Vol. 10, No. 8
MOLECULAR AND CELLULAR BIOLOGY, Aug. 1990, p. 4303-4313
0270-7306/90/084303-11$02.00/0 Copyright © 1990, American Society for Microbiology
IRA2, a Second Gene of Saccharomyces cerevisiae That Encodes a Protein with a Domain Homologous to Mammalian ras GTPase-Activating Protein KAZUMA TANAKA,"2 MASATO NAKAFUKU,3 FUYUHIKO TAMANOI,2 YOSHITO KAZIRO,3 KUNIHIRO MATSUMOTO,4* AND AKIG TOH-El5 Department of Fermentation Technology, Faculty of Engineering, Hiroshima University, Higashihiroshima, 724, Japan1; Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, Illinois 606372; Institute of Medical Science, University of Tokyo, 4-6-1, Shirokanedai, Minatoku, Tokyo 108, Japan3; DNAX Research Institute of Molecular and Cellular Biology, Palo Alto, California 943044; and Department of Biology, Faculty of Science, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113, Japan5 Received 2 March 1990/Accepted 21 May 1990
The IRAI gene is a negative regulator of the RAS-cyclic AMP pathway in Saccharomyces cerevisiae. To identify other genes involved in this pathway, we screened yeast genomic DNA libraries for genes that can suppress the heat shock sensitivity of the iral mutation on a multicopy vector. We identified IRA2, encoding a protein of 3,079 amino acids, that is 45% identical to the IRA] protein. The region homologous between the IRA] protein and ras GTPase-activating protein is also conserved in IRA2. IRA2 maps 11 centimorgans distal to the argl locus on the left arm of chromosome XV and was found to be allelic to gk4. Disruption of the IRA2 gene resulted in (i) increased sensitivity to heat shock and nitrogen starvation, (ii) sporulation defects, and (lii) suppression of the lethality of the cdc25 mutant. Analysis of disruption mutants of IRAI and IRA2 indicated that IRAI and IRA2 proteins additively regulate the RAS-cyclic AMP pathway in a negative fashion. Expression of the IRA2 domain homologous with GAP is sufficient for complementation of the heat shock sensitivity of ira2, suggesting that IRA down regulates RAS activity by stimulating the GTPase activity of RAS proteins.
the lethality of the cdc25 mutation and causes an increased level of intracellular cAMP (29). Thus, the iral disrupted mutant displays phenotypes associated with the activated RAS2va'-19 mutant, which has reduced intrinsic GTPase activity (4). These observations, together with the partial homology of this protein to mammalian GAP (29), suggested that the IRA] protein acts as a negative regulator that stimulates the conversion of the GTP form to the GDP form of RAS proteins by stimulating their GTPase activity. In an attempt to further identify genes involved in regulation of the RAS-cAMP pathway in S. cerevisiae, we have isolated multicopy suppressors of the heat shock sensitivity of the iral mutation (25). In the process, we identified the MSII gene, which encodes a protein homologous to the a subunit of mammalian G protein (25). We found another gene, IRA2, that is also capable of suppressing the heat shock sensitivity phenotype caused by the iral mutation when present on a multicopy plasmid. In a previous paper (30), we reported that a RAS * GTP form is accumulated in either the iral or ira2 mutant, suggesting that the IRA] and IRA2 proteins exert a function similar to that of mammalian GAP. In this communication, we report that IRA2 is a homolog of IRA) and encodes a protein that also shares homology with mammalian GAP. We also describe genetic experiments that examine the effects of IRA2 disruption on the RAS-cAMP pathway.
The ras genes participate in the control of proliferation and differentiation in mammalian cells (2, 10). The ras proteins are localized on the inner face of the plasma membrane, exhibit guanine nucleotide-binding activity, and possess an intrinsic GTPase activity. They are thus members of the family of guanine nucleotide-binding proteins, and their activity is modulated by utilizing a guanine nucleotidebinding and hydrolysis cycle. The GTP-bound form of the protein activates a target protein, and this stimulation is turned off upon hydrolysis of GTP to GDP. It is therefore of interest to identify individual components that interact with ras proteins and regulate their activity. Recently, it has been demonstrated that mammalian cells possess a ras GTPaseactivating protein (GAP) that catalytically accelerates hydrolysis of GTP bound to ras proteins (34). The yeast Saccharomyces cerevisiae contains two homologs of mammalian ras genes, RAS] and RAS2 (8, 22), which are involved in transduction of the signal for growth in response to nutrients (17, 31). The yeast RAS proteins control the activity of adenylyl cyclase, which produces the second messenger, cyclic AMP (cAMP), responsible for the activation of cAMP-dependent protein kinase (A-kinase) (33). The RAS2 protein has been shown to activate adenylyl cyclase when bound to GTP but not when bound to GDP (9). Genetic analysis identified regulatory genes, CDC25 and IRA], that modulate the activity of RAS proteins in S. cerevisiae. The CDC25 protein is proposed to act as a positive regulator that exchanges the GDP bound to RAS proteins for GTP (5, 7, 15, 23). In contrast, genetic evidence indicates that the IRA] protein inhibits the function of RAS proteins in a fashion antagonistic to the function of the CDC25 protein (29). The IRA] gene disruption can suppress *
MATERIALS AND METHODS Strains and culture media. Yeast strains used are described in Table 1. Usually, yeast cells were grown in a rich medium, YPD, which contains 1% yeast extract, 2% polypeptone, 2% glucose, 400 mg of adenine sulfate per liter, and 200 mg of uracil per liter. SD medium, which was used to select the plasmid-containing cells or to determine the auxotrophic
Corresponding author. 4303
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MOL. CELL. BIOL. TABLE 1. Yeast strains used
Strain
Genotype MATa iral-I ura3 trpl his3 lys2 ade8 MATx rasl::URA3 ira2::HIS3 ura3 leu2 trpl his3 MATa ras2::LEU2 ura3 leu2 trpl his3 ade8 MATot glc4-2 ura3 his3 ade8 MA Ta/MA Ta iral::LEU2-alIRAI ura3lura3 leu2lIeu2 trplltrpl his3/his3 MATa cdc25::LEU2 ura3 leu2 trpl his3 (pKT5)a MATa/MATa iral::LEU2-a/IRAI ira2::HIS31IRA2 ura3lura3 leu2/Ieu2 trplltrpl hIhis3 MATa ira2::HIS3 ura3 leu2 trpl his3 MATa ura3 leu2 trpl his3 MATa ira2::HIS3 ras2::URA3 ura3 leu2 trpl his3 MATa argi ura3 leu2 trpi his3 MATa/MATa iral-l/iral-J ura3lura3 Iys211ys2 thr41thr4
C70-3D ...... KMY39-6D ...... KMY46-4D ...... KMY305-5C ...... KT6 ...... KT18-7B ....... KT27 ....... KT31-1A ....... KT31-1C ....... KT31-1D ....... KT69-1B ....... R59-1C-D ....... a A plasmid carrying the
wild-type CDC25 gene (unpublished data).
requirements of strains, was 0.7% yeast nitrogen base without amino acids (Difco Laboratories, Detroit, Mich.), 2% glucose, and appropriate amounts of amino acids and nucleic acid bases. Nitrogen starvation medium contained 0.2% yeast nitrogen base without amino acids and ammonium sulfate (Difco), 2% glucose, and the required amino acids and nucleic acid bases. Cloning of IRA2 and plasmid constructions. Standard techniques for molecular cloning were performed essentially as described elsewhere (14). The IRA2 gene was first cloned from the YEp24-based yeast genomic DNA library (6) as a plasmid, pd46. The original insert DNA was lacking the region encoding the carboxyl terminus of the putative IRA2 protein (see text); thus, the 6-kilobase (kb) HindIII fragment overlapping part of pd46 was cloned from the genomic DNA of strain X2180-1A into the Bluescript vector (Stratagene, San Diego, Calif.) Plasmid pKT14, which contained the
target DNA, was identified by colony hybridization, using the 1.2-kb BamHI-MluI fragment ofIRA2 (Fig. 1) as a probe. A plasmid used for disrupting the IRA2 gene was constructed as follows. First, the 2.7-kb Sacl fragment encoding the amino-terminal region of the IRA2 gene was subcloned into the SacI site of pUC19 (35) to form pFS2. The 1.8-kb BamHI fragment encoding the HIS3 gene was cloned into the BglII site of pFS2 (27). The resulting plasmid, pKZ7, was cut with Sacl and used for transformation of yeast cells to obtain the disruptant of the IRA2 gene by the method of one-step gene disruption (24). Plasmids for overexpressing truncated IRA2 were constructed as follows. Truncated IRA2 proteins were expressed under the control of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene promoter by cloning various restriction fragments of IRA2 into pKT10, a plasmid containing the ARS sequence of 2,um DNA, URA3, and the
HIS3 B P M '
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.
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ORF
iral
dl
d2 d3 I
d4
d5 i
-4
d6 d7
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1Kb FIG. 1. Restriction enzyme maps and deletion analysis of plasmids encoding IRA2. Vector DNA was omitted from the figure. Horizontal lines indicate the DNA sequences remaining after deletion. The IRA2 open reading frame (ORF) is boxed, and its direction is shown with an arrow. The region homologous with GAP (see Fig. 2 and 3) is shown by black boxes. The position where the HIS3 gene fragment was inserted to disrupt IRA2 is also shown. Abbreviations for restriction enzyme sites: B, BamHI; P, PvuII; M, MluI; Sc, Sacl; H, Hindlll; G, BglIl.
VOL. 10, 1990
(28). Northern (RNA) analysis indicated that the level of mRNA from the GAPDH gene promoter was more than 10-fold higher than that of IRA2 mRNA (data not shown). Restriction fragments used were as follows: 2.4-kb BglII-PvuII fragment for [14512255]IRA2, 1.9-kb StuI-PvuII fragment for [1609-2255]IRA2, 1.3-kb EcoRV-EcoRV fragment for [1753-2188]IRA2, 1.4-kb SacI-PvuII fragment for [1804-2255]IRA2, 1.7-kb BgIII-MluI fragment for [1451-2026]IRA2, 1.6-kb BgIII-BglI fragment for [1451-1991]IRA2, and 1.4-kb BgIII-AfIl fragment for [1451-1908]IRA2 (numbers in brackets show the spans of the amino acid sequences of truncated IRA2 proteins). For construction of plasmids containing BglII-PvuII, BglII-MluI, BglII-BgII, and BglII-AfII fragments, synthetic oligonucleotides were added to provide the initiation codon, resulting in the addition of five amino acids, Met-Gly-Thr-Ala-Arg, before the authentic amino acid sequence of the IRA2 protein. In other constructions, internal methionine codons of IRA were used for an initiator codon, and 5' nontranslated sequences provided by the IRA2 DNA sequence were less than 52 base pairs long. At the 3' terminus of each construction, there are three-phase termination codons followed by the transcription terminator of the GAPDH gene to make transcriptional and translational terminations efficient. DNA sequencing. The DNA fragments to be sequenced were cloned in M13mp18, M13mp19 (35), or Bluescript. Unidirectional deletions were constructed in these clones as described previously (11), and single-stranded DNAs were prepared. Nucleotide sequence was determined by the method of Sanger et al. (26) on both strands and overlapped the junction between fragments. cAMP assay. Glucose-induced cAMP formation in starved cells was determined as previously described (20, 29) except that the starvation of cells in buffer and the addition of glucose were at 30°C. Determination of heat sensitivity. For scoring on plated medium, fresh cells grown on YPD plates were replica plated onto YPD and immediately placed at 50°C for 1 h. Plates were incubated for 3 days at 30°C, and heat shock sensitivity was scored. For examination in liquid medium, cells growing exponentially in YPD at 25°C were transferred to 50°C, and small samples were taken at the indicated time and chilled in an ice bath. After appropriate dilution, cells were plated on YPD plates. Three days later at 30°C, colonies were counted and the percent survival was determined. Determination of nitrogen starvation sensitivity. For determination of sensitivity to nitrogen starvation on plated medium, fresh cells grown on YPD plates were replica plated onto nitrogen starvation plates. After 9 days at 30°C, cells were replica plated onto YPD plates and incubated at 30°C for 3 days to observe the fraction surviving. Other methods. Sporulation efficiency (29) and yeast transformation (12) were determined as described previously.
promoter fragment of the GAPDH gene
RESULTS Isolation of the IRA2 gene. A yeast genomic library, constructed in the multicopy vector YEp24, was screened for plasmids that could suppress the heat shock sensitivity phenotype of the iral-J mutation. The iral strain, C70-3D (iral-J ura3), was transformed with library DNA. Plasmids that conferred the ability to form colonies after incubation at 30°C for 24 h, followed by 1-h incubation at 50°C, were selected. Four different sequences in addition to the IRA] gene itself were able to suppress the phenotype of iral-J (25). One of these suppressors, IRA2 (previously called MSI3), is characterized in this report.
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Transformation of R59-1C-D (MATa iral-JIMATa iral-1) with the IRA2 plasmid pd46 restored heat shock resistance to R59-1C-D as well as the ability to sporulate (from 0 to 4.5%). The IRA] gene on YEp24 suppressed the sporulation deficiency of R59-1C-D (from 0 to 34%). IRA2 is a homolog of IRA]. Deletion analysis of pd46 (Fig. 1) suggested that almost the entire DNA sequence (9.4 kb) is required for the suppressor activity of the iral-J mutation, with two exceptions (d2 and d7; see below). This result suggests that the gene on pd46 is large like IRAI (29). In fact, determination of the nucleotide sequence of the cloned DNA revealed a large open reading frame which is truncated at the carboxyl terminus (Fig. 1). To determine the entire nucleotide sequence of the gene, we cloned a 6.0-kb chromosomal HindIII fragment contiguous to the DNA fragment cloned in pd46. The joined nucleotide sequences built up a large open reading frame that can encode a protein of 3,079 amino acids (Fig. 2). Northern hybridization experiments detected a band of about 10 kb in length in the poly(A)+ RNA fraction, consistent with the size predicted from the nucleotide sequence of the gene (data not shown). Computer analysis revealed an extensive and clear amino acid sequence homology between IRA] and IRA2 gene products (45% identity within the total 2,938 amino acid residues of IRAI; Fig. 2). Thus, this gene was designated IRA2. When the amino acid sequences of IRA] and IRA2 proteins were aligned for maximum matching, the IRA2 protein was found to have an extension of 180 amino acid residues at the N terminus. We previously reported (29) that the IRA] protein has two potential sites for A-kinase phosphorylation, Arg-Arg-X-Ser or Thr (13). The IRA2 gene product also has two such sequences, although only one of the two sites is located at the same position in the IRA] and IRA2 proteins (Fig. 2). These sites may be involved in feedback control of cAMP formation conducted by A-kinase (21). When IRA] and IRA2 proteins were divided into three parts, amino terminal (amino acid sequence from positions 1 to 979 of the IRA] protein), middle (980 to 1958), and carboxyl terminal (1959 to 2938), the identities between IRA] and IRA2 proteins were found to be 31, 52, and 53%, respectively, indicating that IRA] and IRA2 proteins are less homologous in their amino-terminal regions. The essential domain of IRA2 corresponds to the region homologous with GAP. In the middle of the IRA] and IRA2 proteins, there is a region showing a weak but significant homology with the carboxyl terminus of bovine GAP (Fig. 2) (29). When these regions of IRA] and IRA2 proteins were aligned with bovine GAP for maximum matching, they were found to be 22% identical and 45% homologous with GAP (Fig. 3). Recently, the carboxyl-terminal 343 amino acids of bovine GAP (amino acid sequence 702 to 1044), which includes the region homologous with IRA proteins, have been shown to be essential for GAP activity (16). As described above, two deletion plasmids, d2 and d7, complemented the iral-J mutation. These two plasmids thus seemed to carry an essential domain of IRA2 which was expressed by readthrough from vector sequences. Because these plasmids contain the region homologous with GAP (Fig. 1), a smaller truncated protein of IRA2, [14512255]IRA2, which includes that region, was expressed and found to suppress the ira2 mutation (Fig. 4). To more precisely define the IRA2 essential region, several deletion fragment of [1451-2255]IRA2 were examined for the ability to complement the ira2 mutation. The amino acid sequence from 1609 to 1991 of the IRA2 protein was sufficient for suppression of the ira2 mutation (Fig. 4). This region corre-
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TANAKA ET AL.
MOL. CELL. BIOL.
IRA2.1 MSQPTKNKKKEHGTDSKSSRMTRTLVNHILFERILPILPVESNLSTYSEVEEYSSFISCRSVLINVTVSRDANAHVEGTL
81 ELIESLLQGHEIISDKGSSDVIESILIILRLLSDALEYNWQNQESLHYNDISTHVEHDQEQKYRPKLNSILPDYSSTHSN 161 GNKHFFHQSKPQALIPELASKLLESCAKLKPNTRTLQILQNHISHVHGNILTTLSSSILPRHKSYLTRHNHPSHCKMIDS IRA1.1
HLLCKISKLKFNTRTLKVLQNNSHHLSGSA-TISKSSILPDSQEFLQKRNYPAYTEKIDL
241 TLGHILRFVAASNPSEYFEFIRKSVQVPVTQTHTHSHSHSHSLPSSVYNSIVPHFDLFSFIYLSKHNPKKYLELIKNLSV 60 TIDYIQRFISASNHVEFTKCVKTKVVAPLLISHT -STELGVVNHLDLFGCEYLTDKNLLAYLDILQHLSS
321 TLRKTIYHCLLLHYSAKAINFWIHARPAEYYELPNLL--------------KDNNNEBSKSLNTLNHTLPEEIHSTFNVN
129 YKKRTIPHSLLLYYASKAFLPWINARPKEYVKIYNNLISSDYNSPSSSSDNGGSNNSDKTSISQLVSLLFDDVYSTPSGS 387 SKITTNQNAHQGSSSPSSSSPSSPPSSSSSDNNNQNIIAKSL -
SRQLSHHQSYIQQQSERKLHSSWTTNSQS-S
209 SLLTNVNNDHHYHLHHSSSSSKTTNTNSPNSISKTSIKQSSVNASGNVSPSQFSTGNDASPTSPMASLSSPLNTNILGYP 459 TSLSSSTSNSTTTDFSTHTQPGEYDPSLPDTPTMSNITISASSLLSQTPTPTTQLQQRLNSAAAAAAAAASPSNSTPTGY 289 LSPITSTLGQANTSTSTTAATTKTDADTPSTMNTNNNNNNNNSANLNNIPQRIPSLDDISSFNSSRKSLNLDDSNSLFLW 539 TAEQQSRASYDAHK--TGHTGKDYDEHFLSVTRLDNVLELYTHFDDTEVLPHTSVLKFLTTLTMFDIDLFNELNATSFKY
369 DTSQHSNASNTNTNMHAGVNNSQSQNDQSSLNYNENINELYSNYTGSELSSHTAILRPLVVLTLLDSEVYDEKNSNSYRK 617 IPDCTMHRPKERTSSFNNTAHETGSEKTSGIKHITQGLKKLTSLPSSTKKTVKFVKHLLRNLNGNQAVSDVALLDTHRAL
449 ISEPIKNINPKD
---
SNTSSWGSASKNPSIRHLTHGLKKLT-LQQGRKRNVKFLTYLIRNLNGGQFVSDVSLIDSIRSI
697 LSPFFTTSAVFLVDRNLPSVLFAKRLIPINGTNLSVGQDWNSKINNSLNV-CLKKNSTTFVQLQLIFFSSAIQFDHELLL 524 LFLMTMTSSISQIDSNIASVIFSKRFYNLLGQNLEVGTNWNSATANTFISHCVERNPLTHRRLQLEFFASGLQLDSDLFL
776 ARLSIDTNANNLNMQKLCLYTEGPRIPPDIPSKKELRKAIAVKISKPFKTLPSIIADILLQEFPYFDEQITDIVASILDG 604 RHLQLEKELNHIDLPKISLYTEGPRVFFHLVSTKKLHEDIAEKTSSVLKRLFCIIADILLKATPYPDDNVTKIIASILDG
856 TIINEYGTKKHPKGSSP-SLCSTTRSRSGSTSQSSNTPVSPLGLDTDICPNNTLSLVGSSTSRNSDNVNSLNS-SPKNLS 684 HILDQFDAARTLSNDDHVSFDAATSVYTEPTEIIHNSSDASLVSSLSQSPLSINSGSNITNTRTWDIQSILPTLSNRSSA 934 SDPYLSHLVAPRARHALGGPSSIIRNKIPTTLT ----SPPGTEKSSPVQRPQTESISATPNAITNSTPLSSAAPGIRSP
764 SDLSLSNILTNPLEAQQNNNANLLAHRLSGVPTTKRYASPNDSERSRQSPYSSPPQLQQSDLPSPLSVLSSSAGPSSNHS 1009 LQKIRT- -RRYSDESLGKFMKSTNNYIQEHLIPKDLNEATLQDARRININIFSIFKRPNSYFIIPHNINSNLQWVSQDFR
844 ITATPTILKNIKSPKPNKTKKIADDKQLKQPSYSRVILSDNDEARKIMMNIPSIFKRNTNWPIRPDANTE----FPKTFT FIG. 2. Amino acid sequence of the putative IRA2 protein (GenBank accession number M33779) and homology with the IRA] protein. The amino acid sequence of IRA2 deduced from the DNA sequence was aligned with that of IRA] for maximum homology. Identical amino acids are shown with colons. Potential sites for A-kinase phosphorylation are underlined. The shaded region shows homology with bovine GAP (see Fig. 3).
sponds to the IRA domain homologous with GAP (Fig. 3). The homology between IRA proteins and GAP thus seems to be significant, suggesting that IRA proteins have a GAP-like activity. Disruption and genetic analysis of the IRA2 gene. To understand the function of the IRA2 gene, we disrupted the chromosomal IRA2. The 1.8-kb BamHI fragment encoding the yeast HIS3 gene was inserted into one of the BglII sites in the IRA2 gene (Fig. 1). This construction fuses the DEDI gene contained in the 1.8-kb fragment (27) to the IRA2 truncated fragment out of frame. A diploid strain, KT6,
harboring the heterozygous iral::LEU2-a mutation was transformed to His' with the 4.5-kb Sacl fragment carrying ira2::HIS3. We confirmed by Southern analysis that several transformants carried the expected change at the IRA2 locus (data not shown). One such transformant, designated KT27, was sporulated and dissected for tetrad analysis. Because RAY-3A-D, which is the original host strain of KT6 and KT27, was constructed by transforming a haploid strain, RAY-3A, with a plasmid carrying the HO gene (29), tetrad segregants obtained from KT27 are isogenic except for the IRA loci. Almost all asci of KT27 (18 of 21 dissected)
VOL. 10, 1990
126TN~ ~
NEGATIVE REGULATOR OF YEAST RAS
4307
1087 NIMKPIFVAIVSPDVDLQNTAQSFMDTLLSNVITYGESDENIS- - - IEGYHLLCSYTVTLPAMGLFDLKINNEKRQILLD 920 DIIKPLPVSILDSNQRLQVTARAFIEIPLSYIATFEDIDNDLDPRVLNDHYLLCTYAVTLFASSLFDLKLENAKREMLLD 1164 ITVKFMKVRSHLAGIAEASHHMEYISDSEKLTFPLINGTVGRALFVSLYSSQQKIEKTLKIAYTEYLSAINPHERNIDDA
1000 IIVKFQRVRSYLSNLAEKHNLVQAIITTERLTLPLLVGAVGSGIFISLYCSRGNTPRLIKISCCEFLRSLRPYQKYVGAL
1244 DKTWVIHNIEPVEAMCHDNYTTSGSIAPQRRTRNNILRPATIPNAILLDSMRMIYKKWHTYTHSKSLEKQERNDFRNPAGI
1080 DQYSIYNIDPIDAMAQDNFTASGSVALQRRLRNNILTYIKGSDSILLDSMDVIYKKWFYPSCSKSVTQEELVDFRSLAGI 1324 LASLSGILFINKKILQENYPYLLDTVS ------------------ ELKKNIDSFISKQCQWLNYPDLLTRENSRDILSVE
1160 LASPSGILSDMQELEKSKSAPDNEGDSLSPESRNPAYEVHKSLKLELTKKNNFFISKQCQWLNNPNLLTRENSRDILSIE
1386 LHPLSPNLLFNNLRLKLKELACSDLSIPENESSYVLLEQIIKMLRTILGRDDDNYVNMLPSTEIVDLIDLLTDEIKKIPA
1240 LHPLSFNLLFNNLGLKIDELHSIDLSKSHEDSSFVLLEQIIIIIRTILKRDDDEKIMLLPSTDLLDAVDKLIEIVEKISI 1466 YCPKYLKAIIQNTKMPSALQHSEVNLGVKNHFHVKNKWLRQITDWFQVSIAREYDFENLSKPLKENDLVKRDNDILYIDT 1320 KSSKYYKGIIQNSKNFRAFEHSEKNLGISNHFHLKNKWLKLVIGWPKLSINKDYDFENLSRPLREMDLQKRDEDFLYIDT
1546 AIEASTAIAYLTRHTFLEIPPAASDPELSRSRSVIPGFYFNILNKGLEKSSSMDYWVll -IIWKV1.NDNVILSLTNLSN .. ..N.. .N... * ** *X * * *X--X- ' ~ ~ ~ ~ ~ ~ ~.'."~ ~ ~ ~ ~. . 2 ............ -* -.-.'. .....
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1400 SIESAKALAYLTHNVPLEIPPSSSKEDWNRSSTVSPGNIIPTILLKGLEKSADLN%VPLISN 1626 TNVDASLLP)IYSGKRRRRNAPLEVPIMVTNYRTYTAKTDLGKLUADEhYTI~EHPQ1SSGAAVCPASDID~AY ......,Y it ,,4.V !P.**
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1480 ANVSKFTLMGYSP.KIRIAPlRSYFID1VTrI..PEIIE.D.'L&XPn.IYIIKNP.IJAFPGS-
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1706 170B 'AC. EPrR'ysHiV....A..QuNE AAGLIMAPETRNATHIVVAQL-IKNEIEK-SSRPDULARNSCAThSL.SMLARSKGNEMLRTLQPLLKKIIQNRDPFEIEK . . .,... ....._AT..H......ltwPzE..WwEE *
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1560 AGGPLNADTRMASHILVTELLKQEIKRAARSDDILRNUSCt.ALSLYTRSRGNKYLIKTlRPYlGI1VDNKESFEIDlK
1786 LKPEDSDARQIELPVKYKINELEUSSNSVS-PP--SPJTRU YT[CEK IIAAGSFVIRPPcPALVSPDSEN * s s -4 4* * .-44K . 1640 KKPGSENSEKHLDLEYKTRLIDAITSSIDDPIELDICKASV TIAV VLRFIGPALYSPDSEN 1866 -IIDISHLSEKRT-PISLA'iVI.'NGENMPSVPAL-.CS XDPLKECSDRI -lLAELCR-TDRTIDIQVRTDPTPIAFDY * -
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1720 I. }t-m{DRPlk sPnXisAK"IYD
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1945 QPLHSFVYLYGLEVRRNVLNEAKHDDGDIDGDDFYKTTFLLIDDVLGQLGQPKHEFSNEIPIYIREHMDDYPELYEFMNR
1800 SFLHKFFYLNEFTIRKEIINESKLP----GEPSPLKNTVMLNDKILGVLGQPSHEIKNEIPPPVVENREKYPSLYEFMSR 2025 HAFRNI---ETSTAYSPSVHESTSSEGIPIITLTMSNFSDRHVDIDTVAYKFLQIYARIWTTKHCLIIDCTEFDEGGLDM 1876 YAFKKVDMKEEEEDNAPFVHEANTLDGIQIIVVTFTNCEYNNFVNDSLVYKVLQIYARNWCSKIIYVVIDCTTFYGGKANF FIG. 2-Continued.
contained four viable spores in which His and Leu phenotypes segregated independently, indicating that the IRA2 gene, like IRA], is dispensable for viability. Furthermore, the iral ira2 progeny were viable. Diploid strains homozygous for ira2 disruption were unable to sporulate. Segregants obtained from KT27 were examined for sensitivity to heat shock or nitrogen starvation. The results indicated that the Leu+ (iral) and His' (ira2) strains were sensitive to heat shock and nitrogen starvation compared with wild-type cells. These phenotypes were more severe in the ira2 mutant than in the iral mutant, and the iral ira2 double mutant was the most sensitive to these stresses (Fig. 5 and 6). These results indicate that the IRA] and IRA2
gene products additively regulate the RAS-cAMP pathway. Next, to test whether IRA] or IRA2 would completely suppress disruption of the counterpart gene, KT27-2B (ira2::HIS3) or KT27-2D (iral::LEU2-a) was transformed with multicopy plasmids pdl3 (IRA]) and pd46 (IRA2) and then examined for sensitivity to heat shock (Fig. 7). IRA] plasmid pdl3 could not suppress the heat shock sensitivity phenotype of the ira2 mutant, whereas IRA2 plasmid pd46 partially suppressed that of the iral mutant. These results suggest that IRA] and IRA2 are not identical in function. In a previous paper (29), CDC25 was shown to be antagonistic to IRAI; the iral mutation suppressed the lethality of the cdc25 mutation, whereas cdc25 suppressed the heat
4308
TANAKA ET AL.
MOL. CELL. BIOL.
2102 RKFISLVMGLLPEVAPKNCIGCYYFNVNETFMDNYGKCLDKDNVYVSSKIPHYPINSNSDEGLMKSVGITGQGLKVLQDI
1956 QKLTTLFPSLIPEQASSNCMGCYYFNVNKSFMDQIASSYTVENPYLVTTIPRCFINSNTDQSLIKSLGLSGRSLEVLKDV 2182 RVSLHDITLYDEKRNRPTPVSLKIGDIYFQVLHETPRQYKIRDNGTLFDVKFNDVYEISRIFEVHVSSITGVAAEFTVTF
2036 RVTLHDITLYDKEKKKFCPVSLKIGNKYFQVLHEIPQLYKVTVSNRTFSIKPNNVYKISNLISVDVSNTTGVSSEFTLSL 2262 QDERRLIPSSPKYLEIVKHPYYAQIRLESEYEMDNNS- ---STSSPNSNNKVKQQKERTILLCHLLLVSLIGLFDESKKH
2116 DNEEKLVFCSPKYLEIVKKPYYAQLKMEEDPGTDPSNDISPSTSSSAVNASYCNVKEVGEIISHLSLVILVGLPNEDDLV 2338 KNSSYNLIAATEASFGLNFGSHFHRSPEVYVPEDTTTFLGVIGKSLAESNPELTAYMpIYVLEALKNNVIPHVYIPHTIC
2196 KNISYNLLVATQEAFNLDFGTRLHKSPETYVPDDMFLALIFKAFSESSTELTPYIWKYMLDGLENDVIPQEHIPTVVC 2418 GLSYWIPNLYQHVYLADDEEGPENISHIFRILIRLSVRETDPKAVYMQYVWLLLLDDGRLTDIIVDEVINHALERDSENR
2276 SLSYWVPNLYEHVYLANDEEGPEAISRIIYSLIRLTVKEPNFTTAYLQQIWPLLALDGRLTNVIVEEIVSHALDRDSENR 2498 DWKKTISLLTVLPTTEVANNIIQKILAKIRSFLPSLKLEANTQSWSELTILVKISIHVPFETSLLVQMYLPEILFIVSLL 2356 DWNKAVSILTSFPTTEIACQVIEKLINMIKSFLPSLAVEASAHSWSELTILSKISVSIFFESPLLSQNYLPEILPAVSLL 2578 IDVGPRELRSSLHQLLMNVCHSLAINSALPQDHRNNLDEISDIFAHQKVKPMFGFSEDKGRILQIFSASSFASKFNILDP
2436 IDVGPSEIRVSLYELLMNVCHSLTNNESLPERNRKNLDIVCATFARQKLNPISGFSQEKGRVLPNFAASSPSSKFGTLDL 2658 PINNILLLNEYSSTYEANVWKTRYKKYVLESVFTSNSFLSARSIMIVGIMGKSYITEGLCKAMLIETMKVIAEPKITDEH 2516 FTKNIMLLMEYGSISEGAQWEAKYKKYLMDAIFGHRSFFSARAKPILGIMSKSHTSLFLCKELLVETMKVFAEPVVDDEQ 2738 LPLAISHIFTYSKIVEGLDPNLDLMKHLFPFSTLFLESRHPIIPEGALLFVSNCIRRLYMAQPENESET-SLISTLLKGR 2596 HFIIIAHVFTYSKIVEGLDPSSELMKELPWLATICVESPHPLLFEGGLLFPVNCLKRLYTVHLQLGFDGKSLAKKLMESR 2817 KFAHTFLSKIENLSGIVWNEDNFTHILIFIINKGLSNPFIKSTAFDFLKMMPFRNSYFEHQINQKSDHYLCYMFLLYFVLN
2676 NFAATLLAKLESYNGCIWNEDNFPHIILGFIANGLSIPVVKGAALDCLQALFKNTYYERKSNPKSSDYLCYLFLLHLVLS 2897 CNQFEELLGDVDFEGEMVNIENKNTIPKILLEWLSSDNENANITLYQGAILFKCSVTDEPSRFRPALIIRHLLTKKPICA 2756 PEQLSTLLLEVGFEDELVPLNNTLKVPLTLINWLSSDSDKSNIVLYQGALLFSCVMSDEPCKFRFALLMRYLLKVNPICV
2977 LRFYSVIRNEIRKISAFEQNSDCVPLAFDILNLLVTIISESNSLEKLHEESIERLTKRGLSIVTSSGIFAKNSDMMIPLDV 2836 FRFYTLTRKEFRRLSTLEQSSEAVAVSFELIGMLVTIISEFNYLEEFNDEMVELLKKRGLSVVKPLDIFDQEHIEKLKGEG
3057 KPED-IYERKRIMTMILSRHSCSA 2916 EHQVAIYERKRLATHILARNSCS FIG. 2-Continued.
shock sensitivity phenotype of iral. To determine whether the ira2 mutation would suppress the growth deficiency of the cdc25 mutation, KT27-1D (ira2::HIS3) was crossed with KT18-7B (cdc25::LEU2) carrying plasmid pKT5 (CDC25TRPI). A diploid clone, which lost pKT5, was selected and subjected to tetrad analysis. Of 45 asci, 1 ascus showed a 4 viable:O nonviable segregation pattern; 25 asci showed a 3 viable:1 nonviable pattern; and 19 asci showed a 2 viable:2 nonviable pattern. All of the Leu+ spores were His'. They showed tiny colonies and were heat shock resistant, indicating that the ira2 and cdc25 mutations are mutual suppressors, although the suppression of cdc25 by ira2 is weak. It was shown that lethality due to the rasi ras2 or cyri mutation could not be suppressed by the iral mutation (29).
Whether this is the case with ira2 was tested. KMY39-6D (rasl:: URA3 ira2: :HIS3) was crossed with KMY46-4D (ras2::LEU2) to obtain KT50, which was subsequently sporulated and dissected. Of 89 asci dissected, no Ura+ Leu+ His- nor Ura+ Leu+ His' spores were obtained (Table 2). Thus, the ira2 mutation cannot suppress the lethality of the rasi ras2 mutation. Also, the inability of the ras2 strain to grow on nonfermentable carbon sources was not suppressed by the ira2 mutation. In addition, the ira2 mutation, as well as iral, could not suppress the lethality of the cyrl:: URA3 mutation (data not shown). Phenotypes of the iral mutant were suppressible by the ras2 mutation but not by the rasi mutation (29). Segregants of KT50 were analyzed to determine whether rasi or ras2
NEGATIVE REGULATOR OF YEAST RAS
VOL. 10, 1990
4309
IRAl 1451 DLNQFPVSLRHKISILNE-NVIIALTNLSN-ANVNVSLKFTLPMGYSPNK *+
GAP
:
++:
+:
+ ++.
+
+
:+::
647 DINRFEITLSNKTKKSKDPDILFMRCQLSRLQKGHATDEWFLLSSHIPLK : + + +
:
:
:+ ++
+::
..
:+
4.6.
IRA2 1597 DRDNYPVFLRHKMSVLND-NVILSLTNLSN-TNVDASLQFTLPMGYSGNR IRAl 1499 DIRIAFLRVFIDIVTNYPVNPEKHEMDK4LAIDDFLKYIIKNPILAFFGS :+
GAP
+
::
+
:+++
:+
+:
:+
+4.
+
697 GIEPGSLR----VRARYSMEKIMPEEE----YSEFKELILQKELHVVYAL :+
4.4.:
:
*+
.4.4.
:
++
IRA2 1645 NIRNAFLEVFINIVTNYRTYTAKTDLGKLEAADKFLRYTIEHPQLSSFGA
IRAl 1549 LACSPADVDLYAGGFLNAFDTRNASHILVTELLKQEIKRAARSDDILRRN + :
GAP
: :
: :+
:
.:+
:
+
+:
739 SHVCGQDRTLLASILLKIFLHERLESLLLCTLNDREISMEDEATTLFRAT +
:.
:.
+
+
+4.4:
::
+:
+:
IRA2 1695 AVCPASDIDAYAAGLINAFETRNATHIVVAQLIKNEIEKSSRPTDILRRN
IRAl 1599 SCATRALSLYTRSRGNKYLIKTLRPVLQGIVDNKESFEIDKMK-PGSENS +
GAP
+ +
:
*64.4.+
4..4
4.
:.+
:4:
:+
:
:
789 TLASTLMEQSMKATATQFVHHALKDSILRIMESKQSCELSPSKLEKNEDV +
:.
4.4
+
+
+++
++:
+
+ :++
++
:+
:
:
IRA2 1745 SCATRSLSMLARSKGNEYLIRTLQPLLKKIIQNRDFFEIEKLK-PEDSDA
IRAl 1648 EKMLDLFEKYMTRLIDAITSSIDDFPIELVDICKTIYNAASVNFPEYAYI +
GAP
+4.
:
:++
:
+ +
:
+
::
+
+:
+ +
839 NTNLAHLLNILSELVEKIFMASEILPPTLRYIYGCLQKSVQHKWPTNTTM + ++
+
+
::+:
+
+
:
4.
:
+
IRA2 1794 ERQIELFVKYNNELLESISNSVSYFPPPLFYICQNIYKVACEKFPDHAII
IRAl 1698 ---AVGSFVFLRFIGPALVSPDSENIIIVTHAH-DRKPFITL-AKVIQSL :+.:::::
GAP
: ::+4
:::
+ +
++ +:::
+
889 RTRVVSGFVFLRLICPAILNPRMFNIISDSPSPIAART-LTLVAKSVQNL +4.: .::
++
:
:::.
:++.
:+:
IRA2 1844 ---AAGSFVFLRFFCPALVSPDSENIIDISHLS-EKRTFISL-AKVIQNI
IRAl 1743 ANGRENIFKKDILVSKEEFLKTCSDKIFNFLSELCKIP
GAP
938 ANLVEFGAKEPYMEGVNPFIKSNKHRMIMFLDELGNVP :::++ : ++
:+:
:+
::
::
+
IRA2 1889 ANGSENFSRWPALCSQKDFLKECSDRIFRFLAELCR-T FIG. 3. Amino acid sequence homology among IRA], GAP (bovine), and IRA2. Colons between amino acids indicate identities. Plus signs represent conservative amino acid substitutions, which are grouped as follows: A, G, P, S, T; L, I, V, M; D, E, N, Q; K, R, H; F, Y, W; and C.
IRA2 1
Complementation of ira2 1
[1451-2255JIRA2 I
_+
2 [1609-2255]m.A2
+
3 [1753-2188]IRA2 4 [1804-2255]IRA2 5 [1451-2026]IRA2 |
6 [1451-1991]IRA2 1 7 [1451-1908]IRA2 1
E
-
+
*
_
+
FIG. 4. Correspondence of the essential domain of IRA2 to the region homologous with mammalian GAP. Plasmids carrying various restriction fragments of the IRA2 gene under control of the GAPDH gene promoter were transformed into KT27-2B (ira2: :HIS3) to test their ability to complement the heat shock sensitivity phenotype of the ira2 mutation. Expressed truncated proteins are shown on the left. The hatched region (1597 to 1925) is homologous with mammalian GAP (see Fig. 3).
4310
TANAKA ET AL.
MOL. CELL. BIOL.
2
1
3
4 xif". t:,:..
A.,I i.'-
..
n, i.:
VI6.-! l.'- - '
M."!
-,
H
L
HS N FIG. 5. Additive phenotype of iral and ira2 mutants. Tetrad segregants obtained from an ascus of KT27 were tested for histidine auxotrophy (-H), leucine auxotrophy (-L), heat shock sensitivity (HS), and nitrogen starvation (-N). 1, KT27-7A (wild type); 2, KT27-7B (iral::LEU2-a); 3, KT27-7C (iral::LEU2-a ira2::HIS3); 4, KT27-7D (ira2::HIS3).
would suppress ira2. The heat shock sensitivity phenotype of the ira2 mutant was suppressed by the ras2 mutation but not by the rasi mutation. The sporulation deficiency of ira2 was suppressed efficiently by the ras2 mutation but only weakly by the rasi mutation. These results indicate that IRA2 negatively regulates the RAS-cAMP pathway through RAS as does IRA1. ira2 is allelic to gkc4. To map the chromosomal location of the IRA2 gene, an IRA2 probe was hybridized to a Southern blot of intact chromosomal DNA separated by orthogonal field alternation gel electrophoresis. The results indicated that the IRA2 gene is located on chromosome XV (data not shown). It was previously reported that the iral mutation is allelic to the glcl mutation (18), which is deficient in glycogen accumulation. This result is consistent with the report that the RAS-cAMP pathway negatively regulates the storage of this carbohydrate (32). Since another mutation deficient in glycogen accumulation, glc4, maps 15 centimorgans distal to the argi locus on the left arm of chromosome XV (19) and since glc4 showed heat shock-sensitive and sporulation-defective (Spo-) phenotypes, ira2 linkage to argi was
tested. KT69-1B (IRA2 argi his3) was crossed with KT271D (ira2::HIS3 ARGI his3) to form a diploid which was subsequently sporulated and dissected. Of 52 tetrads obtained, segregation patterns of the His and Arg phenotypes showed a parental ditype/tetratype/nonparental ditype ratio of 41:11:0; the calculated map distance between ira2::HIS3 and argi was 11 centimorgans. Next, a glc4 mutant, KMY305-5C, was crossed with two strains isogenic except for the IRA2 locus, KT27-2A (IRA2) and KT27-2B (ira2::HIS3), to obtain KT65 to KT66, respectively. KT65 showed a wild-type phenotype with regard to heat shock resistance, sporulation efficiency, and glycogen accumulation. In contrast, KT66 was deficient in all of these phenotypes. Thus, we concluded that ira2 is allelic to g1c4. IRA2 regulates the intracellular cAMP level through RAS2. We examined the effect of the ira2 mutation on the intra-
1
2
3.14
1
2
3
A 100 cis
10 U, O
.O'
B
1 0.1
Time (min) FIG. 6.
Sensitivity
to
heat shock.
Exponentially growing cells
of KT27-13A (0; iral::LEU2-a ira2::HIS3), KT27-13B (0; ira2:: HIS3), KT27-13C (O; iral::LEU2-a), and KT27-7D (U; wild type) at 25°C were tested for heat shock as described in Materials and Methods.
FIG. 7. Suppression of the heat shock sensitivity of ira mutants by overexpression of IRA genes. KT27-2D (iral::LEU2-a; A) or KT27-2B (ira2::HIS3; B) was transformed by IRA] plasmid pdl3 (1), IRA2 plasmid pd46 (2), and control plasmid YEp24 (3). Two transformants were subjected to heat shock at 52°C for 20 min, and then the plate was incubated at 30°C for 4 days.
NEGATIVE REGULATOR OF YEAST RAS
VOL. 10, 1990
TABLE 2. Failure of the ira2 mutation to suppress the lethality of the rasl ras2 mutation No. of spores
Genotypea
Viable
Nonviableb
51 21 39 59 52 51 0 0
0 0 5 3 1 2 30 42
RAS] RAS2 IRA2 RAS] RAS2 ira2 RAS] ras2 IRA2 RASI ras2 ira2 rasl RAS2 IRA2 rasl RAS2 ira2 rasi ras2 IRA2 rasi ras2 ira2
a Genotype of each viable spore was assigned by the auxotrophic marker. Eighty-nine asci of KT50 (rasl::URA31RASl RAS21ras2::LEU2 ira2::HIS31
IRA2) were analyzed. b Genotypes of nonviable spores were inferred form the genotypes of their sister spore clones.
cellular cAMP level. KT31-1A (ira2::HIS3), KT31-1C (wild type), and KT31-1D (ira2::HIS3 ras2:: URA3) were grown to stationary phase. Cells were stimulated by glucose addition, and the intracellular cAMP level was measured as described in Materials and Methods. cAMP levels in the ira2 mutant increased upon glucose addition and remained at an elevated level, as in the iral mutant (29) (Fig. 8). Moreover, this high level of cAMP was suppressed by the ras2 mutation; the ira2 ras2 cells showed a wild-type pattern of cAMP formation. Thus, IRA2 regulates the intracellular level of cAMP by controlling RAS2 activity as in the case of IRA].
4311
DISCUSSION In this work, we have shown that a homolog of the IRA] gene, IRA2, functions through RAS as a negative regulator of the RAS-cAMP pathway in S. cerevisiae. Disruption of the IRA] or IRA2 gene resulted in sensitivity to heat shock and nitrogen starvation, and the double mutant had the additive phenotype. However, the IRA] or IRA2 gene on a multicopy plasmid could not suppress completely the disruption mutation of the counterpart IRA gene, suggesting that their functions are similar but not the same. What is the functional difference between IRA] and IRA2? Although the ras2 mutation clearly suppressed the Spo- and heat shock sensitivity phenotypes of both iral and ira2 mutations, the rasi mutation could not suppress the phenotypes of iral and only weakly suppressed the Spo- phenotype of ira2. Moreover, rasi suppressed the heat shock sensitivity of ira2 in some genetic backgrounds in which iral could not be suppressed by rasi (data not shown). These results suggest that the phenotypes of ira2 were caused by the activation of RAS2 and RAS] proteins, although the contribution by RAS] activation is lower than that of RAS2 activation, possibly because of weaker expression of RAS] than RAS2 (3). In contrast, the phenotypes of iral were caused by the activation of only the RAS2 protein. Thus, some preference appears to exist between RAS and IRA proteins. This speculation is supported by guanine nucleotide analyses recently reported (30). When RAS] or RAS2 protein was overexpressed in the iral mutant, the percentage of the GTP-bound form of RAS] protein was similar to that of the
B
A
200 C1
a.
U
MOLECULAR AND CELLULAR BIOLOGY, Aug. 1990, p. 4303-4313
0270-7306/90/084303-11$02.00/0 Copyright © 1990, American Society for Microbiology
IRA2, a Second Gene of Saccharomyces cerevisiae That Encodes a Protein with a Domain Homologous to Mammalian ras GTPase-Activating Protein KAZUMA TANAKA,"2 MASATO NAKAFUKU,3 FUYUHIKO TAMANOI,2 YOSHITO KAZIRO,3 KUNIHIRO MATSUMOTO,4* AND AKIG TOH-El5 Department of Fermentation Technology, Faculty of Engineering, Hiroshima University, Higashihiroshima, 724, Japan1; Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, Illinois 606372; Institute of Medical Science, University of Tokyo, 4-6-1, Shirokanedai, Minatoku, Tokyo 108, Japan3; DNAX Research Institute of Molecular and Cellular Biology, Palo Alto, California 943044; and Department of Biology, Faculty of Science, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113, Japan5 Received 2 March 1990/Accepted 21 May 1990
The IRAI gene is a negative regulator of the RAS-cyclic AMP pathway in Saccharomyces cerevisiae. To identify other genes involved in this pathway, we screened yeast genomic DNA libraries for genes that can suppress the heat shock sensitivity of the iral mutation on a multicopy vector. We identified IRA2, encoding a protein of 3,079 amino acids, that is 45% identical to the IRA] protein. The region homologous between the IRA] protein and ras GTPase-activating protein is also conserved in IRA2. IRA2 maps 11 centimorgans distal to the argl locus on the left arm of chromosome XV and was found to be allelic to gk4. Disruption of the IRA2 gene resulted in (i) increased sensitivity to heat shock and nitrogen starvation, (ii) sporulation defects, and (lii) suppression of the lethality of the cdc25 mutant. Analysis of disruption mutants of IRAI and IRA2 indicated that IRAI and IRA2 proteins additively regulate the RAS-cyclic AMP pathway in a negative fashion. Expression of the IRA2 domain homologous with GAP is sufficient for complementation of the heat shock sensitivity of ira2, suggesting that IRA down regulates RAS activity by stimulating the GTPase activity of RAS proteins.
the lethality of the cdc25 mutation and causes an increased level of intracellular cAMP (29). Thus, the iral disrupted mutant displays phenotypes associated with the activated RAS2va'-19 mutant, which has reduced intrinsic GTPase activity (4). These observations, together with the partial homology of this protein to mammalian GAP (29), suggested that the IRA] protein acts as a negative regulator that stimulates the conversion of the GTP form to the GDP form of RAS proteins by stimulating their GTPase activity. In an attempt to further identify genes involved in regulation of the RAS-cAMP pathway in S. cerevisiae, we have isolated multicopy suppressors of the heat shock sensitivity of the iral mutation (25). In the process, we identified the MSII gene, which encodes a protein homologous to the a subunit of mammalian G protein (25). We found another gene, IRA2, that is also capable of suppressing the heat shock sensitivity phenotype caused by the iral mutation when present on a multicopy plasmid. In a previous paper (30), we reported that a RAS * GTP form is accumulated in either the iral or ira2 mutant, suggesting that the IRA] and IRA2 proteins exert a function similar to that of mammalian GAP. In this communication, we report that IRA2 is a homolog of IRA) and encodes a protein that also shares homology with mammalian GAP. We also describe genetic experiments that examine the effects of IRA2 disruption on the RAS-cAMP pathway.
The ras genes participate in the control of proliferation and differentiation in mammalian cells (2, 10). The ras proteins are localized on the inner face of the plasma membrane, exhibit guanine nucleotide-binding activity, and possess an intrinsic GTPase activity. They are thus members of the family of guanine nucleotide-binding proteins, and their activity is modulated by utilizing a guanine nucleotidebinding and hydrolysis cycle. The GTP-bound form of the protein activates a target protein, and this stimulation is turned off upon hydrolysis of GTP to GDP. It is therefore of interest to identify individual components that interact with ras proteins and regulate their activity. Recently, it has been demonstrated that mammalian cells possess a ras GTPaseactivating protein (GAP) that catalytically accelerates hydrolysis of GTP bound to ras proteins (34). The yeast Saccharomyces cerevisiae contains two homologs of mammalian ras genes, RAS] and RAS2 (8, 22), which are involved in transduction of the signal for growth in response to nutrients (17, 31). The yeast RAS proteins control the activity of adenylyl cyclase, which produces the second messenger, cyclic AMP (cAMP), responsible for the activation of cAMP-dependent protein kinase (A-kinase) (33). The RAS2 protein has been shown to activate adenylyl cyclase when bound to GTP but not when bound to GDP (9). Genetic analysis identified regulatory genes, CDC25 and IRA], that modulate the activity of RAS proteins in S. cerevisiae. The CDC25 protein is proposed to act as a positive regulator that exchanges the GDP bound to RAS proteins for GTP (5, 7, 15, 23). In contrast, genetic evidence indicates that the IRA] protein inhibits the function of RAS proteins in a fashion antagonistic to the function of the CDC25 protein (29). The IRA] gene disruption can suppress *
MATERIALS AND METHODS Strains and culture media. Yeast strains used are described in Table 1. Usually, yeast cells were grown in a rich medium, YPD, which contains 1% yeast extract, 2% polypeptone, 2% glucose, 400 mg of adenine sulfate per liter, and 200 mg of uracil per liter. SD medium, which was used to select the plasmid-containing cells or to determine the auxotrophic
Corresponding author. 4303
4304
TANAKA ET AL.
MOL. CELL. BIOL. TABLE 1. Yeast strains used
Strain
Genotype MATa iral-I ura3 trpl his3 lys2 ade8 MATx rasl::URA3 ira2::HIS3 ura3 leu2 trpl his3 MATa ras2::LEU2 ura3 leu2 trpl his3 ade8 MATot glc4-2 ura3 his3 ade8 MA Ta/MA Ta iral::LEU2-alIRAI ura3lura3 leu2lIeu2 trplltrpl his3/his3 MATa cdc25::LEU2 ura3 leu2 trpl his3 (pKT5)a MATa/MATa iral::LEU2-a/IRAI ira2::HIS31IRA2 ura3lura3 leu2/Ieu2 trplltrpl hIhis3 MATa ira2::HIS3 ura3 leu2 trpl his3 MATa ura3 leu2 trpl his3 MATa ira2::HIS3 ras2::URA3 ura3 leu2 trpl his3 MATa argi ura3 leu2 trpi his3 MATa/MATa iral-l/iral-J ura3lura3 Iys211ys2 thr41thr4
C70-3D ...... KMY39-6D ...... KMY46-4D ...... KMY305-5C ...... KT6 ...... KT18-7B ....... KT27 ....... KT31-1A ....... KT31-1C ....... KT31-1D ....... KT69-1B ....... R59-1C-D ....... a A plasmid carrying the
wild-type CDC25 gene (unpublished data).
requirements of strains, was 0.7% yeast nitrogen base without amino acids (Difco Laboratories, Detroit, Mich.), 2% glucose, and appropriate amounts of amino acids and nucleic acid bases. Nitrogen starvation medium contained 0.2% yeast nitrogen base without amino acids and ammonium sulfate (Difco), 2% glucose, and the required amino acids and nucleic acid bases. Cloning of IRA2 and plasmid constructions. Standard techniques for molecular cloning were performed essentially as described elsewhere (14). The IRA2 gene was first cloned from the YEp24-based yeast genomic DNA library (6) as a plasmid, pd46. The original insert DNA was lacking the region encoding the carboxyl terminus of the putative IRA2 protein (see text); thus, the 6-kilobase (kb) HindIII fragment overlapping part of pd46 was cloned from the genomic DNA of strain X2180-1A into the Bluescript vector (Stratagene, San Diego, Calif.) Plasmid pKT14, which contained the
target DNA, was identified by colony hybridization, using the 1.2-kb BamHI-MluI fragment ofIRA2 (Fig. 1) as a probe. A plasmid used for disrupting the IRA2 gene was constructed as follows. First, the 2.7-kb Sacl fragment encoding the amino-terminal region of the IRA2 gene was subcloned into the SacI site of pUC19 (35) to form pFS2. The 1.8-kb BamHI fragment encoding the HIS3 gene was cloned into the BglII site of pFS2 (27). The resulting plasmid, pKZ7, was cut with Sacl and used for transformation of yeast cells to obtain the disruptant of the IRA2 gene by the method of one-step gene disruption (24). Plasmids for overexpressing truncated IRA2 were constructed as follows. Truncated IRA2 proteins were expressed under the control of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene promoter by cloning various restriction fragments of IRA2 into pKT10, a plasmid containing the ARS sequence of 2,um DNA, URA3, and the
HIS3 B P M '
H i
{
P_
Sc'-I HG
Sc
PP 5
G HIScH --
.
pd46
ty,
Sc H 1 ~~pKT14 .4
Complementation of
ORF
iral
dl
d2 d3 I
d4
d5 i
-4
d6 d7
+
1Kb FIG. 1. Restriction enzyme maps and deletion analysis of plasmids encoding IRA2. Vector DNA was omitted from the figure. Horizontal lines indicate the DNA sequences remaining after deletion. The IRA2 open reading frame (ORF) is boxed, and its direction is shown with an arrow. The region homologous with GAP (see Fig. 2 and 3) is shown by black boxes. The position where the HIS3 gene fragment was inserted to disrupt IRA2 is also shown. Abbreviations for restriction enzyme sites: B, BamHI; P, PvuII; M, MluI; Sc, Sacl; H, Hindlll; G, BglIl.
VOL. 10, 1990
(28). Northern (RNA) analysis indicated that the level of mRNA from the GAPDH gene promoter was more than 10-fold higher than that of IRA2 mRNA (data not shown). Restriction fragments used were as follows: 2.4-kb BglII-PvuII fragment for [14512255]IRA2, 1.9-kb StuI-PvuII fragment for [1609-2255]IRA2, 1.3-kb EcoRV-EcoRV fragment for [1753-2188]IRA2, 1.4-kb SacI-PvuII fragment for [1804-2255]IRA2, 1.7-kb BgIII-MluI fragment for [1451-2026]IRA2, 1.6-kb BgIII-BglI fragment for [1451-1991]IRA2, and 1.4-kb BgIII-AfIl fragment for [1451-1908]IRA2 (numbers in brackets show the spans of the amino acid sequences of truncated IRA2 proteins). For construction of plasmids containing BglII-PvuII, BglII-MluI, BglII-BgII, and BglII-AfII fragments, synthetic oligonucleotides were added to provide the initiation codon, resulting in the addition of five amino acids, Met-Gly-Thr-Ala-Arg, before the authentic amino acid sequence of the IRA2 protein. In other constructions, internal methionine codons of IRA were used for an initiator codon, and 5' nontranslated sequences provided by the IRA2 DNA sequence were less than 52 base pairs long. At the 3' terminus of each construction, there are three-phase termination codons followed by the transcription terminator of the GAPDH gene to make transcriptional and translational terminations efficient. DNA sequencing. The DNA fragments to be sequenced were cloned in M13mp18, M13mp19 (35), or Bluescript. Unidirectional deletions were constructed in these clones as described previously (11), and single-stranded DNAs were prepared. Nucleotide sequence was determined by the method of Sanger et al. (26) on both strands and overlapped the junction between fragments. cAMP assay. Glucose-induced cAMP formation in starved cells was determined as previously described (20, 29) except that the starvation of cells in buffer and the addition of glucose were at 30°C. Determination of heat sensitivity. For scoring on plated medium, fresh cells grown on YPD plates were replica plated onto YPD and immediately placed at 50°C for 1 h. Plates were incubated for 3 days at 30°C, and heat shock sensitivity was scored. For examination in liquid medium, cells growing exponentially in YPD at 25°C were transferred to 50°C, and small samples were taken at the indicated time and chilled in an ice bath. After appropriate dilution, cells were plated on YPD plates. Three days later at 30°C, colonies were counted and the percent survival was determined. Determination of nitrogen starvation sensitivity. For determination of sensitivity to nitrogen starvation on plated medium, fresh cells grown on YPD plates were replica plated onto nitrogen starvation plates. After 9 days at 30°C, cells were replica plated onto YPD plates and incubated at 30°C for 3 days to observe the fraction surviving. Other methods. Sporulation efficiency (29) and yeast transformation (12) were determined as described previously.
promoter fragment of the GAPDH gene
RESULTS Isolation of the IRA2 gene. A yeast genomic library, constructed in the multicopy vector YEp24, was screened for plasmids that could suppress the heat shock sensitivity phenotype of the iral-J mutation. The iral strain, C70-3D (iral-J ura3), was transformed with library DNA. Plasmids that conferred the ability to form colonies after incubation at 30°C for 24 h, followed by 1-h incubation at 50°C, were selected. Four different sequences in addition to the IRA] gene itself were able to suppress the phenotype of iral-J (25). One of these suppressors, IRA2 (previously called MSI3), is characterized in this report.
NEGATIVE REGULATOR OF YEAST RAS
4305
Transformation of R59-1C-D (MATa iral-JIMATa iral-1) with the IRA2 plasmid pd46 restored heat shock resistance to R59-1C-D as well as the ability to sporulate (from 0 to 4.5%). The IRA] gene on YEp24 suppressed the sporulation deficiency of R59-1C-D (from 0 to 34%). IRA2 is a homolog of IRA]. Deletion analysis of pd46 (Fig. 1) suggested that almost the entire DNA sequence (9.4 kb) is required for the suppressor activity of the iral-J mutation, with two exceptions (d2 and d7; see below). This result suggests that the gene on pd46 is large like IRAI (29). In fact, determination of the nucleotide sequence of the cloned DNA revealed a large open reading frame which is truncated at the carboxyl terminus (Fig. 1). To determine the entire nucleotide sequence of the gene, we cloned a 6.0-kb chromosomal HindIII fragment contiguous to the DNA fragment cloned in pd46. The joined nucleotide sequences built up a large open reading frame that can encode a protein of 3,079 amino acids (Fig. 2). Northern hybridization experiments detected a band of about 10 kb in length in the poly(A)+ RNA fraction, consistent with the size predicted from the nucleotide sequence of the gene (data not shown). Computer analysis revealed an extensive and clear amino acid sequence homology between IRA] and IRA2 gene products (45% identity within the total 2,938 amino acid residues of IRAI; Fig. 2). Thus, this gene was designated IRA2. When the amino acid sequences of IRA] and IRA2 proteins were aligned for maximum matching, the IRA2 protein was found to have an extension of 180 amino acid residues at the N terminus. We previously reported (29) that the IRA] protein has two potential sites for A-kinase phosphorylation, Arg-Arg-X-Ser or Thr (13). The IRA2 gene product also has two such sequences, although only one of the two sites is located at the same position in the IRA] and IRA2 proteins (Fig. 2). These sites may be involved in feedback control of cAMP formation conducted by A-kinase (21). When IRA] and IRA2 proteins were divided into three parts, amino terminal (amino acid sequence from positions 1 to 979 of the IRA] protein), middle (980 to 1958), and carboxyl terminal (1959 to 2938), the identities between IRA] and IRA2 proteins were found to be 31, 52, and 53%, respectively, indicating that IRA] and IRA2 proteins are less homologous in their amino-terminal regions. The essential domain of IRA2 corresponds to the region homologous with GAP. In the middle of the IRA] and IRA2 proteins, there is a region showing a weak but significant homology with the carboxyl terminus of bovine GAP (Fig. 2) (29). When these regions of IRA] and IRA2 proteins were aligned with bovine GAP for maximum matching, they were found to be 22% identical and 45% homologous with GAP (Fig. 3). Recently, the carboxyl-terminal 343 amino acids of bovine GAP (amino acid sequence 702 to 1044), which includes the region homologous with IRA proteins, have been shown to be essential for GAP activity (16). As described above, two deletion plasmids, d2 and d7, complemented the iral-J mutation. These two plasmids thus seemed to carry an essential domain of IRA2 which was expressed by readthrough from vector sequences. Because these plasmids contain the region homologous with GAP (Fig. 1), a smaller truncated protein of IRA2, [14512255]IRA2, which includes that region, was expressed and found to suppress the ira2 mutation (Fig. 4). To more precisely define the IRA2 essential region, several deletion fragment of [1451-2255]IRA2 were examined for the ability to complement the ira2 mutation. The amino acid sequence from 1609 to 1991 of the IRA2 protein was sufficient for suppression of the ira2 mutation (Fig. 4). This region corre-
4306
TANAKA ET AL.
MOL. CELL. BIOL.
IRA2.1 MSQPTKNKKKEHGTDSKSSRMTRTLVNHILFERILPILPVESNLSTYSEVEEYSSFISCRSVLINVTVSRDANAHVEGTL
81 ELIESLLQGHEIISDKGSSDVIESILIILRLLSDALEYNWQNQESLHYNDISTHVEHDQEQKYRPKLNSILPDYSSTHSN 161 GNKHFFHQSKPQALIPELASKLLESCAKLKPNTRTLQILQNHISHVHGNILTTLSSSILPRHKSYLTRHNHPSHCKMIDS IRA1.1
HLLCKISKLKFNTRTLKVLQNNSHHLSGSA-TISKSSILPDSQEFLQKRNYPAYTEKIDL
241 TLGHILRFVAASNPSEYFEFIRKSVQVPVTQTHTHSHSHSHSLPSSVYNSIVPHFDLFSFIYLSKHNPKKYLELIKNLSV 60 TIDYIQRFISASNHVEFTKCVKTKVVAPLLISHT -STELGVVNHLDLFGCEYLTDKNLLAYLDILQHLSS
321 TLRKTIYHCLLLHYSAKAINFWIHARPAEYYELPNLL--------------KDNNNEBSKSLNTLNHTLPEEIHSTFNVN
129 YKKRTIPHSLLLYYASKAFLPWINARPKEYVKIYNNLISSDYNSPSSSSDNGGSNNSDKTSISQLVSLLFDDVYSTPSGS 387 SKITTNQNAHQGSSSPSSSSPSSPPSSSSSDNNNQNIIAKSL -
SRQLSHHQSYIQQQSERKLHSSWTTNSQS-S
209 SLLTNVNNDHHYHLHHSSSSSKTTNTNSPNSISKTSIKQSSVNASGNVSPSQFSTGNDASPTSPMASLSSPLNTNILGYP 459 TSLSSSTSNSTTTDFSTHTQPGEYDPSLPDTPTMSNITISASSLLSQTPTPTTQLQQRLNSAAAAAAAAASPSNSTPTGY 289 LSPITSTLGQANTSTSTTAATTKTDADTPSTMNTNNNNNNNNSANLNNIPQRIPSLDDISSFNSSRKSLNLDDSNSLFLW 539 TAEQQSRASYDAHK--TGHTGKDYDEHFLSVTRLDNVLELYTHFDDTEVLPHTSVLKFLTTLTMFDIDLFNELNATSFKY
369 DTSQHSNASNTNTNMHAGVNNSQSQNDQSSLNYNENINELYSNYTGSELSSHTAILRPLVVLTLLDSEVYDEKNSNSYRK 617 IPDCTMHRPKERTSSFNNTAHETGSEKTSGIKHITQGLKKLTSLPSSTKKTVKFVKHLLRNLNGNQAVSDVALLDTHRAL
449 ISEPIKNINPKD
---
SNTSSWGSASKNPSIRHLTHGLKKLT-LQQGRKRNVKFLTYLIRNLNGGQFVSDVSLIDSIRSI
697 LSPFFTTSAVFLVDRNLPSVLFAKRLIPINGTNLSVGQDWNSKINNSLNV-CLKKNSTTFVQLQLIFFSSAIQFDHELLL 524 LFLMTMTSSISQIDSNIASVIFSKRFYNLLGQNLEVGTNWNSATANTFISHCVERNPLTHRRLQLEFFASGLQLDSDLFL
776 ARLSIDTNANNLNMQKLCLYTEGPRIPPDIPSKKELRKAIAVKISKPFKTLPSIIADILLQEFPYFDEQITDIVASILDG 604 RHLQLEKELNHIDLPKISLYTEGPRVFFHLVSTKKLHEDIAEKTSSVLKRLFCIIADILLKATPYPDDNVTKIIASILDG
856 TIINEYGTKKHPKGSSP-SLCSTTRSRSGSTSQSSNTPVSPLGLDTDICPNNTLSLVGSSTSRNSDNVNSLNS-SPKNLS 684 HILDQFDAARTLSNDDHVSFDAATSVYTEPTEIIHNSSDASLVSSLSQSPLSINSGSNITNTRTWDIQSILPTLSNRSSA 934 SDPYLSHLVAPRARHALGGPSSIIRNKIPTTLT ----SPPGTEKSSPVQRPQTESISATPNAITNSTPLSSAAPGIRSP
764 SDLSLSNILTNPLEAQQNNNANLLAHRLSGVPTTKRYASPNDSERSRQSPYSSPPQLQQSDLPSPLSVLSSSAGPSSNHS 1009 LQKIRT- -RRYSDESLGKFMKSTNNYIQEHLIPKDLNEATLQDARRININIFSIFKRPNSYFIIPHNINSNLQWVSQDFR
844 ITATPTILKNIKSPKPNKTKKIADDKQLKQPSYSRVILSDNDEARKIMMNIPSIFKRNTNWPIRPDANTE----FPKTFT FIG. 2. Amino acid sequence of the putative IRA2 protein (GenBank accession number M33779) and homology with the IRA] protein. The amino acid sequence of IRA2 deduced from the DNA sequence was aligned with that of IRA] for maximum homology. Identical amino acids are shown with colons. Potential sites for A-kinase phosphorylation are underlined. The shaded region shows homology with bovine GAP (see Fig. 3).
sponds to the IRA domain homologous with GAP (Fig. 3). The homology between IRA proteins and GAP thus seems to be significant, suggesting that IRA proteins have a GAP-like activity. Disruption and genetic analysis of the IRA2 gene. To understand the function of the IRA2 gene, we disrupted the chromosomal IRA2. The 1.8-kb BamHI fragment encoding the yeast HIS3 gene was inserted into one of the BglII sites in the IRA2 gene (Fig. 1). This construction fuses the DEDI gene contained in the 1.8-kb fragment (27) to the IRA2 truncated fragment out of frame. A diploid strain, KT6,
harboring the heterozygous iral::LEU2-a mutation was transformed to His' with the 4.5-kb Sacl fragment carrying ira2::HIS3. We confirmed by Southern analysis that several transformants carried the expected change at the IRA2 locus (data not shown). One such transformant, designated KT27, was sporulated and dissected for tetrad analysis. Because RAY-3A-D, which is the original host strain of KT6 and KT27, was constructed by transforming a haploid strain, RAY-3A, with a plasmid carrying the HO gene (29), tetrad segregants obtained from KT27 are isogenic except for the IRA loci. Almost all asci of KT27 (18 of 21 dissected)
VOL. 10, 1990
126TN~ ~
NEGATIVE REGULATOR OF YEAST RAS
4307
1087 NIMKPIFVAIVSPDVDLQNTAQSFMDTLLSNVITYGESDENIS- - - IEGYHLLCSYTVTLPAMGLFDLKINNEKRQILLD 920 DIIKPLPVSILDSNQRLQVTARAFIEIPLSYIATFEDIDNDLDPRVLNDHYLLCTYAVTLFASSLFDLKLENAKREMLLD 1164 ITVKFMKVRSHLAGIAEASHHMEYISDSEKLTFPLINGTVGRALFVSLYSSQQKIEKTLKIAYTEYLSAINPHERNIDDA
1000 IIVKFQRVRSYLSNLAEKHNLVQAIITTERLTLPLLVGAVGSGIFISLYCSRGNTPRLIKISCCEFLRSLRPYQKYVGAL
1244 DKTWVIHNIEPVEAMCHDNYTTSGSIAPQRRTRNNILRPATIPNAILLDSMRMIYKKWHTYTHSKSLEKQERNDFRNPAGI
1080 DQYSIYNIDPIDAMAQDNFTASGSVALQRRLRNNILTYIKGSDSILLDSMDVIYKKWFYPSCSKSVTQEELVDFRSLAGI 1324 LASLSGILFINKKILQENYPYLLDTVS ------------------ ELKKNIDSFISKQCQWLNYPDLLTRENSRDILSVE
1160 LASPSGILSDMQELEKSKSAPDNEGDSLSPESRNPAYEVHKSLKLELTKKNNFFISKQCQWLNNPNLLTRENSRDILSIE
1386 LHPLSPNLLFNNLRLKLKELACSDLSIPENESSYVLLEQIIKMLRTILGRDDDNYVNMLPSTEIVDLIDLLTDEIKKIPA
1240 LHPLSFNLLFNNLGLKIDELHSIDLSKSHEDSSFVLLEQIIIIIRTILKRDDDEKIMLLPSTDLLDAVDKLIEIVEKISI 1466 YCPKYLKAIIQNTKMPSALQHSEVNLGVKNHFHVKNKWLRQITDWFQVSIAREYDFENLSKPLKENDLVKRDNDILYIDT 1320 KSSKYYKGIIQNSKNFRAFEHSEKNLGISNHFHLKNKWLKLVIGWPKLSINKDYDFENLSRPLREMDLQKRDEDFLYIDT
1546 AIEASTAIAYLTRHTFLEIPPAASDPELSRSRSVIPGFYFNILNKGLEKSSSMDYWVll -IIWKV1.NDNVILSLTNLSN .. ..N.. .N... * ** *X * * *X--X- ' ~ ~ ~ ~ ~ ~ ~.'."~ ~ ~ ~ ~. . 2 ............ -* -.-.'. .....
......
*
1400 SIESAKALAYLTHNVPLEIPPSSSKEDWNRSSTVSPGNIIPTILLKGLEKSADLN%VPLISN 1626 TNVDASLLP)IYSGKRRRRNAPLEVPIMVTNYRTYTAKTDLGKLUADEhYTI~EHPQ1SSGAAVCPASDID~AY ......,Y it ,,4.V !P.**
a
b 4.
4
1480 ANVSKFTLMGYSP.KIRIAPlRSYFID1VTrI..PEIIE.D.'L&XPn.IYIIKNP.IJAFPGS-
ADVDLY
1706 170B 'AC. EPrR'ysHiV....A..QuNE AAGLIMAPETRNATHIVVAQL-IKNEIEK-SSRPDULARNSCAThSL.SMLARSKGNEMLRTLQPLLKKIIQNRDPFEIEK . . .,... ....._AT..H......ltwPzE..WwEE *
5:
*'
*
j*X : :
P40 * -*.o440* *
.: ::''
**
4
:
:-' 2
::
~ U 0S:' *@-:'~S
*: .**::- * *:; *.*s
:S
:
:: :::
:
::
::
1560 AGGPLNADTRMASHILVTELLKQEIKRAARSDDILRNUSCt.ALSLYTRSRGNKYLIKTlRPYlGI1VDNKESFEIDlK
1786 LKPEDSDARQIELPVKYKINELEUSSNSVS-PP--SPJTRU YT[CEK IIAAGSFVIRPPcPALVSPDSEN * s s -4 4* * .-44K . 1640 KKPGSENSEKHLDLEYKTRLIDAITSSIDDPIELDICKASV TIAV VLRFIGPALYSPDSEN 1866 -IIDISHLSEKRT-PISLA'iVI.'NGENMPSVPAL-.CS XDPLKECSDRI -lLAELCR-TDRTIDIQVRTDPTPIAFDY * -
'.4` *
4
1720 I. }t-m{DRPlk sPnXisAK"IYD
4
*
* *4
54-4
IMSTNNLITNFT
piSFDY
1945 QPLHSFVYLYGLEVRRNVLNEAKHDDGDIDGDDFYKTTFLLIDDVLGQLGQPKHEFSNEIPIYIREHMDDYPELYEFMNR
1800 SFLHKFFYLNEFTIRKEIINESKLP----GEPSPLKNTVMLNDKILGVLGQPSHEIKNEIPPPVVENREKYPSLYEFMSR 2025 HAFRNI---ETSTAYSPSVHESTSSEGIPIITLTMSNFSDRHVDIDTVAYKFLQIYARIWTTKHCLIIDCTEFDEGGLDM 1876 YAFKKVDMKEEEEDNAPFVHEANTLDGIQIIVVTFTNCEYNNFVNDSLVYKVLQIYARNWCSKIIYVVIDCTTFYGGKANF FIG. 2-Continued.
contained four viable spores in which His and Leu phenotypes segregated independently, indicating that the IRA2 gene, like IRA], is dispensable for viability. Furthermore, the iral ira2 progeny were viable. Diploid strains homozygous for ira2 disruption were unable to sporulate. Segregants obtained from KT27 were examined for sensitivity to heat shock or nitrogen starvation. The results indicated that the Leu+ (iral) and His' (ira2) strains were sensitive to heat shock and nitrogen starvation compared with wild-type cells. These phenotypes were more severe in the ira2 mutant than in the iral mutant, and the iral ira2 double mutant was the most sensitive to these stresses (Fig. 5 and 6). These results indicate that the IRA] and IRA2
gene products additively regulate the RAS-cAMP pathway. Next, to test whether IRA] or IRA2 would completely suppress disruption of the counterpart gene, KT27-2B (ira2::HIS3) or KT27-2D (iral::LEU2-a) was transformed with multicopy plasmids pdl3 (IRA]) and pd46 (IRA2) and then examined for sensitivity to heat shock (Fig. 7). IRA] plasmid pdl3 could not suppress the heat shock sensitivity phenotype of the ira2 mutant, whereas IRA2 plasmid pd46 partially suppressed that of the iral mutant. These results suggest that IRA] and IRA2 are not identical in function. In a previous paper (29), CDC25 was shown to be antagonistic to IRAI; the iral mutation suppressed the lethality of the cdc25 mutation, whereas cdc25 suppressed the heat
4308
TANAKA ET AL.
MOL. CELL. BIOL.
2102 RKFISLVMGLLPEVAPKNCIGCYYFNVNETFMDNYGKCLDKDNVYVSSKIPHYPINSNSDEGLMKSVGITGQGLKVLQDI
1956 QKLTTLFPSLIPEQASSNCMGCYYFNVNKSFMDQIASSYTVENPYLVTTIPRCFINSNTDQSLIKSLGLSGRSLEVLKDV 2182 RVSLHDITLYDEKRNRPTPVSLKIGDIYFQVLHETPRQYKIRDNGTLFDVKFNDVYEISRIFEVHVSSITGVAAEFTVTF
2036 RVTLHDITLYDKEKKKFCPVSLKIGNKYFQVLHEIPQLYKVTVSNRTFSIKPNNVYKISNLISVDVSNTTGVSSEFTLSL 2262 QDERRLIPSSPKYLEIVKHPYYAQIRLESEYEMDNNS- ---STSSPNSNNKVKQQKERTILLCHLLLVSLIGLFDESKKH
2116 DNEEKLVFCSPKYLEIVKKPYYAQLKMEEDPGTDPSNDISPSTSSSAVNASYCNVKEVGEIISHLSLVILVGLPNEDDLV 2338 KNSSYNLIAATEASFGLNFGSHFHRSPEVYVPEDTTTFLGVIGKSLAESNPELTAYMpIYVLEALKNNVIPHVYIPHTIC
2196 KNISYNLLVATQEAFNLDFGTRLHKSPETYVPDDMFLALIFKAFSESSTELTPYIWKYMLDGLENDVIPQEHIPTVVC 2418 GLSYWIPNLYQHVYLADDEEGPENISHIFRILIRLSVRETDPKAVYMQYVWLLLLDDGRLTDIIVDEVINHALERDSENR
2276 SLSYWVPNLYEHVYLANDEEGPEAISRIIYSLIRLTVKEPNFTTAYLQQIWPLLALDGRLTNVIVEEIVSHALDRDSENR 2498 DWKKTISLLTVLPTTEVANNIIQKILAKIRSFLPSLKLEANTQSWSELTILVKISIHVPFETSLLVQMYLPEILFIVSLL 2356 DWNKAVSILTSFPTTEIACQVIEKLINMIKSFLPSLAVEASAHSWSELTILSKISVSIFFESPLLSQNYLPEILPAVSLL 2578 IDVGPRELRSSLHQLLMNVCHSLAINSALPQDHRNNLDEISDIFAHQKVKPMFGFSEDKGRILQIFSASSFASKFNILDP
2436 IDVGPSEIRVSLYELLMNVCHSLTNNESLPERNRKNLDIVCATFARQKLNPISGFSQEKGRVLPNFAASSPSSKFGTLDL 2658 PINNILLLNEYSSTYEANVWKTRYKKYVLESVFTSNSFLSARSIMIVGIMGKSYITEGLCKAMLIETMKVIAEPKITDEH 2516 FTKNIMLLMEYGSISEGAQWEAKYKKYLMDAIFGHRSFFSARAKPILGIMSKSHTSLFLCKELLVETMKVFAEPVVDDEQ 2738 LPLAISHIFTYSKIVEGLDPNLDLMKHLFPFSTLFLESRHPIIPEGALLFVSNCIRRLYMAQPENESET-SLISTLLKGR 2596 HFIIIAHVFTYSKIVEGLDPSSELMKELPWLATICVESPHPLLFEGGLLFPVNCLKRLYTVHLQLGFDGKSLAKKLMESR 2817 KFAHTFLSKIENLSGIVWNEDNFTHILIFIINKGLSNPFIKSTAFDFLKMMPFRNSYFEHQINQKSDHYLCYMFLLYFVLN
2676 NFAATLLAKLESYNGCIWNEDNFPHIILGFIANGLSIPVVKGAALDCLQALFKNTYYERKSNPKSSDYLCYLFLLHLVLS 2897 CNQFEELLGDVDFEGEMVNIENKNTIPKILLEWLSSDNENANITLYQGAILFKCSVTDEPSRFRPALIIRHLLTKKPICA 2756 PEQLSTLLLEVGFEDELVPLNNTLKVPLTLINWLSSDSDKSNIVLYQGALLFSCVMSDEPCKFRFALLMRYLLKVNPICV
2977 LRFYSVIRNEIRKISAFEQNSDCVPLAFDILNLLVTIISESNSLEKLHEESIERLTKRGLSIVTSSGIFAKNSDMMIPLDV 2836 FRFYTLTRKEFRRLSTLEQSSEAVAVSFELIGMLVTIISEFNYLEEFNDEMVELLKKRGLSVVKPLDIFDQEHIEKLKGEG
3057 KPED-IYERKRIMTMILSRHSCSA 2916 EHQVAIYERKRLATHILARNSCS FIG. 2-Continued.
shock sensitivity phenotype of iral. To determine whether the ira2 mutation would suppress the growth deficiency of the cdc25 mutation, KT27-1D (ira2::HIS3) was crossed with KT18-7B (cdc25::LEU2) carrying plasmid pKT5 (CDC25TRPI). A diploid clone, which lost pKT5, was selected and subjected to tetrad analysis. Of 45 asci, 1 ascus showed a 4 viable:O nonviable segregation pattern; 25 asci showed a 3 viable:1 nonviable pattern; and 19 asci showed a 2 viable:2 nonviable pattern. All of the Leu+ spores were His'. They showed tiny colonies and were heat shock resistant, indicating that the ira2 and cdc25 mutations are mutual suppressors, although the suppression of cdc25 by ira2 is weak. It was shown that lethality due to the rasi ras2 or cyri mutation could not be suppressed by the iral mutation (29).
Whether this is the case with ira2 was tested. KMY39-6D (rasl:: URA3 ira2: :HIS3) was crossed with KMY46-4D (ras2::LEU2) to obtain KT50, which was subsequently sporulated and dissected. Of 89 asci dissected, no Ura+ Leu+ His- nor Ura+ Leu+ His' spores were obtained (Table 2). Thus, the ira2 mutation cannot suppress the lethality of the rasi ras2 mutation. Also, the inability of the ras2 strain to grow on nonfermentable carbon sources was not suppressed by the ira2 mutation. In addition, the ira2 mutation, as well as iral, could not suppress the lethality of the cyrl:: URA3 mutation (data not shown). Phenotypes of the iral mutant were suppressible by the ras2 mutation but not by the rasi mutation (29). Segregants of KT50 were analyzed to determine whether rasi or ras2
NEGATIVE REGULATOR OF YEAST RAS
VOL. 10, 1990
4309
IRAl 1451 DLNQFPVSLRHKISILNE-NVIIALTNLSN-ANVNVSLKFTLPMGYSPNK *+
GAP
:
++:
+:
+ ++.
+
+
:+::
647 DINRFEITLSNKTKKSKDPDILFMRCQLSRLQKGHATDEWFLLSSHIPLK : + + +
:
:
:+ ++
+::
..
:+
4.6.
IRA2 1597 DRDNYPVFLRHKMSVLND-NVILSLTNLSN-TNVDASLQFTLPMGYSGNR IRAl 1499 DIRIAFLRVFIDIVTNYPVNPEKHEMDK4LAIDDFLKYIIKNPILAFFGS :+
GAP
+
::
+
:+++
:+
+:
:+
+4.
+
697 GIEPGSLR----VRARYSMEKIMPEEE----YSEFKELILQKELHVVYAL :+
4.4.:
:
*+
.4.4.
:
++
IRA2 1645 NIRNAFLEVFINIVTNYRTYTAKTDLGKLEAADKFLRYTIEHPQLSSFGA
IRAl 1549 LACSPADVDLYAGGFLNAFDTRNASHILVTELLKQEIKRAARSDDILRRN + :
GAP
: :
: :+
:
.:+
:
+
+:
739 SHVCGQDRTLLASILLKIFLHERLESLLLCTLNDREISMEDEATTLFRAT +
:.
:.
+
+
+4.4:
::
+:
+:
IRA2 1695 AVCPASDIDAYAAGLINAFETRNATHIVVAQLIKNEIEKSSRPTDILRRN
IRAl 1599 SCATRALSLYTRSRGNKYLIKTLRPVLQGIVDNKESFEIDKMK-PGSENS +
GAP
+ +
:
*64.4.+
4..4
4.
:.+
:4:
:+
:
:
789 TLASTLMEQSMKATATQFVHHALKDSILRIMESKQSCELSPSKLEKNEDV +
:.
4.4
+
+
+++
++:
+
+ :++
++
:+
:
:
IRA2 1745 SCATRSLSMLARSKGNEYLIRTLQPLLKKIIQNRDFFEIEKLK-PEDSDA
IRAl 1648 EKMLDLFEKYMTRLIDAITSSIDDFPIELVDICKTIYNAASVNFPEYAYI +
GAP
+4.
:
:++
:
+ +
:
+
::
+
+:
+ +
839 NTNLAHLLNILSELVEKIFMASEILPPTLRYIYGCLQKSVQHKWPTNTTM + ++
+
+
::+:
+
+
:
4.
:
+
IRA2 1794 ERQIELFVKYNNELLESISNSVSYFPPPLFYICQNIYKVACEKFPDHAII
IRAl 1698 ---AVGSFVFLRFIGPALVSPDSENIIIVTHAH-DRKPFITL-AKVIQSL :+.:::::
GAP
: ::+4
:::
+ +
++ +:::
+
889 RTRVVSGFVFLRLICPAILNPRMFNIISDSPSPIAART-LTLVAKSVQNL +4.: .::
++
:
:::.
:++.
:+:
IRA2 1844 ---AAGSFVFLRFFCPALVSPDSENIIDISHLS-EKRTFISL-AKVIQNI
IRAl 1743 ANGRENIFKKDILVSKEEFLKTCSDKIFNFLSELCKIP
GAP
938 ANLVEFGAKEPYMEGVNPFIKSNKHRMIMFLDELGNVP :::++ : ++
:+:
:+
::
::
+
IRA2 1889 ANGSENFSRWPALCSQKDFLKECSDRIFRFLAELCR-T FIG. 3. Amino acid sequence homology among IRA], GAP (bovine), and IRA2. Colons between amino acids indicate identities. Plus signs represent conservative amino acid substitutions, which are grouped as follows: A, G, P, S, T; L, I, V, M; D, E, N, Q; K, R, H; F, Y, W; and C.
IRA2 1
Complementation of ira2 1
[1451-2255JIRA2 I
_+
2 [1609-2255]m.A2
+
3 [1753-2188]IRA2 4 [1804-2255]IRA2 5 [1451-2026]IRA2 |
6 [1451-1991]IRA2 1 7 [1451-1908]IRA2 1
E
-
+
*
_
+
FIG. 4. Correspondence of the essential domain of IRA2 to the region homologous with mammalian GAP. Plasmids carrying various restriction fragments of the IRA2 gene under control of the GAPDH gene promoter were transformed into KT27-2B (ira2: :HIS3) to test their ability to complement the heat shock sensitivity phenotype of the ira2 mutation. Expressed truncated proteins are shown on the left. The hatched region (1597 to 1925) is homologous with mammalian GAP (see Fig. 3).
4310
TANAKA ET AL.
MOL. CELL. BIOL.
2
1
3
4 xif". t:,:..
A.,I i.'-
..
n, i.:
VI6.-! l.'- - '
M."!
-,
H
L
HS N FIG. 5. Additive phenotype of iral and ira2 mutants. Tetrad segregants obtained from an ascus of KT27 were tested for histidine auxotrophy (-H), leucine auxotrophy (-L), heat shock sensitivity (HS), and nitrogen starvation (-N). 1, KT27-7A (wild type); 2, KT27-7B (iral::LEU2-a); 3, KT27-7C (iral::LEU2-a ira2::HIS3); 4, KT27-7D (ira2::HIS3).
would suppress ira2. The heat shock sensitivity phenotype of the ira2 mutant was suppressed by the ras2 mutation but not by the rasi mutation. The sporulation deficiency of ira2 was suppressed efficiently by the ras2 mutation but only weakly by the rasi mutation. These results indicate that IRA2 negatively regulates the RAS-cAMP pathway through RAS as does IRA1. ira2 is allelic to gkc4. To map the chromosomal location of the IRA2 gene, an IRA2 probe was hybridized to a Southern blot of intact chromosomal DNA separated by orthogonal field alternation gel electrophoresis. The results indicated that the IRA2 gene is located on chromosome XV (data not shown). It was previously reported that the iral mutation is allelic to the glcl mutation (18), which is deficient in glycogen accumulation. This result is consistent with the report that the RAS-cAMP pathway negatively regulates the storage of this carbohydrate (32). Since another mutation deficient in glycogen accumulation, glc4, maps 15 centimorgans distal to the argi locus on the left arm of chromosome XV (19) and since glc4 showed heat shock-sensitive and sporulation-defective (Spo-) phenotypes, ira2 linkage to argi was
tested. KT69-1B (IRA2 argi his3) was crossed with KT271D (ira2::HIS3 ARGI his3) to form a diploid which was subsequently sporulated and dissected. Of 52 tetrads obtained, segregation patterns of the His and Arg phenotypes showed a parental ditype/tetratype/nonparental ditype ratio of 41:11:0; the calculated map distance between ira2::HIS3 and argi was 11 centimorgans. Next, a glc4 mutant, KMY305-5C, was crossed with two strains isogenic except for the IRA2 locus, KT27-2A (IRA2) and KT27-2B (ira2::HIS3), to obtain KT65 to KT66, respectively. KT65 showed a wild-type phenotype with regard to heat shock resistance, sporulation efficiency, and glycogen accumulation. In contrast, KT66 was deficient in all of these phenotypes. Thus, we concluded that ira2 is allelic to g1c4. IRA2 regulates the intracellular cAMP level through RAS2. We examined the effect of the ira2 mutation on the intra-
1
2
3.14
1
2
3
A 100 cis
10 U, O
.O'
B
1 0.1
Time (min) FIG. 6.
Sensitivity
to
heat shock.
Exponentially growing cells
of KT27-13A (0; iral::LEU2-a ira2::HIS3), KT27-13B (0; ira2:: HIS3), KT27-13C (O; iral::LEU2-a), and KT27-7D (U; wild type) at 25°C were tested for heat shock as described in Materials and Methods.
FIG. 7. Suppression of the heat shock sensitivity of ira mutants by overexpression of IRA genes. KT27-2D (iral::LEU2-a; A) or KT27-2B (ira2::HIS3; B) was transformed by IRA] plasmid pdl3 (1), IRA2 plasmid pd46 (2), and control plasmid YEp24 (3). Two transformants were subjected to heat shock at 52°C for 20 min, and then the plate was incubated at 30°C for 4 days.
NEGATIVE REGULATOR OF YEAST RAS
VOL. 10, 1990
TABLE 2. Failure of the ira2 mutation to suppress the lethality of the rasl ras2 mutation No. of spores
Genotypea
Viable
Nonviableb
51 21 39 59 52 51 0 0
0 0 5 3 1 2 30 42
RAS] RAS2 IRA2 RAS] RAS2 ira2 RAS] ras2 IRA2 RASI ras2 ira2 rasl RAS2 IRA2 rasl RAS2 ira2 rasi ras2 IRA2 rasi ras2 ira2
a Genotype of each viable spore was assigned by the auxotrophic marker. Eighty-nine asci of KT50 (rasl::URA31RASl RAS21ras2::LEU2 ira2::HIS31
IRA2) were analyzed. b Genotypes of nonviable spores were inferred form the genotypes of their sister spore clones.
cellular cAMP level. KT31-1A (ira2::HIS3), KT31-1C (wild type), and KT31-1D (ira2::HIS3 ras2:: URA3) were grown to stationary phase. Cells were stimulated by glucose addition, and the intracellular cAMP level was measured as described in Materials and Methods. cAMP levels in the ira2 mutant increased upon glucose addition and remained at an elevated level, as in the iral mutant (29) (Fig. 8). Moreover, this high level of cAMP was suppressed by the ras2 mutation; the ira2 ras2 cells showed a wild-type pattern of cAMP formation. Thus, IRA2 regulates the intracellular level of cAMP by controlling RAS2 activity as in the case of IRA].
4311
DISCUSSION In this work, we have shown that a homolog of the IRA] gene, IRA2, functions through RAS as a negative regulator of the RAS-cAMP pathway in S. cerevisiae. Disruption of the IRA] or IRA2 gene resulted in sensitivity to heat shock and nitrogen starvation, and the double mutant had the additive phenotype. However, the IRA] or IRA2 gene on a multicopy plasmid could not suppress completely the disruption mutation of the counterpart IRA gene, suggesting that their functions are similar but not the same. What is the functional difference between IRA] and IRA2? Although the ras2 mutation clearly suppressed the Spo- and heat shock sensitivity phenotypes of both iral and ira2 mutations, the rasi mutation could not suppress the phenotypes of iral and only weakly suppressed the Spo- phenotype of ira2. Moreover, rasi suppressed the heat shock sensitivity of ira2 in some genetic backgrounds in which iral could not be suppressed by rasi (data not shown). These results suggest that the phenotypes of ira2 were caused by the activation of RAS2 and RAS] proteins, although the contribution by RAS] activation is lower than that of RAS2 activation, possibly because of weaker expression of RAS] than RAS2 (3). In contrast, the phenotypes of iral were caused by the activation of only the RAS2 protein. Thus, some preference appears to exist between RAS and IRA proteins. This speculation is supported by guanine nucleotide analyses recently reported (30). When RAS] or RAS2 protein was overexpressed in the iral mutant, the percentage of the GTP-bound form of RAS] protein was similar to that of the
B
A
200 C1
a.
U
