Proc. Nati. Acad. Sci. USAVol. 85, pp. 1374-1378, March 5, 1988Biochemistry

Isolation of a second yeast Saccharomyces cerevisiae gene (GPA2)coding for guanine nucleotide-binding regulatory protein:Studies on its structure and possible functions

(GTP-binding proteins/signal transduction/ras proteins/gene disruption/cAMP)

MASATO NAKAFUKU*, TOMOKO OBARA*, Kozo KAIBUCHIt, IKUKO MIYAJIMAt, ATSUSHI MIYAJIMAt,HIROSHI ITOH*, SHUN NAKAMURA*, KEN-ICHI ARAIt, KUNIHIRO MATSUMOTOt, AND YOSHITo KAZIRO***Institute of Medical Science, University of Tokyo, 4-6-1, Shirokanedai, Minatoku, Tokyo 108, Japan; and tDepartment of Molecular Biology, DNAXResearch Institute of Molecular and Cellular Biology, Palo Alto, CA 94304-1104

Communicated by Arthur Kornberg, October 23, 1987

ABSTRACT In a previous paper, we demonstrated that agene coding for a protein homologous to the a subunit ofmammalian guanine nucleotide-binding regulatory (G) pro-teins occurs in Saccharomyces cerevisiae. The gene, designatedGPAI, encodes a protein (GPla) of 472 amino acids with acalculated Mr of 54,075. Here we report the isolation ofanother G-protein-homologous gene, GPA2, which encodes anamino acid sequence of 449 amino acid residues with a Mr of50,516. The predicted primary structure of the GPA2-encodedprotein (GP2a) is homologous to mammalian G proteins[inhibitory and stimulatory G proteins (GI and G., respec-tively), a G protein of unknown function (G), and transducins(Go)] as well as yeast GPla. When aligned with the a subunitof G, (G,.,) to obtain maximal homology, GP2a was found tocontain a stretch of 83 additional amino acid residues near theNH2 terminus. The gene was mapped in chromosome V, closeto the centromere. Haploid cells carrying a disrupted GPA2gene are viable. Cells carrying a high copy number of plasmidGPA2 (YEpGPA2) had markedly elevated levels ofcAMP andcould suppress a temperature-sensitive mutation of RAS2.These results suggest that GPA2 may be involved in theregulation of cAMP levels in S. cerevisiae.

G proteins are a family of guanine nucleotide-binding pro-teins known to be involved in a variety of signal-transducingsystems (for review, see ref. 1). Two G proteins, Gs and G1,are involved in hormonal stimulation and inhibition, respec-tively, of mammalian adenylate cyclase (1, 2), whereas Go(other G protein), which is abundant in brain tissues (3), maybe involved in neuronal responses. Transducins, G,1 and G12,present in retinal rods and cones, respectively, regulate theactivity of cGMP phosphodiesterase and mediate visualsignal transduction (4). There is evidence suggesting thepresence of additional G proteins, which may be involved inthe activation of phospholipase C (5) and phospholipase A2(6) as well as the gating of K + (7) and Ca2+ (8) channels.

Recently cDNA sequences for a subunits of several Gproteins have been determined [e.g., Gs from bovine (9, 10),rat (11), and human (12, 13); Gi from bovine (14), rat (11),mouse (15), and human (16, 17); G. from rat (11) and bovine(18); and G,1 and G12 from bovine (19-22)]. These studieshave revealed that the nucleotide and deduced amino acidsequences are highly conserved among different mammalianspecies.

In view of the strong conservation of the amino acidsequences of each G-protein species among different orga-nisms, we searched for G-protein-homologous genes in yeastand isolated the GPAI gene from Saccharomyces cerevisiae

(23). The GPAI gene encodes a protein of 472 amino acids.This protein is 60% homologous to the a subunit of mam-malian Gi (Gia) at the amino acid sequence level if a stretchof 110 amino acids found only in yeast GPAI is disregarded.Subsequent studies on the function of GPAJ suggest that thegene is involved in mating-factor-mediated signal transduc-tion in yeast haploid cells (24, 25). S. cerevisiae has anothersignal transduction system, which is mediated by nutrientssuch as glucose. Glucose serves as an extracellular signal forthe regulation of adenylate cyclase and inositol phospholipidturnover (26). It has been shown that in S. cerevisiaeRAS2-encoded protein stimulates adenylate cyclase (27, 28),whereas RAS]-encoded protein seems to negatively affectinositol phospholipid turnover (26).

In this paper, we describe the isolation of a secondG-protein-homologous gene (GPA2)§ from S. cerevisiae,which encodes a protein of 449 amino acids. Preliminarystudies on the function of GPA2 suggest that it may beinvolved in the regulation of cAMP levels in S. cerevisiae.

MATERIALS AND METHODSYeast Strains and Media. The genotype of each strain is

described in the text. ras2-101 temperature-sensitive (ts)mutants were isolated as heat shock resistant and tempera-ture-sensitive mutants from RAS2Va'l9 cells (29). YPD me-dium contained 2% polypeptone, 1% yeast extract, and 2%glucose. SD medium contained 0.7% yeast nitrogen basewithout amino acids (Difco) and 2% glucose. SD mediumwas supplemented with auxotrophic requirements (SC me-dium).

Molecular Cloning of the GPA2 Gene. The cloning methodswere essentially the same as those described for GPAI (23).

Construction of Plasmids and Disruption Strains of GPA2.Plasmids pGO303 and pGO304 were constructed by insertingthe 3.2-kilobase (kb) Pst I fragment of pGO5 (see Fig. 1) intothe HindIII site of YCp5O, a low copy number plasmid, andthe BamHI site of YEp24, a high copy number plasmid,respectively. Plasmid pGO202 was constructed by substitut-ing the EcoRV fragment of pGO5 with the blunt-endedBamHI fragment of HIS3 (see Fig. 1). pGO202 cleaved with

Abbreviations: G protein, guanine nucleotide-binding regulatoryprotein; Gs and Gi, G proteins that mediate stimulation and inhibi-tion, respectively, of adenylate cyclase; G0, a G protein of unknownfunction; Gt, transducin; Ga, Qk., and G., a subunits of G,, Gi, andG., respectively; Gi2u, rat C6 glioma cell Gj,; ts, temperaturesensitive.*To whom reprint requests should be addressed.§The sequence reported in this paper is being deposited in theEMBL/GenBank data base (Bolt, Beranek, and Newman Labora-tories, Cambridge, MA, and Eur. Mol. Biol. Lab., Heidelberg)(accession no. J03609).

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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Sma I was used to transform his3 yeast strains, and trans-formants were selected by histidine prototrophy.

Assay for cAMP Content. Yeast cells were collected in thestationary phase of growth, washed twice with water, andthen suspended in a buffer containing 10 mM Mes (pH 6.0)and 0.1 mM EDTA for 2 hr at 37TC. Glucose was then addedto a final concentration of 25 mM. After incubation forvarious periods of time, 1-ml aliquots were taken from theculture and transferred to 1 ml of 10% (wt/vol) trichloroace-tic acid. The cAMP content was measured using a cAMPdetermination kit (Amersham).

PSIlI PstpMN5v\/ii

Pstl HirxLL.,Clal SrQaL PvuIlClal Hincil Hincll AatIl Hind Dral PstlpG05wv\/ I I I I I---------0 ^

EcoRV EcoRV *b

: :~~~~~~eiG0202I

RESULTS

Isolation and Identification of a Second G-Protein a-

Subunit Homologous Gene (GPA2) from S. cerevisiae. Wepreviously isolated the GPAI gene (23) from a yeast genomicDNA library by cross-hybridization with cDNAs for ratbrain Gia (now referred to as Gi2a) and the a subunit of G.(GO.) (11).

Since no other yeast DNA hybridized with GPAJ, we

rescreened the yeast genomic library using rat brain Gi2a andG0a cDNAs as probes. A clone, pMN5, containing -9 kb ofyeast genomic DNA was found to possess a different restric-tion map from that of the GPAI gene. The 3.2-kb Pst Ifragment harboring this sequence (GPA2) was subcloned atthe Pst I site of pSP65 to yield pGO5. The detailed physicalmap of pGO5 is shown in Fig. 1.

Nucleotide sequence analysis of pGO5 revealed that theGPA2 gene exists in the Pvu II-Dra I fragment ofpGO5. Thegene encodes a protein of 449 amino acids (including theinitiator methionine) with a calculated M, of 50,516 (Fig. 2).The sequence around the initiator methionine codon (AT-CATGG) is favorable according to the Kozak rule (30); it has

FIG. 1. Restriction map of the pG05 plasmid and its derivativepG0202, which was constructed for the GPA2 gene disruptionexperiments. The wavy and straight lines show vector DNA andinserted yeast DNA, respectively. The amino acid coding region isrepresented by a hatched bar. The open bar indicates the insertedHIS3 gene.

a purine in position -3 and a guanosine in position +4.Upstream of the ATG codon, no definite "TATA"-liketranscription initiation signal was found up to position - 239.The open reading frame ends at the TGA stop codon(positions 1348-1350).Primary Structure of Yeast GP2a Protein. The deduced

amino acid sequence of yeast GP2a (GPA2-encoded protein)revealed homology with that of mammalian G protein asubunits and also with yeast GPla. The amino acid se-quences of yeast GPla and GP2a are compared with those ofrat brain Gi2, and the a subunit of Gs (Gsa) in Fig. 3. Thereare several regions in which the homology is especiallypronounced-i.e., the regions responsible for GTP hydroly-sis (residues 125-140 of GP2a and 35-50 of Gi,2), andguanine ring binding (residues 359-374 of GP2a'and 264-279

-239 CAGCTGCGCCCAAATGATTCTTCTTTACCTCTTGCATACTGCACCGAAAAAAAAAGT1AATiAAGiCAGCAG TCGAIIITACITAb$AAK-VIU.VAI VAV A AI I"V";-IU W WIW^

-120 TTAGGTAGGTTTGGTTCTCACTACCCAAAGAGCAATCGATAGwGTATCAsAAGTGAGCAATTGCTATCACAGCGAGCCTTATTGTTACAGCACAAATCACGCGTATTTTCAAGCAAATATC1 ATG GGT CTC TGC GCA TCT TCA GAA AAG AAC GGC AGC ACT CCT GAC ACG CAG ACC GCC AGC GCT GGT AGT GAC AAC GTT GGC AAA GCG AAG1 Net Gly Lou Cys Ala Sor Ser Glu Lys Asn Gly Ser Thr Pro Asp Thr Gin Thr Ala Sor Ala Gly Ser Asp Asn Val Gly Lys Ala Lys

91 GTA CCA CCA AAG CAG GAG CCA CAG AAG ACT GTG AGA ACA GTC AAC ACA GCA AAT CAA CAA GAA AAG CAA CAA CAG AGG CAG CAG CAG CCG31 Val Pro Pro Lys Gin Glu Pro Gin Lys Thr Val Arg Thr Val Asn Thr Ala Asn Gin Gin Glu Lys Gin Gin Gin Arg Gin Gin Gln Pro

181 TCT CCG CAT AAT GTT AAG GAC CGC AAG GAG CAM AAC GGG AGC ATT AAT AAC GCG ATA TCT CCC ACG GCT ACG GCA AAT ACA AGC GGA TCG61 Ser Pro His Asn Vai Lys Asp Arg Lys Glu Gin Asn Gly Ser Ile Asn Asn Ala Ile Ser Pro Thr Ala Thr Ala Asn Thr Ser Gly Ser

271 CAA CAG ATC AAT ATC GAC TCT GCC CTG AGA GAC AGG TCG AGT MC GTT GCA GCA CAA CCA TCA TTG TCG GAC GCT TCA AGT GGC AGC AAC91 Gin Gin le Asn Ile Asp Ser Ala Lou Arg Asp Arg Ser Ser Asn Val Ala Ala Gin Pro Ser Lou Ser Asp Ala Ser Ser Gly Ser Asn

361 GAC AAA GAA CTG AAA GTG CTA CTG CTG GGT GCC GGT GAA AGT GGT AAG TCC ACG GTA TTG CAG CAG TTG AAG ATT TTA CAC CAG AAC GGG121 Asp Lys Glu Lou Lys Val Lou Lou Lou Gly Ala Gly Glu Ser Gly Lys Ser Thr Val Lou Gin Gin Lou Lys Ile Lou His Gin Asn Gly

451 TTT AGC GAG CAG GAA ATT AAA GAG TAC ATC CCC TTG ATC TAT CAG AAT CTA TTG GAA ATT GGC AGG AAC CTC ATC CAG GCG AGA ACA AGG151 Phb Ser Glu Gin Glu Ile Lys Glu Tyr lie Pro Lou l1b Tyr Gin Asn Lou Lou Glu l11 Gly Arg Asn Lou 1le Gin Ala Arg Thr Arg

541 TTT AAC GTC AAC TTG GAA CCG GAG TGT GAA CTG ACG CAA CAA GAC CTG TCG AGA ACC ATG TCC TAT GAA ATG CCC AAT AAC TAC ACG GGC181 Phe Asn Val Asn Lou Glu Pro Glu Cys Glu Lou Thr Gin Gin Asp Lou Ser Arg Thr Not Ser Tyr Glu Not Pro Asn Asn Tyr Thr Gly

631 CAA TTC CCG GAA GAT ATC GCG GGC GTA ATA TCT ACG TTG TGG GCC TTG CCC TCA ACA CAA GAT TTA GTC AAT GGG CCT AAC GCA TCG AAG211 Gin Phe Pro Glu Asp 11o Ala Gly Val Ile Ser Thr Lou Trp Ala Lou Pro Ser Thr Gin Asp Lou Val Asn Gly Pro Asn Ala Ser Lys

721 TTC TAT CTA ATG GAC TCG ACT CCT TAC TTC ATG GM AAT TTC ACC AGG ATC ACT TCG CCC AAT TAC AGA CCC ACC CAG CAG GAC ATA TTA241 Pho Tyr Lou Not Asp Str Thr Pro Tyr Phe Not Glu Asn Phe Thr Are Ile Thr Ser Pro Asn Tyr Arg Pro Thr Gin Gin Asp Ile Lou

811 AGA TCG AGA CAG ATG ACG TCA GGG ATT TTT GAC ACC GTC ATT GAT ATG GGG TCG GAT ATC AAG ATG CAT ATT TAC GAC GTG GGT GGA CAG271 Arg Ser Arc Gin Not Thr Ser Gly Ile Phe Asp Thr Val Ile Asp Not Gly Ser Asp Ile Lys Not His Ile Tyr Asp Val Gly Gly Gin

901301

991331

1081361

1171391

1261421

CGT TCC GAA AGA AAA AAA TGG ATA CAC TGC TTC GAC AAT GTC ACT CTG GTC ATA TTT TGC GTT TCT CTA TCG GAG TAC GAC CAG ACG CTGArg Ser Glu Arg Lys Lys Trp 11o His Cys Phe Asp Asn Val Thr Lou Val Ile Phe Cys Val Ser Lou Ser Glu Tyr Asp Gin Thr Lou

ATG GAG GAC AAG AAC CAG AAC AGG TTT CAG GAM TCG CTG GTG CTT TTC GAT AAT ATT GTC AAC AGT AGA TGG TTC GCG CGC ACG TCT GTCNot Glu Asp Lys Asn Gin Asn Arg Phe Gin Glu Ser Lou Val Lou Phe Asp Asn 11b Val Asn Sor Arg Trp Phe Ala Arg Thr Ser Val

GTA CTC TTT CTG AAT AAA ATC GAC CTT TTT GCT GAA AAA CTA AGG AAA GTG CCT ATG GAA AAT TAC TTC CCA GAC TAC ACC GGC GGG TCAVal Lou Phe Lou Asn Lys Ile Asp Lou Phe Ala Glu Lys Lou Arg Lys Val Pro Not Glu Asn Tyr Phe Pro Asp Tyr Thr Gly Gly Ser

GAC ATC AAC AAG GCT GCT AAG TAC ATA CTC TGG AGG TTT GTT CAG TTA AAC AGG GCG AAT CTA AGC ATA TAT CCT CAC GTG ACA CAG GCCAsp 11e Asn Lys Ala Ala Lys Tyr I1e Lou Trp Arg Phe Val Gln Lou Asn Arg Ala Asn Lou Ser Ile Tyr Pro His Val Thr Gin Ala

ACA GAC ACG TCG AAT ATA AGA TTA GTA TTT GCC GCC ATC AAA GAA ACA ATT TTG GAA AAT ACA TTG AAA GAC TCT GGA GTG TTA CAA TGAThr Asp Thr Ser Asn h1o Arg Lou Val Phe Ala Ala Ile Lys GMu Thr Ile Lou Glu Asn Thr Lou Lys Asp Ser Gly Val Lou Gin End

1351 ATGCACAGCTAMACAGAGACAACTGCATGCCTCTTCTCCCCTTTATTATCACCTTTMA 1412

FIG. 2. Nucleotide and predicted amino acid sequences of the GPA2 gene. Numbering of the nucleotide sequence begins at the firstnucleotide in the open reading frame. The deduced amino acid sequence is shown below the nucleotide sequence.

Biochemistry: Nakafuku et al.

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1376 Biochemistry: Nakafuku et al.

GPla: 1- 37: M G C TVSTQTIGDESDEPFLQNK - - - RANDVIEQSLQLEKQR - - - - - - - - - - - -

GP2a: 1-55: MGLCA-SSEKNGSTPDT --- QTASAGSDNV-GKAKVPPKQEPQKTVRTVNTANQQEKQQQGi2a: 1- 29: M G C T V S A - - - - - - - - EDIKAAAERSKMI DKNLREDGGEK - - - - - - - - - - - - - - - -

Gsa: 1- 36: IM GI _ C L G N S K T E D Q R N - - - - ELEJK A Q R E A N K K I E K Q L Q K D KV - - - - - - - - - - - - - - - - -

GPla:GP2a: 56-115: R Q Q Q P S P H N V K D R K E Q N G S I N N A I S P T A T A N T S G S Q Q I N I D S A L R D R S S N V A A Q P S L S D A

Gi2a: - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -_Gsa: - - - - - - - - - - - - - - - - - - - - -

GPla: 38- 78: - - - - D K N E IGP2a:116-160: S S G S N D K E LGi2a: 30- 70: - - - - A A R E VGsa: 37- 92: - - - - Y R A T H

[KLLLLGAGESGKSTVLKQLKLLHQGGTSHM6---------fERLQYAKVLLLGAGESGKSTVLQQLKIQLNHQNGFISEOI----------- EIKEYIKLLLLGAGESGKSTIVKIQMXIIHEDGYSEE_IE--_______- ECRQYRIRLLLLGAGESGKSTIVK QMR I LHVNG FNGLEJGGEEDPQAARSNSDGIE KATKV

GPla: 79-136: Q V[I W A6DA IQ S M K I L7I I Q A R - K L G I Q L DC D D P I N - K D L FA C K R I L L K A K AL D Y I N A S V AGG7P2a.161-210: P LIllIY QINIL LIEI I G R N LII - 0 A R T R F N V N L E P E C E L TIQI - Q D L S R T M S Y E M P N N Y T G--------

Gi2a: 71-118: A VVI Y S NIT S I M A IIV K A M G - N L Q I D F A D P Q R A DD-A R L F A L S C A A E E Q G

Gsa: 93-140: Q D I K N LN L K E A I E T I V A AM S - N L V P P V E L A N P E N Q F R V D Y I L S VM N V P N F - - - - - - - - - - -

GPla:137-197: G S D F L N D Y V L K Y S E R Y E T R R R V Q S T G R A K A A F D E D G N I S N V K S D T D R D A E T V T Q N E D A D R

GP2a: - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

Gi2a: - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

Gsa:

GPla:197-255: N N S SRI N L Q DICK DL N Q E GD D Q M F V R K TSR EI Q G Q N R RN LI H ED1I A K A I K QLWN ND-KG IGP2a:211-231: - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Q F P EDIAGVISTLWALP S T Q DGi2a:119-137: - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - M L P EDLSGVIRRLWA-D - H G VGsa:141-159: - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - D F P P F YE H A K ALwE - D - E G V

GPla:256-314: K Q C F A R N E F Q L E G S A A F D N I E K F A S N Y V C T D E D I L K G R I K|T T G I T E T E F N I G S - S K

GP2a:232-291: L V N G P N A S K FY L M D S T P Y F M E N F T RI T S P N YR P T Q Q D I L R S R Q MTS G I|FDTV I D M G S D I KGi2a:138-196: Q A C F G R S R E Y Q LN D S A A YYLN D L E R I A Q SDY I P TQQDV L R T R V KITTGI|VETH F T F K D - L H

Gsa:160-218: RACYER SNEYQ LIDCAQY FLDKIDVIKQADYVPSDQDLLRCRVLTSGIFETKFQVDK - V N

GPla:315-374: F K V L AG G Q RSE R K K w I|H|C F EGITA V L FVLAMSEYDQ M EDE R VNHM H|E S IMLFD T L

GP2a:292-351: M|H IYDVG G QR S E R K K W H|C F D|N|V T L V I F|C|V S LSEY DQTLM|E D|K N QN R F E S L|V|L F D NIIIVIN

Gi2a:197-256: F|KM|FDV|G G Q R|S|ER K K WI|H|C FE|G|V TAI IFCV AL|SAY DLVLA|ED|E EM N R|M HE SMKL F|D SIICINGsa:219-278: F F[DJVG G Q R|D|E R R K W IQLC F N DIV AII I F VWSW SIY NIM VRL2JNRTEN Q TJLQL |EA.LN1LFK SJWN

GPla:375-421: SKWF K D TmFI L F L N K I DLF EE K VK - - S M PfR KYF PDYQGRVGDA A L - - - - - - - - - - - K

GP2a:352-417: SIRWIFARTISIVVLFLNKIIDLIFAIEKLIR--KVPMENYFPDYTGG-SDINKIAA - - - - - - - - - - - KGi2a:257-302: NiK W|F T D T|SI|I L F L N KKD LF EE K I T - - Q S PLT I CF PE YT G A N KY DEAAS - - - - - - - - - - - -

Gsa:279-338: NRL R T IMJV-IL FL N Q A|E K V LAG K S KWED Y|F P E FARY T T P E ATP E P GE D PR VT R A K

GPla:422-472: Y F E K I -FL S N K - - T N K P - - IjVVRR CAT DTQ T K FVL S A V TDLmT Q Q N|L K K I GII -

GP2a:418-449: YILWR -FIVQILINR--ANLLSS-I -IYFHVITIQATTIDTSNIIRLIVIFAAIKIEITIIILENTILK|DSGIVILQGi2a:303-355: -YIQSKIFEDILNKRKDTKE E-IITIITHFCATIDTIKNVQFIVIFDAVTIDIVIIKNNILKIDCGILF

Gsa:339-394: Y F I RD EFL RIST AS GD G RH Y CYPH FT C A VDTEN IR R VJF N DC RDIW QR M HLRQYELL-

FIG. 3. Alignment of the predicted amino acid sequence of yeast GP2a with those of yeast GPla (23) and rat brain GQa (now referred toas Gj2a) and Gsa (11). Identical or conservative amino acid residues are enclosed within solid lines. The standard one-letter symbols for aminoacids are used. Conservative amino acid substitutions are grouped according to Dayhoff et al. (31): C; S, T, P, A, and G; N, D, E, and Q; H,R, and K; M, I, L, and V; and F, Y, and W.

of Gi2a) and a region common to all G proteins (residues296-312 of GP2a and 201-217 of GQ2a). The sequences of theGTP hydrolysis sites of GPla, GP2a9, Q, and Gsa areidentical at 12 of 16 positions, with conservative substitu-tions at the other 4 positions. In the guanine ring binding siteof the four proteins, 9 out of 16 amino acids are identical,with conservative substitutions at the other 7 positions, andin the region common to all G proteins, identical amino acidsequences containing 14 contiguous amino acids are identi-cal in GPla, GP2a, and Gi2,.The overall homology in nucleotide and amino acid se-

quences of yeast GPla, yeast GP2a, rat Gi2a, and rat Gsa isremarkable. Disregarding the unique sequences present inGPla (residues 126-235) and GP2a (residues 37-119), theproteins are 60% homologous if conservative amino acidsubstitutions are considered to be homologous. The homol-ogy is smaller than that between rat Gina and GOa (85%) butis comparable to that between rat Gin2 and Gsa (60%).

Detection of the Transcript of GPA2. To examine theexpression of the GPA2 gene, we prepared poly(A)+ RNAfrom growing cultures of two haploid cell types (a and a) anda diploid cell type (a/a). RNA blot hybridization analysisusing the 2.0-kb Pvu II-Pst I fragment of pGO5 (see Fig. 1)as a probe revealed a single band of about 1.9-kb in all threecell types (data not shown).

Disruption of the GPA2 Gene. To examine whether theGPA2 gene is essential for yeast cell growth, we performeda one-step gene replacement experiment. Using pGO202, a

diploid strain (GPA2/GPA2, his3/his3) was transformed toHIS+, which results in the replacement of one wild-typeallele of GPA2 with disrupted Agpa2::HIS3. The heterozy-gous diploid strains (GPA2/Agpa2::HIS3) were sporulated,and the spores were dissected to obtain haploid progenies. Inevery tetrad analyzed, all four spores developed into normalcolonies. DNA was prepared from a set of four haploidprogenies, digested with HindIII and analyzed by Southernhybridization. The results indicated that two HIS + proge-nies possessed the disrupted GPA2 gene (data not shown).From this, we concluded that the GPA2 gene is not essentialfor the growth of yeast cells.Mapping of the GPA2 Gene. The map position of GPA2

was determined from chromosome blotting and tetrad anal-ysis. A GPA2 probe hybridized to a Southern blot of intactchromosomal DNA separated by orthogonal-field-alter-nating gel electrophoresis (32) at a band corresponding tochromosome V. This assignment was confirmed by tetradanalysis. A three-factor cross involving ura3, can), andAgpa2::HI53 localized gpa2 on chromosome V approxi-mately 18 centimorgans from ura3 and 61 centimorgans fromcan). The tetrad data showed a parental ditype/nonparentalditype/tetratype (PD/NPD/T) ratio of 18:0:10 for gpa2 andura3 and 9:3:16 for gpa2 and can), whereas the ura3 andcan) combination showed a 13:2:13 ratio. These data clearlyindicate that the order of genes on chromosome V isgpa2-centromere-ura3-canl.

Possible Involvement of the GPA2 Gene in Regulation of

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1~~t.

B C

1?~~h

5 10 30 5 10 30 5Time in Minutes

-D

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FIG. 4. Kinetics of glucose-induced cAMP formation. The cells were stimulated with 25 mM glucose and incubated at 30'C (A, B, and C)or 370C (D) for various periods of time. (A) Wild-type strain (SP1). (B) GPA2-disrupted strain (KMG13). (C) SP1 with pGO304 (YEpGPA2).(D) ras2ts strain (KMY14-5D) without (e) and with (o) pGO304. The isogenic mutant KMG13 was constructed starting from SP1 (a trpl ura3Ieu2 his3 ade8 canl). The genotype of KMY14-5D is a ras2-101 trpl ura3 leu2 his3 ade2 ade8 lys2.

cAMP Levels. In mammalian systems, hormonal regulationof adenylate cyclase activity is mediated by two G proteins,Gs and G, (1, 2), whereas in S. cerevisiae, RAS2 is involvedin the activation of adenylate cyclase (27, 28). To examinethe possibility that GPA2 may also be involved in theregulation ofcAMP levels, we studied the effect of GPA2 oncAMP formation. In the experiments shown in Fig. 4, thekinetics of cAMP formation in response to glucose wasmeasured. In a wild-type yeast strain, the addition of glucosestimulated cAMP formation transiently (Fig. 4A). The GPA2disruption (Agpa2: :H1IS3) did not affect glucose-inducedcAMP formation (Fig. 4B). On the other hand, introductionof YEpGPA2 into the wild-type strain remarkably increasedthe level of glucose-induced synthesis of cAMP, and thishigh level of cAMP was maintained for 30 min (Fig. 4C).This effect was not observed when YCpGPA2 or YEpGPA1was introduced to wild-type cells (data not shown).The effect of introduction of YEpGPA2 into various

mutants that affect cAMP formation was further examined.Although the introduction of YEpGPA2 did not suppress thegrowth defect of a rasl::HIS2 ras2::LEU2 double mutant,cdc25-1 (ts) (33) and cyrl-2 (ts) (34), we found that thisplasmid can suppress a ras2ts mutant, ras2-101 (ts) (Fig. 5).In the ras2-10J (ts) mutant, glucose did not induce anycAMP formation at the restrictive temperature (Fig. 4D).YEpGPA2 restored glucose-induced cAMP formation in theras2-101 (ts) mutant at high temperature (Fig. 4D).

DISCUSSIONWe have isolated two G-protein-homologous genes-i.e.,GPAI (23) and GPA2-from S. cerevisiae. The products,GPla and GP2a, contain 472 and 449 amino acid residues,respectively, and therefore are considerably larger in sizethan the mammalian G proteins. When GPla and GP2a arealigned with rat brain Gi2a to obtain maximal homology, theextra sequences of 110 and 83 amino acids, which are uniqueto GPla and GP2a, respectively, are found in the NH2-terminal portion (Fig. 3). The extra sequence of GPla isinserted between Gly-118 and Met-119 of Gi2a, whereas thatof GP2a is found closer to the NH2-terminus, betweenLys-29 and Ala-30 of Gi2a.A comparison of the primary structures of GPAI and

GPA2 indicates that they share 53% and 60% homology innucleotide and amino acid sequences, respectively. The

extent of homology between yeast and mammalian G pro-teins is about the same as those between the two yeast Gproteins. As shown in Fig. 3, there are three regions in whichthe homologies are especially pronounced. The sites that areresponsible for GTP hydrolysis (amino acid residues 125-140of GP2a) and the guanine ring interaction (residues 359-374of GP2a) are conserved in all G proteins and also in otherGTP-binding proteins such as elongation factor Tu and theras family. On the other hand, the site that corresponds toresidues 296-312 of GP2a is unique to the G protein family.

Besides the conservation of their structures, G proteinshave microheterogeneities in regions (i) close to the NH2terminus, (ii) at a position about one-third of the way towardthe COOH terminus, and (iii) near the COOH terminus. Theextra sequences in GP1a and GP2a occur in the NH2-terminal region, which is presumed, based on analogy withother G proteins, to be the binding site for amplifiers. Thesequences unique to GPla and GP2a may play an importantrole in the interaction with their putative amplifiers.

In this study, we show that a GPA2 null mutant is viableand has no defect in glucose-induced cAMP formation. Onthe other hand, a multicopy plasmid carrying GPA2 in-creases the level of cAMP and suppresses the growth defectin a ras2ts strain at high temperature. These results suggestthat GPA2, in addition to RASI and RAS2, regulates thelevel of cAMP in S. cerevisiae. Since the level of cAMP isdetermined by adenylate cyclase and phosphodiesteraseactivities, GPA2 on multicopy plasmids may directly acti-vate adenylate cyclase or inhibit phosphodiesterase. If the

A B

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3

FIG. 5. Effect of GPA2 on the ability of ras2ts strain to grow atrestrictive temperature. Patches of ras2ts strain, KMY14-5D, carry-ing YEp24 (patches 1), pGO303 (YCpGPA2) (patches 2), or pGO304(YEpGPA2) (patches 3) were grown on selection medium(SC/uracil) at 25°C. The master plate was replica plated andincubated at 25°C (A) or 37°C (B). Plates are shown 2 days afterreplicating.

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function of GPA2 were to regulate adenylate cyclase, thenonlethal phenotype of the GPA2 null mutant suggests thatan additional G protein may be involved in the activation ofadenylate cyclase in S. cerevisiae. In view of the suppres-sion by multicopy GPA2 of the growth defect in a ras2tsstrain but not in a rasl- ras2- strain, GPA2-encodedprotein may activate adenylate cyclase in a RAS protein-dependent manner. On the other hand, the lack of lethality ofthe GPA2 null mutant is consistent with the idea that GPA2maintains the cAMP level by inhibiting phosphodiesteraseactivity, which is not essential for growth (35, 36). Alterna-tively, the effect of GPA2 could be indirect and it may not benormally involved in the regulation of cAMP level. Inmammalian cells, hormonal stimuli dissociate f8y subunitsfrom the a subunit of the G protein. Excess f3y subunits areinhibitory for activation of adenylate cyclase by the GTP-bound form of Gsa (37). Likewise, GPA2-encoded proteinexpressed at high level may form a complex with free frysubunits, which are otherwise inhibitory to other G pro-tein(s) involved in cAMP formation, although the occurrenceof fry subunits in yeast is not clear at present. This possibilityis less likely in view of the lack of appreciable effect of amulticopy plasmid carrying GPAI on glucose-induced cAMPformation and on the growth defect of the ras2ts strain.

In a previous paper (24), we presented evidence that theGPAI gene is specifically expressed in the haploid cells, andits product, GPla, is essential for mating-factor-mediatedsignal transduction. It is likely that GP1a interacts with themating factor receptors (STE2 and STE3 gene products) in amanner analogous to the interaction of mammalian G pro-teins with ,3-adrenergic type receptors. Therefore, it may bereasonable to assume that GP2a also interacts with a recep-tor molecule in the cytoplasmic membrane of yeast cells totransmit a signal to an amplifier molecule. Identification ofthe receptor and the amplifier for GP2a must await furtherinvestigation.

We thank Charles Brenner for carrying out the chromosomalassignment of the GPA2 gene and Naoki Nakayama for helpfuldiscussions.

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