THE JOURNAL OF BIOLQGICAL CHEMISTRY 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 269, No. 27, Issue of July 8, pp. 1807618082, 1994 Printed in U.S.A.

Transcriptional Control of Yeast Plasma Membrane H+-ATPase by Glucose CLONING AND CHARACTERIZATION OF A NEW GENE INVOLVED IN THIS REGULATION*

(Received for publication, January 27, 1994, and in revised form, March 29, 1994)

Mariano Garcia-Arranz, Ana M. MaldonadoS, Mm'a J. Mazbn, and Francisco Portill00 From the Departamento de Bioquimica, Facultad de Medicina, Uniuersidad Autonoma de Madrid, Znstituto de Znvestigaciones Biomedicas del Consejo Superior de Znuestigacioned Cientificas, Arturo Duperier, 4, 28029 Madrid, Spain

The expression of the ATPase gene (PMAl) is regulated by glucose (Rao, R., Drummond-Barbosa, D., and Slay- man, C. W. (1993) Yeast 9, 1075-1084) and by the TUF/ RAPl/GRFl transcription factor (Capieaux, E., Wgnais, M.-L., Sentenac, A, and Goffeau, A. (1989) J. BioZ. Chem. 264,7437-7446). In this work, we describe the isolation of mutations on seven genes that affect the levels ofATPase. One of these genes ( M A 1 ) was cloned by complementa- tion and shown to encode a protein with six putative transmembrane stretches. Expression of APAl gene is regulated by the carbon source and requires the protein GCR1. Deletion of APAl causes a defective regulation of the PMAl expression by glucose but has not noticeable effect on the expression of other TUF-regulated genes. Nevertheless the expression of glucose-repressible HxT3 and SNF3 genes is significantly reduced. These results suggest a model in which M A 1 acts on a glucose-signal- ing pathway that controls the expression of several genes that are transcriptionally regulated by glucose.

The plasma membrane H+-ATPase plays an essential role in the physiology of yeast; it creates the electrochemical proton gradient that drives the uptake of nutrients by secondary ac- tive transport, and it regulates the intracellular pH (reviewed by Goffeau and Slayman (1981), Goffeau and Green (19901, Serrano (19911, and Gaber (1992)).

Different environmental factors modulate the yeast plasma membrane H+-ATPase (Serrano, 1983; Tuduri et al., 1985; Lentzen et al., 1987; Eraso and Gancedo, 1987; Rosa and Sa- Correia, 1991; Benito et ul., 1992; Amigo et ul., 1993) and glu- cose metabolism is one of the most important regulatory fac- tors. Glucose regulation takes place at two levels. At the post- transcriptional level, glucose metabolism induces activation of ATPase activity (Serrano, 1983). The activation of the ATPase by glucose is based on the modulation of an inhibitory interac- tion of the carboxyl terminus with the active site of the enzyme (Portillo et al., 1991) and is mediated by a SerPThr phospho- rylation of the enzyme (Chang and Slayman, 1991). At the transcriptional level, glucose increases the synthesis of P M A l mRNA (Rao et al., 1993). This regulation seems to be controlled by the glucose-modulated TUF/RAPl/GRFl transcriptional factor (Capieaux et al., 1989).

PB91-0063 and Glaxo, S.A. The costs of publication of this article were * This work has been supported by Spanish Grants from DGICYT-

defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankrM/EMBL Data Bank with accession number(s1 X78326.

$ Supported by a predoctoral fellowship from the Gobierno Vasco. 8 To whom correspondence should be addressed. Tel.: 34-1-5854616;

Fax: 34-1-5854587.

We have initiated a search for mutants affected in the regu- lation of the plasma membrane H+-ATPase. We have obtained mutations on seven genes that cause a decrease of the level of ATPase. One of these genes, APAl, was cloned by complemen- tation. We present in this report the molecular analysis of the A P A l gene and data indicating that APAl could function on the regulation of the glucose-dependent expression of P M A l . Fur- thermore, we show that deletion of A P A l does not affect the expression of other TUF-regulated genes, thus providing evi- dence for a new pathway that controls PMAZ expression in addition to the earlier described regulation by the T U F M l / GRFl transcriptional factor.

MATERIALS AND METHODS Strains, Media, and Microbiologic Techniques-Saccharomyces cer-

euisiae strains BWG1-7A(MATaadel-100 his4-519 leu2-3,112 uru3-52) (Guarente et al., 19821, A10700B (MATa thr4) (Portillo and Mazbn, 1986), DFY642 (MATa leu2-3,112 ura3-52) (Uemura and Fraenkel, 1990), DFY645 (MATa Agcrl::LEU2 leu2-3,112 ura3-52) (Uemura and Fraenkel, 19901, and its derivatives described in the present work were grown in medium with 2% glucose, 2% galactose or 2% lactate plus 2% glycerol, 0.7% yeast nitrogen base without amino acids (Difco). Auxotro- phic mutants were supplemented with adenine (40 pg/ml), uracil (40 pg/ml), L-histidine (30 pg/ml), L-leucine (30 pg/ml), or L-threonine (200 pg/ml). When indicated the glucose medium was buffered with 50 mM Mes' adjusted to pH 6.0 with Tris (SD6.0) or with 50 mM succinic acid adjusted to pH 3.0 with Tris (SD3.0). Hygromicin B resistance was tested in YPD (1% yeast extract, 2% peptone, and 2% glucose) supple- mented with 150 pg/ml antibiotic. Solid media contained 2% agar. For drop test cells were grown for 2 days on solid media and then 5 pl of a dilution (-lo4 cells) were dropped on solid media. Growth was scored after 2 days at 30 "C. Standard yeast genetic manipulations were per- formed as described by Sherman et al. (1986).

Isolation ofMutants-The selection scheme used to isolate mutations affecting the levels of plasma membrane ATPase was similar to the scheme used by McCusker et al. (1987). Four hundred independent overnight cultures of strain BWG1-7A were grown from individual single colonies. For each culture a drop of -10' cells was plated on hygromicin B media. Single spontaneous HygR were picked from each drop and tested for growth on SD3.0. Mutants growing slowly on acid media were selected for further analysis.

Cloning and Sequence Analysis of APAl Gene-Yeast carrying the apal-3 mutant allele was transformed (Ito et al., 1983) with 50 pg of DNA from a YCp50-based genomic library (Rose et al., 1987). About 12,000 transformants were selected in SD6.0 medium without uracil. Transformed cells were pooled and plated in SD3.0 medium. After 4 days at 30 "C only six colonies grew on acidic media.

A 3-kb KpnI-Sal1 fragment (Fig. 1) containing the entire MA1 gene was subcloned into M13mp18 and M13mp19 (Vleira and Messing, 19821, and the two strands were sequenced using the Sequenase kit (U. S. Biochemical Corp.) with synthetic oligonucleotide primers. Sequence analysis was carried out by using the UWGCG programs (Devereaux et al., 1984).

kb, kilobase(s1. ' The abbreviations used are: Mes, 4-morpholineethanesulfonic acid;

18076

c o n t a i n i n g M A 1 gene. Thr t h r n linos FIG. 1 . R e s t r i c t i o n m a p of plasmids

indicate vvrtor srqwnces and thr. thirk linrs indicate yeast sequences. The oprn rrnding frame of APAl is indicatrd hy a n orrnw. R , Ilnmlll; llg, BglIl: C, ( ' 1 0 1 ; I I , Hpo l : Hd. NindIII: K, KpnI: P. PvuII; S. Snl l ; A', XhoI; A%. X h l . pRS316 is n single-copy plasmid (Sikomki :and Hirter. 19x91. YICp.75'2 is a multicopy vector 11lill rt nl. . 1986) and YEpR57R is a multicopy vector usrtl fnr ImrX fusion I Myers r t n l . . 1986). p\ lGl l9 \vas usrd to construct yrnst crlls drlrted for Al.l.11.

Regulation of PMAl Expression 18077 H C Xb K HHSHBg X B C

pMGIOO 3 H Kb

K H H S

pMG115 (pRS316)

I

I I

, \

\

, , 1 Kb

pMG 122 - K ;a:! !Id P H d P U S

pMG119

pMG 1 23

Drlrtion of thc APA I Grnr-The 3-kh KpnI-Sal1 fragment (Fig. 1) was suhclonrd into a pUCl8 plasmid (Yanisch-Perron rt n l . , 1985) in which the Ifindlll site had hrrn drstroyed. The resulting plasmid \\*as cut with N/ndlII and ligated with a IlindIII fragment containing the CRA.3 grne ohtained from the ~J~J2.14 plasmid (.Jonrs and Prakash. 1990). The resulting plasmid (pMG119; Fig. 1) was cut with Kpnl and SnlI hrforr transformation of an autodiploid constructed hy transformation of strain R\\'C;1-7Awith the H O grnr (Hrrskowitz and densrn, 1991 1. T h r transformrd diploid was sporulated, and tetrads werr dissrrtrd.

IYorthrrn Annlwis-Total K S A was ohtained as descrihrd (Carlson and Rotstein, 19821. separated on lr; agarose gels containing 2.2 11

formnldrhyde. transferrrd to Sylon memhranes, and hyhridizrd to the diffrrrnt laheled prohrs. I'rohes were ohtainrd hv using the polymerase chain reaction utilizing synthctic oligonuclrotido as primrrs. Thr prim- ers designs wrrr hascd on t h r puhlished rrnding framr srquences avail- able in the EMRIJGrnRankT\'.

Construrtiotl o fAPAI-hcZ and PMAI-ImrZ Fusions and p-Galorto- sidnsr Assa.s-Plasmid pMG123 (Fig. 1 ) was constructed hy inserting the Kpnl-I'cwIl fragmrnt of APAI locus into Kpnl-SnlnI-digestrd YEp357R (Myers rt n l . , 19861. Plasmid carrying a /'.!fAl-lmrZ fusion' was constructrd hv inserting the coding region of ImrZ ohtained as a RnmHl fragmrnt from plasmid p3fC1871 (('asadahan rt 01.. 1983) into thr RotnHl-digrsted pRSI.1 (Cid r t nl.. 19871.

Transformnnt crlls containing either Af'A1-ImcZ or PMAI-lmrZ fu- sion were grown up to exponential phase fahsorhance at 660 nm of -0.5) in the appropriatrd medium. Snmplrs of 2 ml wrrr crntrifugrd and frozen in liquid nitrogen. The p-galactosidase activity was detrrminrd in cell-free rxtracts as descrihrd rlsrwhrrr miller, 19721.

Rinrhrmirnl Mrthorls-Yrast plasma memhranes were purified from glucosr-mrtaholizing cells hy differential and sucrose gradirnt ccntrifu- gation (Scrrano. 19831. ATPase activity was assayed as drscrihrd ISrrrano. 198x1. Protrin concentration \\'as drtrrminrd hy the method of Rradford ( 1976) with t h e I h - R a d protein assay reagrnt and hovinr I g G as the standard. Rahhit pnlyrlonal antibody against the yeast ATPase (Serrano rt n l . , 19861 was nflinity-purifird Ofonk ct a / . , 1991 ). Plasma mrmhrane proteins were separated hy SI)S-polyacrylnmidr gel electro- phoresis on 81 ncrylamidr using the systrm of Lnemmli f 19701. \Vest- r r n hlot with second antihody conjugatrd to nlkalinr phosphatase I IGo- Rad1 was as drscrihrd prrviously t13lake rt nl., 19841. Intracrllular pH was measured as in Eraso et 01. I 1987). For transport studirs crlls wrrr grown in medium with either glucose or galactose. harvested in the rxponential phase fser ahove). washrd twicr with water, and incuhatcd at 10 mglml in 50 m>I Mes-Tris huffer, pI1 6.0. after 10-min prrincuha- tion at 30 'C transport of glucosr (final concentration 110 m\r) was mrasurrd as descrihrd by Risson and Frarnkrl (19831.

RESULTS

Isolafion and Characfrritafion of Mutants Affrcted in Yrasf Plasma Mrmhranr Ha-ATPasr Acfivif.s"Four hundred inde- pendent mutants were isolated in a selection for hygromicin R-resistant yeast cells. Thirty-four out of these 400 mutants exhibited slow growth in acid media and a low level of plasma membrane H'-ATPase activity. "" ~.

? P. Eraso and R. Srrrano. unpublished results.

W T

apa 1-3

apa 2-4 apa 3- 1

apa 4-1

apa 5-2 apa 6-6

apa 7- 1

ON: ATPase ac!w!y SD 6 0 SD 3.0 (umolmn '.rq pxer ' 1

0.87

0.24

0.40 0.20 0.22 0.20 0.30

0.39

FIG. 2. Growth p h e n o t y p e rand ATPase ac t iv i ty of representa- t i v e a p a l a p a 7 m u t a n t alcllrs. Crlls werr dropped on platrs con- taining glucow merlin htlfTcrvtl a t pi-I 6 . 0 or 3.0 and Incuhatrtl at 30 C for 2 rhys. A'T'l'asr activity tvas nwasurrd in purifird plasma mrmhranr from glucosr growing crlls. i'alues ar r th r avr ragr of two diffrrrnt rxperimrnts. Thr spcbcifir activity diffrrrd in less than 105.

Genetic analysis of the mutants showed that seven were allelic to PMAl and that the remaining mutant alleles com- prised seven different genes designated arbitrarily M A 1 to M A 7 (genes affrctingplasma membrane ATPase activity). Fig. 2 shows the growth and the H'-ATPase activity of representa- tive mutants.

Only the detailed characterization of the APAl locus will be discussed in this report.

Isolnfion of APAl-The M A 1 gene was isolated from a YCp50 yeast genomic library by complementing the low pH sensitivity phenotype of the npal-.? mutant. Six transformants able to grow on acidic medium and exhihiting wild type levels of ATPase activity were isolated. All six transformants showed cosegregation of growth, ATPase activity, and uracil prototro- phy. In each case plasmid was rescued from yeast and amplified in Eschwichia coli. Restriction analysis of these clones revealed that all six plasmids contained the same insert.

Plasmid pMGlO0 (Fig. 1) containing a 12-kb insert in YCp.50 was subcloned to determine the extent of the complementary activity that was confined to a 3-kb KpnI-Sal1 fragment (Fig. 1). The possibility that the insert contained an extragenic sup- pressor of apa I was discarded by inserting a copy of the cloned DNA containing an URA.?' gene in the yeast genome and ana- lyzing linkage to the APA l locus. The deleted copy ofARAI used for the linkagr analysis is described below.

Spqurncr Analysis of APAI Crnr-DNA sequence analysis of the 3-kb KpnI-Sal1 fragment revealed a single open reading frame of 1035 base pairs (Fig. 3).

The 5' non-coding region shows a canonical TATA box at

18078

-150 -660

-570

-480 -390 -300

-210

-120

-30

61

151

241

331

421

FIG. 3. Nucleotide and deduced amino acid sequences of APAl. Puta- 601 tive membrane-spanning segments are underlined.

511

691

781

871

961

1051

1141

1231

1321

1411

1501

1591

1681 1771

1861

1951 2041

2131

Regulation of PMAl Expression

G G T A C C C G T A C A T C T T G G C A T T C G A C A ~ ~ T C A T T W L G A T C A ~ T ~ ~ G G C G R A T T A A T T C G T T ~ T C C T T G f f W L T A

R G A T T C G A C T T C T A C R A A U V L G T A C T C A A W U V \ T C C T G T A A T T T C T G

G G G T T A A G A T A G C A A C G T A T A T G A ~ T C C T T A T T T T C T T T T

A A R C A T T G A T A C A T A G T G G C G A C T A A A T A A T A R T T T T

U L T G A T A C C U C G C G A A A ~ T T C T T C - ~ T T C G A A T ~ T A T T t C G T C ~ ~ G T ~ ~ T C A G T T

T T C U L G A C T G G G T T G G C T T A C G C A T T T G G C T T A C G U L T T T T T T T ~ T C A C A G G T T C T T A G T ~ T A A T T G T A T ~

TATTTATCTGWLGTCTTCTGTTTGTTGTTTAGC~GTTTWLAGTTTTATTTAACTTATTACC~AGTTAGGTACTGTGTA~TCC~

T A T C T G G T G T t T T T T T A A T T T G G T C A T T G G T T T T T G T

TTTCCGmGTTTACGAAACATAAACAGTCRTGAACA~ACCAULTCTACTGTTATAGCAG~GTTGCCWLCCAGTTtCAGTt~GRAC

M N T T T S T V I A A V A D Q F Q S L N

TCTTCTTCTmRTGTTTCTTWLAGGTTCATGTTCCTTC~~GATAAC~TTCGGTATTWLATTATGGC~TTTTCTCCAAAGTGTTT

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

GAATACTTTAGTGGCTATCCAGCTGAG~TTC~GTTTATTCACAATAAGA~TCTTGGCTAACG~ATCATGCTGTTAGTATTATT

E Y F S G Y P A E Q F E F I H N K T F L A N G Y H A V S I I A T C G T T T A T T A C A T T A T T A T C T T T G G T ~ C A A ~ A T C T

I V Y Y I I I F G G Q A I L R A L N A S P L K F K L L F E ~ CACARCTTGTTT~CTTATTTCTCTAGTTTTATffiTTGCTWLTGTTAWLACAG~GGTTCCTATffiTTTATCACAACGGTCTATTC

H H L F L T S I S L V L W L L M L E Q L V P M V Y H N G L F T G G T C P A T C T G C T C T A A G G A C ~ G C A C C A A A A T T A G C

W S I C S K E A F A P K L V T L Y Y L N Y L T K F V E L I D A C T G T G T T T T T A G T T T T G ~ G ~ T T A T T G T T T T T G C A C A C T T A C ~ ~ C ~ T G C C A C C G C T T T G T T G T G C T A C A C T ~ T T A

T V F L V L R R K K L L F L H T Y H H G A T A L L C Y T Q L ATTGGTCGTACTTCTGTTGAACGGGTAGTTATCffACT~CTT~T~TCACGTTATCATGTACTGGTACTACTTtTTG~TTCAT~

I G R T S V E R V V I L L N L G V H V I M Y W Y Y F L S S C

GGTATTAGAGTTTGGTGGAAG~TGGG~CT~T~C~TTATTCAAT~TGATTGACTTff iTATTTGTTTACTTTGCTACCTAT

G I R V W W K Q W V T R F Q I I Q F L I D L V F V Y F A T Y A C A T T C T A T G C T C A U U L A T A C ~ G G A C G G T A T T T T A C C A T T G

T F Y A H K Y L D G I L P N K G T C Y G T Q A A A A Y G Y L A T T C T A A ~ T C T T A T T T G C T T T T G T T T A m C C T T C T A C R T C

I L T S Y L L L F I S F Y I Q S Y K K G G K K T V K K E S E

G T T T C C G G C T C C G T T G C A T C C G G T T C T T T t T A C f f i T G T ~ G A C C T C T A A C A ~ A A G ~ C T C T T C C A G ~ ~ T T ~ T A ~ G C ~ G

V S G S V A S G S F Y G V K T S N T K V S S R K A * A A T T T T T G A C A G C W L A T G A ~ T T A A R T G

AATGATAGATTATTGGCTTGGTGTTTTTCCCAT~GATTAATCATTT~TTAGCTGCCUULA~TTGCAC~TA~GTGAffiAGTAG

T W L T A U L T T A C A A T T A T A T T A T A T A T A G C A G C T ~ T T T ~ ~ C A G R C ~ G T T C C T T T G T A T G T C C T A T ~ W L T

ACTWLCTTGCAGTTTGTAIlAGTGTCCAGTCGTCGT~TCCAC~TAGTTGC~CWLATTA~ATTGAAT~TTT~TTTT~TAATCTCTTGA

G T T A A A T C G O W L T G T A C T C T G G G R A T G A G C A T T T A A A A A T

~TTGTAATATCGGCCCAGATCATArrTAAGTATTGGGACCAWLACGCGTTACACACAGWLACATGGATATCAAGTAA~CTT~T

GRAGTTCATTGGTTGTTCTAGCTCTtCAATAATAGAGTC~CTGTTATTATTGTCTTCTT~CffiCCACATCTCTAATG~TTGCTT

TGTTCACAATGACGACCTGTAAGATATGffiTACGATGGATGGA~TTAffGTACT~GTACTATATAAGTC~C~AATGATTTGTAA

EAAGATAGRTCTATTTCCTTTGATATGGTAWLGTAGAG~CGAT~TGAT~TC~AAGA~CC~TGATAG~TATTATTGCTATTCTG

T G G T T G T T G T T C C G G T R R T A C A G A A T T T T C T C C A G T A G T C G T

TGTCGTTATCATGTTCGTTATCATTATT~CGCTGTTGT~CGCTTCTATTGTTGATGTTGTTCGTCTGATTAT~GTGTWLATGTTCA

TGTTGCTATGATTGWLAGTATTATWLCTGTTGTTAACGTTAACGTTGCCTATTGTGTGTAAATT~CCTTTTCAATAAACT~TTGATG

TAGAAAGTTTGTCGAC

20

50

80

110

140

110

200

230

260

290

320

345

location -217, a UAS,, at position -747 and two CTTCC mo- tifs at locations -357 and -546, respectively. The UAS, and the pentamer sequence CTTCC have been found in the up- stream regulatory region of many genes including glycolytic and translational machinery component genes (Huet et al., 1985; Lue and Kornberg, 1987). The UAS,, is the DNA bind- ing site for the multifunctional protein TUF/RAPl/GRFl (Leer et al., 1985; Huet et al., 1985) and the CTTCC motif is recog- nized by the DNA binding protein GCRl (Baker, 1991; Huie et al., 1992). In genes that contain both motifs, GCRl functions complexed with TUF/RAPl/GRFl to activate transcription (Tornow et al., 1993).

At the 3' end a polyadenylation signal AATAAA is detected 211 base pairs downstream from the stop codon. At positions 1260, 1300, and 1329 there is a sequence TAG. . . TAT- GT . . . TTT for transcription termination in yeast (Zaret and Sherman, 1982).

The sequence predicts a 39.5-kDa protein of 345 amino acids. The codon bias index (Bennetzen and Hall, 1982) of the pre- dicted protein is 0.47 which corresponds to proteins expressed at low level (Sharp and Cowe, 1991). The hydropathy plot gen- erated by using the residue-specific hydrophobicity index of Kyte and Doolittle (1982) and a window size of 19 showed that the protein contains 6 stretches of hydrophobic residues large enough to be considered membrane-spanning segments. An-

other interesting feature of the M A 1 sequence is a perfect leucine zipper motif starting at position 106 and located in the predicted transmembrane stretch 2 that could trigger dimer- ization of the protein within the membrane.

A search of the EMBL/GenBankTM found that the predicted APAl amino acid sequence is homologous (50% identity) to the previously sequenced gene YCR34w located on chromosome I11 (Oliver et al., 1992). This gene was predicted to code for a nonessential plasma membrane protein of unknown function (Goffeau et al., 1993). No relevant homology to any other se- quence of the data bank was found.

Expression ofAPAl Gene-Efficient expression ofAPAl gene required glucose as a carbon source and the presence of the GCRl protein as demonstrated both by Northern analysis (Fig. 4A) and the activity of the reporter P-galactosidase gene fused to the APAl promoter (Fig. 4B). Northern analysis revealed a mRNA length of about 1.3 kb in accordance with the length of the open reading frame. The APAl transcript was detected in the wild type strain growing on glycerol plus lactate but the amount increased when cells were growing on glucose. Deletion of the GCRl gene reduced the amount of transcript but the residual level was still increased by the presence of glucose. Similar results were obtained when the activity of the reporter gene was assayed.

Regulation of PMAl Expression 18079

0.25

0.10

0.05

0.00 GCRl Agcrl

FIG. 4. Northern analysis of M A 1 expression ( A ) and MA1- Lac2 activity ( B ) in GCRl and Agcrl strains. Yeast cells growing in medium with glycerol plus lactate or glucose were harvested during exponential phase and processed to obtain total RNA or to assay P-ga- lactosidase activity as decribed under "Materials and Methods." Values of P-galactosidase activity are the average of three different experi- ments and bars represent the standard deviation.

Construction and Characterization of APAl Null Muta- tion-A deletion of APAl was created by removal of the entire protein coding sequence and replacement of this segment with the selectable marker URA3 (Fig. 1, see "Materials and Meth- ods"). This construct, Aapal::URA3, was used to transform a diploid yeast strain. After sporulation and tetrad dissection the deletion ofAPAl was checked by Southern blot analysis. Tetrad analysis (12 asci analyzed) showed that APAl was essential for growth on glucose medium buffered at acidic pH. The low pH- sensitive phenotype segregated 2:2 and was ligated to the URA3 marker.

To study the biochemical phenotype of the APAl null allele a set of isogenic strains was created by transforming the Aapal::URA3 mutant strain with a wild-type copy of APAl on either a centromeric (pMG115) or multicopy (pMG122) plasmid (Fig. 1).

First, we examined the ATPase activity of the isogenic strains growing on glucose medium buffered at neutral pH. Under these conditions the ATPase activity was significantly reduced in the apal deletion mutant as compared with its isogenic strains (Table I). The presence ofAPAl on a multicopy plasmid had not noticeable effect on the level of activity.

Yeast plasma membrane H+-ATPase is essential for internal pH homeostasis (McCusker et al., 1987; Ulaszewski et al., 1987; Portillo and Serrano, 1989; Vallejo and Serrano, 1989). Accord- ingly, the Aapal::URA3 mutant had a more acidic intracellular pH than the wild type strain (Table I). This defective intracel- lular pH regulation may account for the low pH sensitive phe- notype of Aapal mutants.

Deletion of APAl did not result in changes in the apparent affinity constant for ATP or the pH optimum of the ATPase (Table I). These data suggest that the reduced ATPase activity in the apal null mutant is not due to altered properties of the enzyme but rather results from a decrease in the concentration of the enzyme in the plasma membrane.

M A 1 Null Mutation Affects PMAl Expression-The reduc- tion in the amount ofATPase in Aapal::URA3 strain suggested by the aforementioned results was confirmed by Western anal-

ysis of membrane preparations from MA1 and Aapal strains using specific antibodies against the yeast plasma membrane ATPase (Fig. 5). The level of ATPase protein in total mem- branes and purified plasma membrane fraction from apal null mutant was much lower than in the isogenic wild type strain. The fact that the amount of Pmal was reduced also in the total membrane fraction indicates that ATPase was not accumulated in other membrane system. Total membranes from APAl and Aapal strains fractionated by sucrose gradient centrifugation under isopicnic conditions showed that the distribution of ATPase was identical in both strains (data not shown). These results suggest that the reduced level of ATPase in plasma membrane of the apal null mutant is not due to a defect in the intracellular targeting of Pmal to the plasma membrane but rather results from either a reduction in the synthesis or the stability of the protein.

To determine if the decreased amount of ATPase in the Aapal::URA3 mutant strain was due to a decreased amount of transcript, Northern analysis of total RNA from MA1 and Aapal strains was performed (Fig. 6). The apal null mutant had reduced levels of PMAl mRNArelative to the isogenic wild type strain. The effect ofAPAl deletion on PMAl transcription was also studied by analyzing the expression of a PMAl-LacZ fusion gene on a monocopy plasmid in APAl and Aapal strains growing on SD6.0 medium. The level of P-galactosidase activity in the apal null mutant was 0.57 2 0.07 pmol.min".mg pro- tein" while in the MA1 isogenic strain it was 1.2 f 0.10 pmol.min".mg protein" (both results are the average of three different experiments). These results suggest that deletion of APAl affects the level of plasma membrane H+-ATPase by modifying the synthesis of PMAl mRNA.

Arecent report (Rao et al. 1993) has shown that yeast plasma membrane ATPase transcription is regulated by glucose. It was of interest to determine whether APAl was involved in the observed regulation ofPMA1 transcription by glucose or not. To address this question Northern analysis of total RNA from APAl and Aapal::URA3 strains growing on glucose or galac- tose media was performed (Fig. 7A). The PMAl transcript was more abundant when wild type cells were growing on glucose. In the Aapal strain growing on glucose the expected increase in PMAl mRNA was not observed. Similar results were obtained when the expression of a PMAl-LacZ fusion gene on a mono- copy plasmid was studied (Fig. 7B). These results suggest that MA1 is involved in the regulation by glucose of PMAl gene transcription.

Expression of n o Glucose Dansporter Genes Is Affected by apal Null Mutation-To ascertain whether the observed regu- lation of PMAl expression by APAl was general to TUF-regu- lated genes or not, we examined the mRNA level of several genes the expression of which is regulated by the TUF/RAPlt GRFl factor (Huet et al., 1985; Leer et al., 1985). Northern analysis of RNA isolated from APAl and apal null mutant strains (Fig. 8) showed that expression of TEFl, RP51, POLl, KRSl, PDCl, GAPl, and PGKl was not significantly affected by the deletion ofAPA1. Only minor changes in EN01 expres- sion were observed.

The fact that PMAl encodes a plasma membrane protein prompted us to study the expression of genes coding for other plasma membrane proteins. Northern analysis (Fig. 8) showed that APAl deletion had no effect on the expression of GAL2 and TRKl genes; however, a remarkable alteration in the mRNA levels of HXT3 and SNF3 genes was observed. HXT3 and SNF3 are members of the large glucose transporters gene family. Neither HXT3 nor SNF3 are essential for viability, since ex- pression of any of the glucose transporter genes is sufficient for normal growth on glucose medium (KO et al., 1993). It should be

18080 Regulation of PMAl Expression TABLE I

Effect of the apal null mutation on the kinetic properties of yeast plasma membrane ATPase and on intracellular pH

Relevant genotype ATPase activity' PHopt Intracellular

pH

,umol.min".mg protein" mM ,umol.min".mg protein" AFAl 1.15 0.40 3.00 6.5 7.00 Aapa1::URAJ 0.35 0.40 0.95 6.5 6.60 Aapal::URA3 + YCp.APA1 1.10 0.45 2.85 6.5 6.94 Aapa1::URAS + YEp.APA1 0.90 0.40 2.70 6.5 7.00

ATPase activity was measured in purified plasma membrane at pH 6.5 with 2 mM ATP.

kd 1 2 3 4 5 6 7 8 9 A

106 D

80 D

49.5 D

32.5 D

27.5 D

TM PM TM PM TMPM TM PM ""

APAl Aapal APAlAapal FIG. 5. Analysis of the amount of Pmal in the plasma mem-

brane of APAl and apal null mutant strains. SDS-polyacrylamide gel electrophoresis (lanes 2-5) and Western blot analysis (lanes 6-9) of total membranes (2") and purified plasma membrane ( P M ) from APAl and Aapal strains. Each lane contained 10 pg of protein. Antibody against Pmal was affinity-purified. Lane 1 shows the position of pre- stained standards of the indicated size in kilodaltons.

25sD Y a PMAl

n a ACT1

FIG. 6. Northern blot analysis of PMAl expression in APAl and apal strains. Total RNA was isolated from yeast cells growing to mid-exponential phase in SD6.0. A 5-kb Hind111 fragment containing the entire PMAl gene (Serrano et al., 1986) was used as probe. The filter was stripped and rehybridized with a yeast actin probe as an internal control.

noted that expression of HXTl , another member of the family, was unaffected by the APAl deletion.

When glucose uptake was measured in APAl and Aapal strains growing on repressive and nonrepressive carbon sources it was observed that glucose uptake ability of the apal mutant was decreased (Fig. 9). This may be due to the observed effect of the APAl deletion on HXT3 and SNF3 gene expression.

To discard that the observed effect of the apal null mutation on the glucose-regulated PMAl expression was due to a defect on glucose uptake caused by low levels of HXT3 and SNF3 transporters, we measured the ability of either HXT3, SNF3, or

1.2 SGal 0 SD

0.8

APAl Aapal FIG. 7. Northern blot analysis of PMAl expression (A) and

PMAl-LacZ activity ( B ) in APAl and Aapal strains growing in medium with galactose or glucose. Yeast cells were grown to mid- exponential phase and samples were processed as described under "Ma- terials and Methods." Values of p-galactosidase activity are the mean of three different experiments.

HXTl on multicopy plasmid to suppress the slow growth on acidic media phenotype caused by APAl deletion (Fig. 9). Nei- ther HXT3, SNF3, nor HXTl was able to suppress completely the low pH sensitivity phenotype of apal null mutant strain. When the uptake of glucose was measured it was observed that the defect on glucose uptake caused by apal null mutation was suppressed by HXTl on multicopy plasmid. These results sug- gest that the control of PMAl gene expression by APAl is functionally independent of glucose uptake.

DISCUSSION In this report we described the isolation and characterization

of a gene, APAl, that affects the level of yeast plasma mem- brane ATPase activity. The most prominent feature of the APAl sequence is the presence of six stretches of amino acids pre- dicted to be membrane-spanning segments. Unfortunately, we were not able to confirm the membrane location of Apal since the antibodies raised against the carboxyl terminus of Apal failed to detect the protein in yeast homogenates? probably because of the low amount of Apal in the cell.

M. J. Maz6n and F. Portillo, unpublished results.

Regulation of PMAl Expression 18081

have shown both by direct measurements of the steady state level of PMAl mRNA and by indirect quantification of the expression with a Lac2 reporter gene. The control of mRNA synthesis by APAl does not appear to be specific for PMAl since the steady state level of, at least, HXT3 and SNF3 mRNAs was also affected by APAl deletion.

We do not know the function of the Apal protein. Although some general features of the sequence resemble the cation- channel protein family there are no obvious structural motifs that suggest a function for Apal. We can only speculate as to the mechanism of the gene expression control exerted by ApA1. The fact that an APAl deletion affects the expression of either glucose-inducible genes (as PMAl) or glucose-repressible genes (as HXT3 and SNF3) (Celenza et al., 1988; KO et al., 1993) suggests that Apal could act as a member of a glucose-signal- ing pathway. Several such pathways have been proposed in yeast (Gancedo et al., 1985; Thevelein, 1992; Rao et al., 1993). Although we have not been able to identify the cellular location of the MA1 protein, computer prediction suggests that Apal could be a plasma membrane protein. IfApal is located in the plasma membrane, then it seems reasonable to think that it could be part of a glucose-sensing complex which through yet unknown mechanisms leads to either transcriptional activation or repression of glucose-regulated genes. The molecular analy- sis of other APA genes will help to clarify this hypothesis.

TEF1

RP51

POL1 PC,

KRSl mu

PDC1 ccu GAP1 .I...

PGKl W

ENOl rU

GAL2 r(ir TRKl

HXT3 - 8 0

SNF3 * HXT1

ACTl . * * *

FIG. 8. Northern analysis of RNA isolated from APAl and Aapal strains. Total RNA was prepared from cultures growing loga- rithmically and 10 pg were applied to each lane. Filters were hybridized to DNA probe of: elongation factor la , TEFI; ribosomal protein 51, RP51; largest subunit RNA polymerase A, POLl; lysyl-tRNA synthe- tase, KRSl; pyruvate decarboxylase, PDCl; glyceraldehyde-3-phos- phate dehydrogenase, GAPI; phosphoglycerate kinase, PGKl; enolase 1, ENOl; galactose transporter, G M 2 ; potassium transporter, TRKl; glucose transporters, SNF3, HXT3, and HXTI. ACTl mRNA was probed as an internal control for the amount loaded onto the gel.

GROWTH ON:

SD 6.0 SD 3.0

Aapal + YEpoHXT3

Aapal + YEP-SNF3

(pmol-rnin"mg wet weight") GLUCOSE UPTAKE

Galactose Glucose 10.5of0.98 4.30f0.77

4.42M.37 3.30f0.55

4.41fo.87 3.40k0.93

4.21fo.52 3.20+0.40

Aapal + YEpHXTl 7.3020.70 7.9920.36

FIG. 9. Growth phenotype and uptake of glucose in M A 1 and Aapal strains transformed with a multicopy plasmid carrying different glucose transporter genes. Yeast cells were grown in me- dium with galactose or glucose, harvested during logarithmic phase and glucose uptake assayed as described under "Materials and Methods." Values are the mean of three different experiments standard deviation.

Physical analysis indicated that APAl encoded a 1.3-kb tran- script the expression of which is carbon source- and GCR1-

Acknowledgments-We thank Dr. P. Eraso for the PMAI-Lac2 fusion, Dr. D. G. Fraenkel for the Agcrl mutant strain, Dr. R. F. Gaber for the glucose transporter genes, and Dr. A. Rodriguez-Navarro for the TRKI gene. We acknowledge Drs. P. Eraso, C. Gancedo, J. M. Gancedo, and R. Serrano for critical reading of the manuscript.

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