was constructed by the

method of Rothstein (1983). The 4-kb BamHI-XhoI fragment con- taining the VMAZ3 gene was cloned into pBluescript KS' (Strata- gene). The 1.1-kb BglII fragment within the VMAl3 coding region was replaced by 0.9-kb BamHI-BglII fragment from pJJ281 (TRPI ). The resulting plasmid was digested with BamHI and X b I , and the mutant allele of the VMAl3 gene introduced into YPH500 to yield yeast strain RH302. Disruption of the VMAZ3 locus was confirmed by Southern blot analysis. Chromosomal DNA from YPH500 and RH302 was digested with ApaLI, separated on agarose gels and blotted onto nylon membrane filters in 0.4 M NaOH. Blots were hybridized with the 1.1-kb ApaLI-SpeI fragments within the VMAl3 gene labeled with horseradish peroxidase, and bound DNA probe was detected using a chemiluminescence substrate (Amersham Corp.). Hybridiza- tion and washing conditions were according to the supplier's protocol.

SDS-Polyacrylamide Gel Electrophoresis and Western Blot Analy- sis-Protein extracts of whole cells and vacuolar membrane vesicles were prepared as described by Kane et al. (1992). SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was performed by the method of Laemmli (1970). Immunoblots were prepared and probed as described. Blots were probed with primary antibodies diluted 1:lOOO. Alkaline phosphatase-conjugated goat anti-mouse or anti-rabbit antibodies (Promega) were used as secondary antibodies, and bound alkaline phosphatase was visualized by the addition of 5-bromo-4-chloro-3- indolyl phosphate (Wako Chemicals) and p-nitro blue tetrazolium (Wako Chemicals). Apparent molecular masses of proteins were determined relative to prestained molecular weight standards (Be- thesda Research Laboratories; Amersham Corp.).

Antibodies-Antibodies that recognize Vmal3p were generated by subcloning a SpeI-XbaI 1.5-kb fragment containing a portion of the VMA13 gene (codons 160-478) into the E. coli expression vector pEXP2 (pMN21) (Roberts et al., 1989). The Vmal3p antigen pro- duced from this plasmid was purified from E. coli (Raymond et al., 1990) and injected into New Zealand White rabbits. Rabbit anti- Vmal3p antibodies were affinity purified against antigen expressed from plasmid p ~ H 2 1 as described (Raymond et al., 1990). Monoclonal antibodies that recognize the 100-kDa (7B1), 69-kDa (R70), and 42- kDa (7A2) V-ATPase subunits were prepared as described (Hirata et al., 1990; Kane et al., 1989, 1992). Anti-60-kDa subunit and anti- Vma4p polyclonal antibodies were prepared as described (Yamashiro et al., 1990; Ho et al., 1993).

EDTA Wash and Alkaline Carbonate Extraction of Vacuolar Mem- brane Vesicles-Vacuolar membrane vesicles (1 mg of protein) were suspended in a solution (1 ml) containing 1 mM EDTA and 10 mM Tris-HC1, pH 7.5. The suspension was centrifuged at 37,000 X g for 30 min at 4 "C. This washing step was repeated three times. The EDTA-washed membrane vesicles were resuspended in the same buffer at a protein concentration of 5 mg/ml, diluted with 10 volumes of cold 100 mM sodium carbonate, pH 11.5, and incubated on ice for 30 min. The membrane vesicles were pelleted by centrifugation at 100,000 x g for 1 h at 4 "C. Proteins in the supernatant fractions were precipitated by adding 10% trichloroacetic acid. The precipitates and the membrane pellets were solubilized and subjected to SDS- PAGE and Western blot analysis (Kane et al., 1992).

ChloroformlMethanol Extraction of Vacuolar Membrane Vesicles- EDTA-washed vacuolar membranes vesicles were resuspended in 1 mM EDTA and 10 mM Tris-HC1, pH 7.5 (1 mg protein/ml), and five volumes of chloroform/methanol (2:1, v/v) added. The suspension was placed on ice for 1 h with occasional vortexing. After the incu- bation on ice, the suspension was cleared by centrifugation, and the upper aqueous phase and interphase were removed. The solvent in the organic phase was evaporated, the pellet resuspended (Ohya et

18288 Vmal3p Is a 54-kDa V-ATPase Subunit al., 1991), and the solubilized proteins separated by SDS-PAGE.

Other Methods-Preparation of vacuolar membrane vesicles and purification of the yeast V-ATPase were done as described previously (Uchida et al., 1985,1988). V-ATPase, a-mannosidase, and dipeptidyl aminopeptidase B (DPAP-B) activities were assayed as previously described (Uchida et al., 1985; Kane et al., 1989).

RESULTS

Isolation of the VMA13 Gene-The vmal3 mutant was originally identified as a Ca2+-sensitive mutant (ckill) that was also respiratory deficient (Pet-) (Ohya et al., 1986,1991). Recently, Ohya et al. (1991) found that the mutant completely lacks V-ATPase activity. The VMA13 gene was isolated from a yeast genomic library by complementation of the Pet- phenotype of the mutant cells. A vmal3 mutant strain, NUY34 (vmal3-1, leu2), was transformed with a yeast ge- nomic DNA library carried on the yeast multicopy vector YEpl3 (LEU2) and plated onto YPG plates. Ten plasmid- dependent Pet+ transformants, all of which contained the same plasmid, pNUVA450, were isolated. pNUVA450 also restored the growth of NUY34 on YPD supplemented with 100 mM CaC12. Fig. 1 shows the restriction map of pNUVA450 and various subclones. Subcloning and complementation analysis using a single-copy vector, pRS315, indicated that the 1.1-kb SpeI-ApaLI region of the insert (pNUVA454) was sufficient for complementation (Fig. 1). To confirm that the complementing clone was indeed the VMA13 gene, we ana- lyzed linkage between the chromosomal locus of the cloned DNA and the vmal3-1 mutation by integration mapping. The 2.6-kb XhoI-SpeI fragment from pNUVA456 (Fig. 1) was cloned into the vector pRS305, which contains a LEU2 marker. The resulting plasmid was linearized with BglII and introduced into a wild-type strain, YPH499 (leu2). A Leu+ transformant, NUY45, was isolated and further analyzed. The integration of the cloned fragment into the chromosomal locus of the gene was confirmed by Southern blot analysis (data not shown). NUY45 was mated with NUY32' (vmal3-1 leu2), and diploid cells derived from this were cross-sporulated. All tetrads analyzed (44 asci) were parental ditype (2 Vma+Leu+: 2 Vma-Leu-), indicating that the cloned gene is very tightly linked to the VMA13 locus. We thus concluded that we cloned the authentic VMAl3 gene.

Nucleotide Sequence of the VMA13 Gene-The nucleotide sequence of the 1.1-kb SpeI-ApaLI fragment in pNUVA454 was determined for both strands. This region of the DNA

A j)BQs,,Bg p qc c .y pNUVA450 -f-

pNUVA476 pUUVA453 = pNuvA466 pNUVA458 -

-P ~~

FIG. 1. Physical map of the VMAlS gene and various sub-

BglII; s, Sun; Sc, SacI; Sp, SpeI; P, PsB. The hatched and open boxes clones. A , restriction sites are indicated Ap, ApaLI; B, BamHI; Bg,

represent the complementing and noncomplementing subclones, re- spectively. B, sequence strategy for the VMA13 gene. Arrows indicate the direction and the extent of the sequences determined.

contained a single open reading frame of 299 codons starting 62-base pair downstream of the SpeI site (Fig. 2). However, there was no in-frame stop codon upstream of the putative initiating ATG codon in the fragment, and more extensive sequence analysis revealed that the open reading frame that we first assigned is part of a larger open reading frame of 478 codons. This large open reading frame is predicted to encode a polypeptide of 54,415 Daltons. The SpeI-ApaLI VMAl3 fragment was also capable of complementing the Pet- and Cls- growth defects of a null una13 strain (RH302, see below), suggesting that the NHZ-terminal third of the gene product is dispensable for its activity. There were no putative membrane spanning domains in the Vmal3 polypeptide (Vmal3p) as predicted by hydropathy analysis of Kyte and Doolittle (1982) (data not shown). There were no proteins exhibiting signifi- cant sequence similarity to Vmal3p in the NBRF or SWISS protein database.

Disruption of the VMA13 Gene-The chromosomal locus of the gene was disrupted by the method of Rothstein (1983). The 4-kb BamHI-XhoI fragment containing the VMA13 gene was cloned into the vector pBluescript KS+. The 1.1-kb BglII fragment within the coding region of the VMA13 gene was replaced with the 0.9-kb BamHI-BglII fragment from pJJ281 that contains the TRPl gene (Fig. 3A). The disrupted allele of the vmal3 gene (Avmal3) was released from the vector by BamHI-XhoI digestion and introduced into a wild-type dip- loid strain, YPH501. A Trp' transformant was picked, and substitution of one of the chromosomal VMA13 gene was confirmed by Southern blot analysis (Fig. 3B). The VMA13/ A v m l 3 diploid cells were sporulated, and tetrads were dis- sected. All tetrads analyzed (21 asci) yielded four viable spores. This result indicates that the disruption of the gene was not lethal to haploid yeast cells.

Haploid Avmal3 cells (RH302) exhibited growth pheno- types identical to those strains disrupted for any of the other V-ATPase subunit genes (Avmal, Avma2, Avma3, Avma4, Avma5, and Avma6) (Anraku et al., 1992; Kane and Stevens, 1992; Ho et al., 1993; Bauerle et al., 1993). Cells carrying the Avmal3 allele did not grow in YPG medium, which contains glycerol as sole carbon source, YPD medium supplemented with 100 mM CaC12, or YPD buffered to neutral pH (data not shown). The growth deficiencies associated with loss of VMA13 function suggest that Avmal3 cells may lack the V- ATPase activity.

Vacuolar membranes isolated from Avmal3 cells lacked any bafilomycin Al-sensitive ATPase activity (Table I), indicating that Vmal3p is essential for expression of V-ATPase activity. Morphology of the vacuoles in the mutant cells appeared normal, but the mutant vacuoles failed to accumulate the fluorescent dye quinacrine (data not shown), which concen- trates within acidic membrane compartments (Weisman et al., 1987), indicating that the mutant is also defective in vacuolar acidification.

VMA13 Encodes a 54-kDa V-ATPase Subunit-To deter- mine whether Vmal3p is a subunit of the yeast V-ATPase, we generated antiserum against E. coli-expressed Vmal3p. Fig. 4 shows the result of Western blot analysis to detect Vmal3p in whole cell extracts and vacuolar membrane frac- tions. The antiserum recognized a polypeptide with an appar- ent molecular mass of 54 kDa in both fractions. The identity of the polypeptide was confirmed by the lack of the 54-kDa cross-reacting species in Avmal3 mutant extracts. This result indicates that Vmal3p is associated with vacuolar mem- branes.

We next examined whether Vmal3p is a component of the V-ATPase enzyme complex. The enzyme was isolated by the

Vmal3p Is a 54-kDa V-ATPase Subunit 18289

1

42

132

222

312

402

492

582

672 1

762 31

852 61

942 91

1032 121

1122 151

1212 181

1302 211

1392 24 1

1482 271

1572 301

1662 331

1752 361

1842 391

1932 421

2022 451

2112

2202

M G A T K I L M D S T H F N E I R S I I R S R S V A W D A L

A R S E E L S E I D A S T A K A L E S I L V K K N I G D G L

S S S N N A H S G F K V N G K T L I P L I H L L S T S D N E

D C K K S V Q N L I A E L L S S D K Y G D D I V K F F Q E D

P K Q L E Q L F D V S L K G D F Q T V L I S G F N V V S L L a=I

V Q N G L H N V K L V E K L L K N N N L I N I L Q N I E Q M

D T C Y V C I R L L Q E L A V I P E Y R D V I W L H E K K F

M P T L F K I L Q R A T D S Q L A T R I V A T N S N H L G I

Q L Q Y H S L L L I W L L T F N P V F A N E L V Q K Y L S D

F L D L L K L V K I T I K E K V S R L C I S I I L Q C C S T

R V K Q H K K V I K Q L L L L G N A L P T V Q S L S E R K Y

S D E E L R Q D I S N L K E I L E N E Y Q E L T S F D E Y V

A E L D S K L L C W S P P H V D N G F W S D N I D E F K K D w n

N Y K I F R Q L I E L L Q A K V R N G D V N A K Q E K I I I

Q V A L N D I T H V V E L L P E S I D V L D K T G G K A D I

M E L L N H S D S R V K Y E A L K A T Q A I I G Y T F K *

41

131

221

311

401

491

581

671

761 30

851 60

941 90

1031 120

1121 150

1211 180

1301 210

1391 240

1481 270

1571 300

1661 330

1751 360

1841 390

1931 420

2021 450

2111 478

2201

2279

FIG. 2. Nucleotide sequence of the VMAI3 gene and the amino acid sequence of the predicted gene product. A 2.3-kb region defined by BarnHI and ApaLI sites was sequenced as described under “Experimental Procedures.”

method of Uchida et al. (1985). Proteins in vacuolar mem- brane vesicles were solubilized with the zwitterionic detergent ZW3-14 and size-fractionated by centrifugation through a glycerol density gradient. Fig. 5 shows the results of SDS- PAGE visualized by Coomassie Brilliant Blue staining (Fig. 5A) and Western blot (Fig. 5 B ) analyses of the glycerol gradient fractions. Fractions that contained maximal ATPase or DPAP-B activities are indicated by arrows. Vmal3p co- sedimented with the ATPase activity and with the other known V-ATPase subunits indicating that this polypeptide is a component of the purified V-ATPase complex.

Vmal3p Is a Peripheral Membrane V-ATPase Subunit- The DNA sequence of the VMAl3 gene predicts that the encoded protein is hydrophilic, lacking either a signal se-

quence or membrane spanning domain. To determine whether Vmal3p behaves as a peripheral or integral membrane com- ponent of the yeast V-ATPase complex, vacuolar membranes were subjected to alkaline sodium carbonate extraction. As with the 69-, 60-, 42-, and 27-kDa V-ATPase subunits (Kane et al., 1989, 1992), Vmal3p was removed from the vacuolar membrane by the high pH carbonate treatment whereas the 100-kDa integral membrane subunit remained in the mem- brane, as demonstrated by Western blot analysis (Fig. 6). Thus, Vmal3p adds to the growing list of V-ATPase subunits that are peripherally associated with the vacuolar membrane.

Assembly of the V-ATPase Subunits in Aumal3 Cells-We were interested in determining what effect the loss of the VMA13 gene had on the synthesis and localization of the

18290 Vmal3p Is a 54-kDa V-ATPase Subunit

- VMA13 + wlld 0 0

4 . 6 k b p 41.25kbp

ApaLl 200bps U FIG. 3. Disruption of the VMA23 gene. A , construction of the

null mutant allele of the VMA13 gene. A 1.1-kb EglII fragment within the V M A l 3 coding region was replaced with TRPl gene. Details of the construct are described under “Experimental Procedures.” B, Southern blot analysis of the V M A l 3 locus in wild-type and the null vrnal3 mutant cells. Chromosomal DNA from YPH500 (wild-type) and RH302 (Avrnal3:TRPI) cells were digested by ApaLI, resolved in an agarose gel, blotted onto a nylon membrane, and probed with the 1.1-kb SpeI-ApaLI fragment of the VMA13 gene.

TABLE I Vacwlar enzyme activities in wild-type and null vmal3 mutant cells

Vacuolar enzyme activities were assayed as described under “Ex- Derimental Procedures.”

cr-Mannosidase ATPase” Whole-cell Vacuolar Vacuolar

extract membrane membrane nmol/min/mg protein

YPH5OO (Vma’) 0.12 1.44 690 RH302 (Aumal3) 0.88 15.9 0.4

Activity that is sensitive to 5 p~ bafilomycin A,.

A \KT m a l a

FIG. 4. Detection of V m a l 3 p in whole cell ( A ) or vacuolar membrane vesicle ( B ) protein extracts. Whole cell or vacuolar membrane vesicle extracts were prepared from YPH500 and RH302 cells. Whole cell extracts prepared from 2 X 10’ cells (-50 pg of protein) ( A ) , or 5 pg of vacuolar membrane vesicle protein ( E ) , was loaded for each sample. Protein extracts were separated by SDS- PAGE on 10% polyacrylamide gels, and blots were probed with anti- Vmal3p antibody.

remaining V-ATPase subunits. The steady-state levels of four peripheral membrane VI subunits (69-, 60-, 42-, and 27-kDa) and one integral membrane V, subunit (100-kDa) were ana- lyzed. We also examined whether these subunits were assem- bled onto the vacuolar membrane in Avmul3 cells. Assembly of another integral membrane V, subunit, the 17-kDa proteo- lipid, into the vacuolar membrane was also assessed by ex- amining the proteins present in chloroform/methanol extracts of vacuolar membrane vesicles.

Western blot analysis revealed that cells lacking Vmal3p have normal levels of the remaining V-ATPase subunits (Fig. 7A), and these polypeptides are assembled onto the vacuolar

A W a ) 205

4

2

0 (bcnom’ ATPase DPAP-B P)

FIG. 5. Detection of V m a l 3 p in glycerol gradient fractions. Solubilized vacuolar membrane vesicles were applied to a 20-50% glycerol gradient and fractionated as previously described (Uchida et al., 1985). Twenty-two fractions of -500 pl each were collected from the bottom of the centrifuge tube. Each fraction was assayed for ATPase and DPAP-B activities. The fractions exhibiting maximal enzyme activities are shown by arrows. The proteins present in each fraction were prepared for gel electrophoresis as described (Uchida et al., 1985). A constant percentage of each fraction was separated by SDS-PAGE on 10% polyacrylamide gels. Total proteins present in each fraction were detected by staining with Coomassie Brilliant Blue ( A ) . Western blots of the same glycerol gradient fractions were probed with anti-Vmal3p antibodies (B) .

V P S ” ,+1 OOkDa 0- *

- -+Vmal3p

FIG. 6. Alkaline sodium carbonate treatment of vacuolar vesicles. EDTA-washed vacuolar membrane vesicles were treated with 100 mM sodium carbonate (pH 11.5) as described under “Exper- imental Procedures,” and the treated vesicles centrifuged to yield supernatant ( S ) and pellet (P) fractions. A comparable amount of untreated vesicles ( V ) is shown as a control. Proteins were separated by SDS-PAGE on 10% polyacrylamide gels and the blots probed with anti-Vmal3p, anti-lOO-kDa, or anti-69-kDa antibodies. The anti-100- kDa antibody recognized two polypeptides of 100 and 75 kDa. The 75-kDa species (asterisk) is a proteolytic product of the 100-kDa subunit (Kane et al., 1992).

membrane (Fig. 723). The level of the 17-kDa proteolipid was also normal in vacuolar membranes from Avmul3 cells (Fig. 7C). These results indicate that Vmal3p is not essential for the synthesis of V-ATPase subunits or their targeting onto the vacuolar membrane in yeast. However, we have noticed that the level of association of the VI subunits with the vacuolar membrane varied among successive membrane prep- arations (10-100% of wild-type; data not shown), although the-levels of V, subunits in the vacuolar membranes did not vary (data not shown). This result suggested that the VI subunits may not be as tightly associated with the vacuolar membrane in Avmul3 cells as in wild-type cells.

Stability of the V-ATPase “Complex”from Avmnl3 CelLs- When the vacuolar membrane vesicles prepared from the Avma13 mutant cells were treated with a buffer containing 1 mM EDTA, between 10-20% of the 69- and 60-kDa subunits was detached from the membrane, as evidenced by Western

Vmal3p Is a 54-kDa V-ATPase Subunit 18291

”

“

am a.

7 -442kDa

427kDa

.)I)

I“I 417kDa

FIG. 7. Detection of V-ATPase subunits in whole cell pro- tein extracts and vacuolar membrane vesicles. A , detection of V-ATPase subunits in whole cell extracts prepared from wild-type (YPH500, WT) and Aumald (RH302) cells. Protein extracts prepared from 3 X lo7 cells (100 kDa), 2 X lo7 cells (69 and 42 kDa), and 2 X lo6 cells (60 and 27 kDa) were loaded for each sample. Extracts were separated by SDS-PAGE on 10% acrylamide gels and immunoblots were probed with antibodies specific for individual V-ATPase sub- units as described under “Experimental Procedures.” B, detection of V-ATPase subunits in vacuolar membrane vesicles. Vacuolar mem- brane vesicles were prepared from YPH500 and RH302 cells and were analyzed by SDS-PAGE followed by immunoblotting. 20 pg (100 kDa), 10 pg (69 and 42 kDa), or 1 pg (60 and 27 kDa) of protein was loaded for each sample. C, detection of 17-kDa proteolipids in vacuolar membrane fractions. Vacuolar membrane vesicles from YPH500 and RH302 cells were extracted with chloroform/methanol (21) as de- scribed under “Experimental Procedures.” Proteins in the organic phase were resolved by SDS-PAGE on 13.5% acrylamide gels, and detected by staining with silver. Protein bands with higher apparent masses are aggregates of the proteolipid.

wild vmal3A EDTA EDTA

v n v m

69kDa

6OkDa

FIG. 8. Effect of low salt wash on the 69- and 60-kDa subunits on the wild-type and Avmal3 mutant vacuolar mem- brane vesicles. Wild-type and Aumald mutant vesicles were sus- pended in a solution containing 10 mM Tris-HC1, pH 7.5, and 1 mM EDTA, and the suspension was centrifuged to give supernatant (S) and pellet (P) fractions. A comparable amount of untreated vesicles is also shown ( V ) . Proteins were separated by SDS-PAGE on 10% polyacrylamide gels and blots probed with anti-100-kDa or anti-69- kDa antibodies.

blot analysis (Fig. 8). This is in contrast to the results obtained for the wild-type enzyme, in which these peripheral membrane subunits remain stably associated with vacuolar membranes (Fig. 8). These results suggest that although the V, subunits attach to the vacuolar membrane in Avmal3 cells and form some type of inactive V-ATPase complex, the nature of this complex is fundamentally different from the wild-type enzyme.

DISCUSSION

Previous characterization of the yeast V-ATPase has sug- gested that this enzyme is composed of a t least eight subunits, including the 69-, 60-, 42-, and 27-kDa peripheral membrane V1 polypeptides, the loo-, 36-, and 17-kDa V, polypeptides, and the uncharacterized 32-kDa polypeptide (Kane et al., 1989). The genes encoding seven of these V-ATPase subunits have been cloned, including the VMAl (69-kDa), VMA2 (60- kDa), VMA3 (17-kDa), VMA4 (27-kDa), VMAS (42-kDa), VMA6 (36-kDa), and VPHI (100-kDa) genes (Hirata et al., 1990; Nelson and Nelson, 1989; Umemoto et al., 1990; Foury, 1990; Ho et al., 1993; Manolson et al., 1992; Bauerle et al.,

1993). In addition, six genes that are known to be essential for the ATPase activity of this complex have been described (VMAll , 12, 13,21,22, and 23) (Ohya et al., 1991; Umemoto et al. 1991; Hirata et al., 1993; Ho et al., 1993). The VMAll gene encodes a homologue of the VMA3-encoded 17-kDa subunit, which is essential for the assembly of the V-ATPase and may be a component of the enzyme complex (Umemoto et al., 1991). A detailed molecular characterization of the VMAl2 gene indicates that the Vmal2 protein is required for assembly of the yeast V-ATPase, yet it is not a subunit of the active enzyme complex (Hirata et al., 1993). In this paper, we report on the VMA13 gene and its requirement for yeast V- ATPase function.

The VMA13 gene was isolated by complementation of the vmal3-1 (clsll ) mutation and sequenced. VMA13 is predicted to encode a hydrophilic polypeptide of 54 kDa, lacking either a signal sequence or significant identity to any polypeptide in the data base. We next assessed the localization of Vmal3p by using antiserum raised against E. coli-expressed Vmal3 polypeptide. The 54-kDa Vmal3p was found to be a peripheral vacuolar membrane protein that cosedimented with the yeast V-ATPase complex when the solubilized enzyme was isolated by glycerol gradient centrifugation. The VMA13 gene product is essential for the ATPase activity of the enzyme, as evi- denced by the absence of V-ATPase activity in Avmal3 mu- tant cells. By these criteria we conclude that the 54-kDa polypeptide is a peripheral membrane V1 subunit of the yeast V-ATPase. This polypeptide was not originally identified as a subunit by Kane et al. (1989), most likely because this 54- kDa polypeptide migrates on SDS-PAGE near some of the 69- and 60-kDa V-ATPase subunit degradation products. Thus, the characterization of Vmal3p reveals that the yeast V-ATPase is composed of at least nine polypeptides. However, whether additional polypeptides are subunits of this complex enzyme remains to be determined.

To begin to assess the function of VmalBp, we investigated the consequence of the loss of this polypeptide on the remain- ing V-ATPase subunits. While cells devoid of Vmal3p lacked V-ATPase activity and function, all the remaining V-ATPase subunits analyzed were present on the vacuolar membrane in these cells. However, our results indicate that the V1 V- ATPase subunits are associated less tightly to Avmal3 vacu- olar membranes than to vacuolar membranes isolated from wild-type cells. I t is possible that this difference in stability of the V-ATPase complex reflects a requirement for Vmal3p to form a fully stable and active V-ATPase enzyme complex.

Despite the relative instability of the V-ATPase complex lacking Vmal3p, it is clear that Vmal3p is not essential for assembly of V-ATPase subunits onto the vacuolar membrane. This result is in contrast to what is found in cells lacking other V-ATPase subunits. Mutants deficient for any other V1 subunit (69-, 60-, 42-, and 27-kDa) fail to assemble the re- maining peripheral membrane polypeptides onto the vacuolar membrane (Umemoto et al., 1990; Kane and Stevens, 1992; Ho et al., 1993). Similarly, the absence of any single V, subunit (loo-, 36-, and 17-kDa) results in the destabilization of the remaining V, subunits as well as a failure to assemble the V1 subunits onto the vacuolar membrane (Umemoto et al., 1990; Kane et al., 1992; Manolson et al., 1992; Bauerle et al., 1993). Therefore, Vmal3p is unique among yeast V-ATPase subunits in that it is not required for assembly of the enzyme complex, yet it is required for function of the V-ATPase.

The composition of V-ATPase complexes between species is reported to be relatively well conserved (Table 11). Two V1 subunits, the catalytic “70-kDa” and the regulatory “BO-kDa” subunits are present in all V-ATPases characterized. These

18292 Vmal3p Is a 54-kDa V-ATPase Subunit

TABLE I1 Comparison of V-ATPase subunit compositions

Source of V-ATPase Molecular masses of subunits

Yeast vacuolar membranes" 100 69 Bovine clathrin-coated vesicles' 100 73 Red beet vacuolar membranes' 100 67 Neurospora crassa vacuolar membranes' 100d 67

' Kane and Stevens, 1992; Ho et aL, 1993; Bauerle et al., 1993. ' Reported in this manuscript. Forgac, 1992; Puopolo et al., 1992. Assignment as a subunit is not final.

Bowman et al., 1992. e Parry et al., 1989.

two subunits contain ATP-binding sites and are highly con- served (SO-SO% amino acid identity) between species. The 17- kDa proteolipid subunit, which binds dicyclohexyl carbodi- imide and is predicted to form a part of the proton pore, is also a well conserved component of the V-ATPase. Other subunits of the V-ATPase are not as well conserved between species (-3040% identity). It is thought, therefore, that these less well conserved subunits might determine the unique qualities of each V-ATPase including the enzyme's subcellular location as well as the pH gradient that is generated by each enzyme. The Vmal3 54-kDa yeast V-ATPase subunit has no significant identity to any protein in the data base. However, there are V-ATPase "subunits" of similar sizes found in the V-ATPases from bovine clathrin-coated vesicles (50-kDa; Puopolo et al., 1992), Neurospora crmsa vacuoles (51-kDa; Bowman et al., 1992), and red beet vacuoles (52-kDa; Parry et al., 1989). All of these subunits have proven to be peripheral components of the enzyme complexes like Vmal3p. These polypeptides, when sequenced, may reveal identity with the VMA13 gene product, It will also be interesting to learn whether, like the yeast Vmal3p subunit, the -50-kDa V- ATPase subunits are essential for activity but not assembly of the bovine, N . crassa, and red beet V-ATPase complexes.

The characterization of the VMA13 gene product as a subunit of the yeast V-ATPase further defines the composi- tion of this multisubunit enzyme. However, because Vmal3p is essential for ATPase activity but not assembly of the V- ATPase, this polypeptide should prove useful in the functional analysis of individual subunits as well as dissection of the assembly pathway of this complex enzyme.

Acknowledgments-We thank Drs. J. S. Jones and L. Prakash (University of Rochester) for pJJ plasmids, Drs. R. S. Sikorsky and P. Hieter (John Hopkins University) for YPH strains and pRS plasmids, and Dr. K. Altendorf for bafilomycin A,. We thank the University of Oregon Animal Care Facility for assistance in preparing polyclonal antibodies,

REFERENCES Anraku, Y., Umemoto, N., Hirata, R., and Ohya, Y. (1992) J. Bioenerg. Biom-

Ausubel, F. M., Brent, R., Kingstone, R. E., Moore, D. D., Seidman, J. G., embr. 24,395-405

Smith, J. A,, and Struhl, K. (1987) Current Protocols in Molecular Biology,

kDa 60 54' 42 36 32 27 17 58 50 40 34 33 19 17 -lod 55 52 44 42 32 29 16 57 51 48 30 16

Bauerle, C., Ho, M. N., Lindorfer, M. A., and Stevens, T. H. (1993) J. Eiol. Green Publishing Associates, New York

Chem. 268,12749-12757 Bowman, B. J., Vazquez-Laslop, N., and Bowman, E. J. (1992) J. Bioenerg.

Forgac, M. (1989) Physiol. Reo. 69, 765-796

Foury, F. (1990) J. Bid. Chem. 2 6 6 , 18554-18560 Forgac, M. (1992) J. Btoenerg. Bwmembr. 24,341-350

Hirata, R., Ohsumi, Y., Nakano, A,, Kawasaki, H., Suzuki, K., and Anraku, Y. Hirata, R., Ho, M. N., Umemoto, N., Ohya, Y., Stevens, T. H., and Anraku, Y.

Ho, M. N., Hill, K. J., Lindorfer, M. A,, and Stevens, T. H. (1993) J. Biol.

Holm, C., Meeks-Wagner, D. W., Fangman, W. L., and Botstein, D. (1986)

Ito, H., Fukuda, Y., Murata, K., and Kimura, A. (1983) J. Bacterial. 153 , 163-

Jones, J. S., and Prakash, L. (1990) Yemt 6,363-366 Kane, P. M., and Stevens, T. H. (1992) J. Bioenerg. Biomembr. 2 4 , 383-393 Kane, P. M., Yamashiro, C. T., and Stevens, T. H. (1989) J. Biol. Chem. 2 6 4 ,

Kane, P. M., Kuehn, M. C., Howald, I., and Stevens, T. H. (1992) J. Biol.

Kornfeld, S., and Mellman, I. (1989) Annu. Reu. Cell Biol. 5 , 483-525 Kyte, J., and Doolittle, R. F. (1982) J. Mol. Biol. 157, 105-132

Manolson, M. F., Proteau, D., Preston, R. A., Stenbit, A., Roberts, B. T., Hoyt, Laemmli, U. K. (1970) Nature 227,680-685

Bid. Chem. 2 6 7 , 14294-14303 M. A., Preuss, D., Mulholland, J., Botstein, D., and Jones, E. W. (1992) J.

Biomembr. 24,361-370

(1990) J. Biol. Chem. 265,6726-6733

(1993) J. Biol. Chem. 268,961-967

Chem. 268,221-227

Gene (Amst.) 4 2 , 169-173

168

19236-19244

Chem. 267,447-454

Ohya, Y., Ohsumi, Y., and Anraku, Y. (1986) J. Gen. Microbiol. 132,979-988 Nelson, H., and Nelson, N. (1989) FEBS Lett. 247,147-153

Ohya, Y., Umemoto, N., Tanida, I., Obta, A., Iida, H., and Anraku, Y. (1991)

Parry, R. V., Turner, J. C., and Rea, P. A. (1989) J. Biol. Chem. 264 , 20025-

Pearson, W. R., and Lipman, D. J. (1988) Proc. Natl. Acad. Sci. U. S. A. 85,

Puopolo, K., Sczekan, M., Magner, R., and Forgac. M. (1992) J. Eiol. Chem.

J. Biol. Chem. 266,13971-13977

20032

2444-2448

2 R 7 5171-5176 Raymond, C. K., O'Hara, P. J., Eichinger, G., Rothman, J. H., and Stevens, T. Roberts, C. J., Pholig, G., Rothman, J. H., and Stevens, T. H. (1989) J. Cell

- - . , - - . - - - , Y

H. (1990) J. Cell E d . 1 1 1,877-892

Biol. 108.1363-1373 Rothstein,R.J.~(1983) MethodsEnzymol. 101 , 202-211 Sanger, F., Nicklen, S., and Coulson, A. R. (1977) Proc. Natl. Acad. Sei. U. S. A.

74.5463-5467 Sherman, F. (1991) Methods Enzymol. 1 9 4 , 3-21 Sikorski, R. S., and Hieter, P. (1989) Genetics 1 2 2 , 19-27 Uchida, E., Ohsumi, Y., and Anraku, Y. (1985) J. Biol. Chem. 2 6 0 , 1090-1095 Uchida, E., Ohsumi, Y., and Anraku, Y. (1988) J. Eiol. Chem. 263,45-51 Umemoto. N.. Yoshihisa. T.. Hirata, R., and Anraku, Y. (1990) J. Biol. &tm.

2 6 5 , 1&147-18453 Umemoto, N., Ohya, Y., and Anraku, Y. (1991) J. Biol. Chem. 2 6 6 , 24526-

3A.523 W$&&, L. S., Bacallao, R., and Wickner, W. (1987) J. Cell Biol. 106,1539-

Yamashiro, C. T., Kane, P. M., Wolczyk, D. F., Preston, R. A., and Stevens, T.

Yoshlhlsa, T., and Anraku, Y. (1989) Ezochem. Eiophys. Res. Commun. 163 ,

104 I

H. $1990) Mol. Cell. Biol. 10 , 3737-3749

90&915