CDC42 and Three Newly Identified Genes Including the Ras-Related

Home , CDC42

Proc. Natl. Acad. Sci. USA Vol. 86, pp. 9976-9980, December 1989 Genetics Multicopy suppression of the cdc24 budding defect in yeast by CDC42 and three newly identified genes including the ras-related gene RSRI (cell polarity/cell cycle/morphogenesis/Saccharomyces cerevisiae/guanine nucleotide-binding protein) ALAN BENDER AND JOHN R. PRINGLE Department of Biology, The University of Michigan, Ann Arbor, MI 48109 Communicated by Leland Hartwell, July 31, 1989

ABSTRACT Genes CDC24, CDC42, and CDC43 are re- Johnson, and J.R.P., unpublished data), and overproduction quired for the establishment ofcell polarity and the localization of the CDC42 product can produce a mislocalization of of secretion in Saccharomyces cerevisiae; mutants defective in budding sites like that seen in some cdc24 mutants (D. these genes fail to form buds and display isotropic expansion of Johnson and J.R.P., unpublished data). Sequencing of the cell surface. To identify other genes that may be involved CDC42 (D. Johnson and J.R.P., unpublished data) revealed in these processes, we screened yeast genomic DNA libraries for that it is a member of the rho family (11) of ras oncogene- heterologous genes that, when overexpressed from a plasmid, related genes and encodes typical domains for GTP binding can suppress a temperature-sensitive cdc24 mutation. We and hydrolysis. Moreover, its C-terminal sequence suggests identified four such genes. One of these proved to be CDC42, that the CDC42 product, like the ras products, may be which has previously been shown to be a member of the rho modified and thence membrane-associated. The available (ras-homologous) family of genes, and a second is a newly observations suggest a tentative model in which the products identified ras-related gene that we named RSR1. RSR1 maps of CDC24, CDC42, and related genes may mark the budding between CDC62 and ADE3 on the right arm of chromosome site and provide orientational signals to elements of the VII; its predicted product is 50% identical to other proteins cytoskeleton. in the ras family. Deletion ofRSRI is nonlethal but disrupts the Testing and amplifying this model will require identifica- normal pattern of bud site selection. Although both CDC42 and RSRI can suppress cdc24 and both appear to encode GTP- tion of other genes whose products interact with those of binding proteins, these genes do not themselves appear to be CDC24 and CDC42. We report here the identification of four functionally interchangeable. However, one of the other genes genes* that, when overexpressed, can suppress a Ts- mu- that was isolated by virtue of its ability to suppress cdc24 can tation in CDC24. This approach was suggested by the ob- also suppress cdc42. This gene, named MSBI, maps between servation that such "multicopy suppression" is observed not ADE9 and HIS3 on the right arm of chromosome XV. infrequently during attempts to clone genes by complementation. In at least some cases, it seems clear that the genes so The cell-division cycle of the yeast Saccharomyces cerevi- identified are indeed related in function to the gene harboring siae involves a sequence of morphogenetic events including the original mutation [AAS3/aasl or aas2 (12), STE5/ste4 the selection of a nonrandom budding site and the subsequent (13), CDCII/cdcl2 (2), SIR31sir4 (14), suc1+/cdc2- (15), polarization of secretion and localization of new cell-wall SCGJ/sst2 (16), SEC4/secl5 (17), cdc2+/cdc13- (18), and deposition to the bud (1, 2). The product of gene CDC24 avtA+, tyrB+, and alaB+/alaA- (19)]. seems centrally involved in these morphogenetic processes. Under appropriate conditions, mutants carrying various tem- MATERIALS AND METHODS perature-sensitive (Ts-) cdc24 alleles (i) form apparently normal buds but at abnormal positions; (ii) form buds of Strains and Plasmids. The yeast strains used are listed in abnormal shape; or (iii) fail to bud and display an apparently Table 1. The YEp24 library (25) and the YCp50 library (26) complete delocalization of secretion and cell-surface depo- contain yeast Sau3A genomic DNA fragments inserted into sition (1, 3, 4). Moreover, the failure to bud at restrictive the BamHI sites of YEp24 (a high-copy-number plasmid temperatures is preceded by a failure to localize either actin containing the URA3 selectable marker and the 2-,m-plas- or the CDC3 and CDCIO products to the presumptive bud- mid origin of replication) and YCp5O (a low-copy-number ding site (ref. 5; H. Kim, B. Haarer, and J.R.P., unpublished plasmid containing URA3, CEN4, and the ARSI origin of data). replication). pSL113 is YEp13 [a high-copy-number plasmid Although the original cdc24 mutants were isolated as Ts- containing the LEU2 selectable marker and the 2-,m-plasmid lethal morphogenetic mutants (1, 6), another cdc24 mutant origin of replication (20)] with an inversion of the Xho I-Sal was found among a collection of Ca2'-sensitive mutants (7). I fragment and was provided by G. Sprague (University of This observation and the presence of putative Ca2+-binding Oregon, Eugene). YEp1O3(CDC24) contains CDC24 in a sites in the predicted CDC24 amino acid sequence (8) suggest YEp24 derivative (27). YEp24(CDC43-18-4) contains CDC43 that the CDC24 product interacts with Ca2 , a hypothesis in YEp24 (C. Jacobs, D. Johnson, and J.R.P., unpublished that is attractive because of the association between Ca2+ data). gradients or currents and cell polarization in a variety of cell Media and Growth Conditions. Standard rich (YPD) and types (9, 10). defined (SC) media were used (28). Other media were SC-U Ts- mutations in CDC42 and CDC43 yield phenotypes (as SC but lacking uracil), SC-L (as SC but lacking leucine), similar to those of cdc24 mutants (ref. 2; A. Adams, D. and SC+Sorb, SC-U+Sorb, and SC-L+Sorb (as SC,

The publication costs of this article were defrayed in part by page charge Abbreviation: Ts-, temperature-sensitive. payment. This article must therefore be hereby marked "advertisement" *The sequence reported in this paper has been deposited in the in accordance with 18 U.S.C. §1734 solely to indicate this fact. GenBank data base (accession no. M26928).

9976 Downloaded by guest on September 24, 2021 Genetics: Bender and Pringle Proc. Natl. Acad. Sci. USA 86 (1989) 9977 Table 1. Yeast strains used in this study Strain Genotype Source (ref.) Y51 MATa cdc244 met] Segregant from PTRD5-BD1-lA* x DC5 (20) Y147 MATa cdc24-4 ura3 leu2-3,112 his3 Segregant from Y51 x SY1263 (21) Y146 MATa cdc244 ura3 Segregant from Y51 x SY1263 (21) DJTD2-16D MATa cdc42-1 ura3 leu2 trpl his4 (D. Johnson and J.R.P., unpublished data) TD4[YIp5(CDC42)-3] MATa/CDC42:: URA3::CDC42 ura3 leu2 trpl his4 (D. Johnson and J.R.P., unpublished data) Y246 MATa/MATa ura3/ura3 leu2-3,112/leu2-3,112 his3/HIS3 This workt Y276 MA Ta/MATa rsrl:: URA3/RSRl ura3/ura3 rsrl:: URA3 derivative of Y246 leu2-3,112/leu2-3,112 his3/HIS3 Y301 MATa rsrl:: URA3 ura3 leu2-3,112 his3 Segregant from Y276 Y124 MATa ura3 leu2-3,112 Segregant from SY1229 (21) x C276-4B (22) Y300 MATa rsrl:: URA3 ura3 leu2-3,112 Segregant from Y276 Y251 MATa ade2 ade3 ura3 Ieu2 trpl his7 This workt Y355 MATa rsrl:: URA3 ade3 ura3 leu2 trpl Segregant from Y251 x Y300 MP62 MATa cdc62-1 ura3 Ieu2 his3 (23) Y270 MATa msbl::LEU2 ura3 leu2-3,112 his3 This work§ 855 MATa ade9 leu2-3 lys2 (24) *A MATa cdc244 met] strain derived by a series of crosses from JPT19 (ref. 1; D. Johnson, personal communication). tThe diploid formed by mating SY1229 (21) to a segregant from the cross of SY1229 to C2764B (22). tA segregant from 4795-303 x 4795-408 (both strains obtained from L. Hartwell, University of Washington, Seattle). §A segregant from Y246 that had been transformed with msbl ::LEU2 (see text). SC-U, and SC-L, respectively, but containing 1 M sorbi- fragments from one or another of four discrete genomic tol). regions (Fig. 1), none of which contained CDC24 itself. One Yeast Transformations and Suppression Assays. To isolate of these fragments proved to contain the known gene CDC42 genes that could suppress a cdc24 mutation, strain Y147 was (see below), but the others appear to contain newly identified transformed by the spheroplast method (28); transformation genes that we provisionally call RSRJ, MSBI, and MSB2, as plates (SC-U+Sorb) were incubated for 2 days at 20-24°C described below. and then shifted to 36°C. To retest the suppression ability of The 26 transformants that did grow when restreaked at isolated plasmids, yeast transformation plates were incu- 36°C on SC- U medium presumably included those harboring bated at 20-24°C until colonies appeared. Individual trans- authentic CDC24-containing plasmids, but were not investi- formants were then streaked at 20-24°C on SC-U or SC-L, gated further. The relative rarity of CDC24-containing plas- and cells from single colonies were then streaked at restric- mids (especially in the case of the YEp24 library) presumably tive temperature (36°C) on the appropriate medium. reflects the toxicity of CDC24 overexpression, as noted DNA Sequencing. DNA was sequenced by the dideoxy previously (27). chain-termination method (29). Both strands were com- Interactions Between CDC24 and CDC42. The class of DNA pletely sequenced, and all sites used for cloning were over- fragment we isolated most often, and the one that suppressed lapped by other cloned segments. a cdc24 mutation the best, had a restriction pattern indistin- DNA-DNA Blot Hybridization. After separation by electro- guishable from that of CDC42, another gene that is required phoresis, DNA fragments were blotted to nitrocellulose (30). The filter was hybridized to a 32P-labeled DNA probe for 24 Number of times Ability to hr at 42°C in a solution containing 50% (vol/vol) formamide, isolated suppress 5x SSC, 50 mM sodium phosphate (pH 6.5), 200 ,ug of Gene Restriction map YEp24 YCp5O cdc24 cdc42 sonicated salmon sperm DNA per ml, 0.1% NaDodSO4, and library library 5 x Denhardt's reagent (30). The filter was washed for 24 hr xxG~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ at 650C in 2x SSC/0.1% NaDodSO4. CDC42 _E 31 6 +++ ++++ EE E RESULTS SaSpO V Sp Multicopy Suppression ofcdc24. To search for heterologous RSR1 H GHH 13 1 + - genes that can suppress a Ts- cdc24 mutation when overex- E EB G E pressed, we transformed strain Y147 with yeast genomic MSB1 HX H B X GG 3 0 ++ ++ DNA libraries constructed in the high-copy-number plasmid E E E E YEp24 and in the low-copy-number plasmid YCp5O. After H X HHB H selection on SC-U+Sorb plates at 36°C (sorbitol was in- MSB2 1kb 2 0 +/- - cluded to provide osmotic support for spheroplasts during E E E transformation), we obtained 50 Ts' transformants from the FIG. 1. DNA segments that suppress cdc24. See text for expla- YEp24 library and 32 from the YCp5O library. Forty-nine of nation of gene names. The boxed areas of the composite restriction the transformants from the YEp24 library and 7 ofthose from maps indicate the smallest fragments to which the suppressing the YCp5O library failed to grow when restreaked on SC-U activities have been localized. All BamHI (B), EcoRI (E), Bgl 11(G), plates at 36°C but grew when restreaked on SC-U+Sorb HindIII (H), and Xho I (X) sites are shown. Sac I (Sa), Ssp I (Sp), plates at 36°C. We reasoned that the failure of these trans- Cla I (C), and EcoRV (V) sites are shown only for the expanded at region of the RSRI map. Scale bar represents 1 kilobase (kb). The formants to grow in the absence of sorbitol 36°C could be numbers of independent isolates of each gene from the two libraries an indication of incomplete suppression of the cdc24 muta- are indicated. The abilities ofthe genes carried on YEp24 to suppress tion by some other gene. From each of these 56 transfor- cdc24-4 (strain Y147) and cdc42-1 (strain DJTD2-16D) mutations mants, plasmid DNA was recovered into Escherichia coli for were assayed at 36°C on SC-U+Sorb plates (see Materials and further analysis. By restriction analysis and colony hybrid- Methods). These abilities ranged from allowing no detectable growth ization, all of the transformants were found to contain DNA (-) to allowing essentially normal growth (+ + + +). Downloaded by guest on September 24, 2021 9978 Genetics: Bender and Pringle Proc. Natl. Acad. Sci. USA 86 (1989) for bud emergence. These fragments could also complement lished data) and the ability of the MSBJ gene to suppress both the Ts- cdc42-1 mutation, even in the absence of sorbitol. cdc24 and cdc42 mutations (see below). To explore this Colony hybridization using authentic CDC42 DNA as probe possible interaction further, we asked whether the high- indicated that these fragments did in fact contain CDC42 copy-number, CDC24-containing plasmid YEp1O3(CDC24) (data not shown). In addition, we assayed the ability of the could suppress the cdc42 mutation in strain DJTD2-16D. wild-type CDC42 and cdc42-2' [which contains an amber However, no suppression was observed in either the absence mutation at codon 70 (D. Johnson and J.R.P., unpublished or the presence of sorbitol. data)] in YEp24 to suppress cdc24. As expected, only the Characterization of the ras-Related Gene RSR1. To identify wild-type CDC42 could suppress cdc24. the gene responsible for suppression of cdc24 within the Because CDC42 could suppress cdc24 even in the low- second-most-frequently isolated class of DNA fragments, we copy-number vector YCpSO (Fig. 1), it appeared that only one tested the suppression activity of various subclones. This or a few extra copies of CDC42 were required for the activity localized to a 1.0-kb Sac I-EcoRV segment (Fig. 1). suppression. To examine this further, we constructed cdc24 Fig. 2 shows the DNA sequence of the 1.3-kb Sac I-Ssp I strains that contained one or two copies of CDC42 by mating segment that includes this suppressing region. This segment a cdc244 ura3 mutant (strain Y146) to strain TD4[YIp5- contains a sequence that is identical to the coding region of (CDC42)-3], which contains a second complete copy of an alanine tRNA gene (31). It also contains two open reading CDC42 integrated together with URA3 at the CDC42 locus. frames of >60 codons. One of these has 272 codons and is From 10 tetrads dissected, 19 segregants were recovered that indicated in Fig. 2; the second has 111 codons and starts at failed to grow on SC at 36°C and hence carried the cdc244 nucleotide 693 (Fig. 2) on the same strand. mutation. Of these, 11 could grow on SC+Sorb at 36°C. By searching the PIR protein data bank (National Biomed- These 11 segregants were all Ura+, whereas all of the 8 ical Research Foundation Protein Identification Resource, segregants that failed to grow on SC+Sorb at 36°C were release 14.0) with the Molecular Biology Information Re- Ura-. Thus, two copies of CDC42 suffice for the suppression source EUGENE/SAM search package (45), we found that the of cdc244 on sorbitol-containing medium. 272-amino acid (30-kDa) predicted protein is 57% identical These observations suggest that the CDC24 and CDC42 over its first 120 amino acids to the human c-Ha-ras protein gene products interact, a suggestion that is supported also by (32) and 58% identical over the same stretch to the S. the similarities of the cdc24 and cdc42 mutant phenotypes cerevisiae RASI and RAS2 proteins (33) (Fig. 3). We thus call (refs. 1-3 and 5; A. Adams, D. Johnson, and J.R.P., unpub- this gene RSRI, for ras-related. Of the ras-related proteins of which we are aware, the RSRl-predicted protein is most 1 GAGCTCGATCTCGAAAAAAAGAACCATTGCGTTCGTTCTTAACTACGCCAAGAAGTTTTT similar to the human rapla gene product (34); the two 61 GAAATTTCATCCTCTCCACTGAACAATAATACTATTATTTAGTAACGATATAGAACATTT proteins are 65% identical over their first 125 amino acids and 121 ACATACAACGTTCTAATATTTTGGCTTCTATCATCGCTTAGAAATATTTGGCTAGGAAAC 56% identical over the entire length (184 amino acids) of the rapla protein. The RSRI protein is very similar to other 181 ATTAGGACTAATGAGAGACTATAAATTAGTAGTATTGGGTGCTGGTGGTGTCGGTAAATC M R D Y K L V V L G A G G V G K S ras-related proteins in regions known to be important for 241 CTGCTTAACCGTGCAGTTTGTACAGGGAGTTTATTTGGATACGTATGATCCAACGATCGA GTP binding (ref. 35; thin overlines in Fig. 3), putative C L T V Q F V Q G V Y L D T Y D P T I E effector function (refs. 36 and 37; thick overline in Fig. 3), and 301 AGATTCTTACAGGAAAACCATCGAGATCGATAACAAGGTATTCGACCTGGAAATTTTAGA membrane attachment [cysteine near or at the C terminus D S Y R K T I E I D N K V F D L E I L D (38)]. However, the RSRI protein, like the rapla protein, 361 TACAGCGGGTATAGCACAATTTACTGCAATGAGAGAATTATACATAAAGTCAGGAATGGG T A M R E L Y I K S G M G lacks the consensus glutamine at position 61. Like RASI and T A G I A Q F RAS2, RSRI encodes a longer C-terminal region than do 421 GTTCCTGTTGGTATATTCAGTAACAGATCGGCAATCTTTGGAAGAATTAATGGAGCTAAG F L L V Y S V T D R Q S L E E L M E L R most ras homologues, but the RSRI product is not similar to 481 AGAACAGGTCCTTAGGATCAAAGATTCCGATCGCGTTCCAATGGTTCTAATAGGTAACAA the RASI or RAS2 proteins in this region. E Q V L R I K D S D R V P M V L I G N K

541 GGCTGATCTAATCAATGAAAGGGTAATAAGTGTGGAAGAAGGTATAGAGGTAAGCAGTAA RSR1 MRDYKLVVLGAGGVGKSCLTVQFVQGVYLDTYDPTIEDSYRKTIEID A D L I N E R V I S V E E G I E V S S K H-ras -TE -----V------A--I-LI-NHFV-E------QVV-- 47 rapla --E ------S------A------IFVEK------QV-V- 601 ATGGGGTAGAGTTCCCTTTTATGAAACAAGCGCTTTGTTGAGGAGCAATGTGGATGAAGT RAS1 MQGNKSTI----I--V-G-----A--I--I-SYFV-E------QVV-- W G R V P FY ET S A L L R S N V D E V RAS2 MPLNKSNI-E-----V-G--D----A--I-LT-SHFV-E------QVV-- 661 GTTCGTTGATTTAGTTAGGCAAATCATTCGGAATGAAATGGAAAGTGTTGCAGTTAAAGA F V D L V R Q I I R N E M E S VA V K D RSR1 NKVFDLEILDTAGIAQFTAMRELYIKSGMGFLLVYSVTDRQSLEELMELRE H-ras GETCL-D ------QEEYS---DQ-MRT-E ---C-FAINNTK-F-DIHQY-- 98 721 CGCAAGAAATCAAAGTCAACAATTTAGCAAAATCGAGTCTCCATCAACCAGGCTCCCTAG rapla CQQCM------TE -----D--M-N-Q--A---- I-AQSTFND-QD--- RN S F S K I E S P S T R L P S A Q Q Q RAS1 D--S I-D------QEEYS----Q-MRT-E------S-N-FD--LSYYQ DE-SI-D------QEEYS----Q-MRN-E------I-SKS--D---TYYQ 781 TTCTGCGAAACAGGATACGAAACAATCAAACAATAAGCAATCATCAAAAGGTTTATATAA RAS2 S A K Q D T K Q S NNK Q S S K G L Y N 841 CAAATCTTCACAAGGACAAGCTAAAGTTAAACAATCTACTCCGGTTAATGAAAAGCACAA RSR1 QVLRIKDSDRVPMVLIGNKADLINERVISVEEGIEVSSKWGRVPFYETSAL K SS Q G Q A K V K Q S T P V N E K H K H-ras -IK-V----D---V-V---C--AA -TVESRQAQDLARSY GI-YI----K 147 rapla -I--V--TED --- I-V---C--ED--- VGK-Q-QNLARQ-CNCA-L-S--K 901 ACCGTCACATGCCGTTCCGAAATCTGGTTCTAGCAACAGGACAGGAATTAGCGCTACTTC RAS1 -IQ-V----YI-V-VV---L--E---QV-Y-D-LRLAKQL NA--L----K P S H A V P K S G S S N R T G I S A T S RAS2 -I--V--T-Y--I-VV --- S--E--KQV-YQD-LNMAKQM NA--L----K 961 ACAACAAAAGAAAAAGAAGAAAAACGCTTCCACTTGCACTATTCTATAGTCACTTAATTT Q Q KK KKKN A S TC T I L RSR1 LRSNVDEVFVDLVRQIIRNEMESVAV (81 aa) SQQKKKKKNASTCTIL H-ras T-QG-EDA-YT---E-RQHKLRKLNP PDESGPGCMSCK-VLS 189 1021 TATTATAAATGAATCAAGATATCAGATAAAAAGACTTTACTTGAAATAGTTTTATTATAG rapla SKI--N-I-Y------N-K TPVE---PKKKS-LL- RAS1 QAI ----A-YS-I-LVRDDGGKYNSM (112aa) -ANAR-ESSGGC-I-C 1081 TTCTAAAAGGTTTAGTTTAAAGTATTAGCATACGTTGTATAAGTTTTTAAAGAAATCAAT RAS2 QAI--E-A-YT-A-LVRDEGGKYNKT (125aa) TSEAS-SGSGGC-I-S 1141 TAATAATGTTTGAAAATAAATTTAAACCCAAAAAAAATGAAATGTTAAAAATATGGACGC FIG. 3. Similarities of the predicted amino acid sequences of the 1201 AACCGGAATCGAACCGATGACCTCTTCCTTGCAAGGGAAGCGCGCTACCAACTGCGCCAT RSRJ, human c-Ha-ras (32), human rapla (34), and S. cerevisiae 1261 GTGCCCGCAATCTATGGGATTTTACGGTAGATGCTGCGTTACGTATAAAAAATATT RASI and RAS2 (33) gene products. The coordinates for the c-Ha-ras product are indicated. Dashes indicate identity with RSRI; blanks FIG. 2. Nucleotide sequence of the RSRI region and the pre- indicate gaps inserted to maximize alignment of the sequences. No dicted amino acid sequence of the RSRI product. The following attempt was made to mark similar amino acids. GTP-binding domains restriction sites (see Fig. 1) are indicated by overlining: Sac I (0), Ssp are indicated by thin overlines. The putative effector region is I (163), Cla I (326), EcoRV (1038), and Ssp 1 (1311). The alanine marked by a thick overline. Amino acid 61 (see text) is marked with tRNA gene (see text) is indicated by underlining. a star. aa, Amino acids. Downloaded by guest on September 24, 2021 Genetics: Bender and Pringle Proc. Natl. Acad. Sci. USA 86 (1989) 9979

1 2 3 4 5 6 Table 2. Linkage data for RSR1 and MSBI Tetrad type Map _ RSR1 Marker pair PD T NPD distance cdc62: rsrl 37 63 0 32 rsrl : ade3 22 73 3 50 cdc62: ade3 21 58 18 rsrl::URA3 ade9: msbl 96 6 0 3 msbl : his3 89 13 0 6 ade9 . his3 83 19 0 9 FIG. 4. DNA-DNA blot hybridizations of strains with and with- out the RSRI disruption. Lanes 1 and 2, a/a strain Y246 before and Tetrad data for RSRI are from a cross between strains Y355 and after transformation with the rsrl:: URA3 fragment (see text); lanes MP62, Ural was scored as rsrl. Tetrad data for MSBJ are from a 3-6, four segregants from a complete tetrad from the transformed cross between strains Y270 and 855. Leu+ was scored as msbl. Map diploid. distances were calculated using Perkins's formula [Xp = 50(T + 6N)/(P + N + T), where P, T, and N are the numbers of PD (parental Confirmation that it is actually RSRI which is responsible ditype), T (tetratype), and NPD (nonparental ditype) tetrads, respectively] and (for the rsrl-ade3 interval) the graphical correction for for the suppression of cdc24 by the RSRI-containing frag- larger map distances (40). ments was provided by the observation (to be described in detail elsewhere) that an in vitro generated A-to-T mutation RSRJ was mapped to the right arm of chromosome VII by at position 238 (Fig. 2) results in a loss of cdc24-suppressing hybridization to a A phage library of mapped yeast genomic activity. This mutation is 455 base pairs (bp) upstream of the DNA inserts (L. Riles and J. Dutchik, personal communica- first ATG of the 111-codon open reading frame but changes tion). RSRJ was then further localized between ADE3 and a lysine codon to an asparagine codon in RSRJ. CDC62 by tetrad analysis using a cross between an rsrl:: The similarities in sequence between CDC42 (D. Johnson URA3 ade3 strain and a cdc62 strain (Table 2). This map and J.R.P., unpublished data) and RSRJ suggested that position is distinct from those of CDC43 and CDC42 (41). RSRJ-containing plasmids might also suppress a cdc42 mu- Preliminary Characterization ofthe GenesMSB1 and MSB2. tation. However, no such suppression was detectable using The other two cdc24-suppressing genes have been less in- a high-copy-number plasmid and sorbitol-containing plates. tensively studied as yet; they are tentatively named MSBI In an attempt to identify the physiological role ofRSRJ, we and MSB2 (multicopy suppression of a budding defect). disrupted it by gene replacement in an a/a diploid (strain Several lines of evidence suggest that neither MSBI nor Y246) by using a fragment of DNA that had the 710-bp Cla MSB2 is allelic to CDC43. First, the authentic CDC43 in the I-EcoRV fragment of RSRI (see Figs. 1 and 2) replaced with high-copy-number plasmid YEp24(CDC43-18-4), which com- a 1.1-kb HindIII-Sma I DNA fragment containing the URA3 plemented a Ts- cdc43 mutation, failed to suppress either a gene (39). In this construction, only 46 N-terminal codons of cdc24 (strain Y147) or a cdc42 (strain DJTD2-16D) mutation RSRI remain. DNA-DNA blot hybridizations confirmed the either in the presence or in the absence of sorbitol at 36°C. success of this construction (Fig. 4). Yeast genomic DNA Second, the restriction maps of the MSBI and MSB2 regions that had been digested with EcoRI and HindIII was probed are clearly distinct from that of CDC43 (D. Johnson and with the 0.9-kb Ssp I-EcoRV fragment of RSRJ DNA (see J.R.P., unpublished data). Third, using an insertion of LEU2 Fig. 1). In the construction of the rsrl:: URA3 disruption, a DNA at the MSBI locus-(to be described in detail elsewhere), HindIII site was introduced adjacent to the Cla I site of we determined that MSBJ maps between ADE9 and HIS3 on RSRJ. Thus, digestion with EcoRI and HindIII and hybrid- the right arm ofchromosome XV (Table 2), a position distinct ization to the Ssp I-EcoRV probe reveals a 3.0-kb fragment from that of CDC43 (41). The multidrug-resistance gene from the RSRI DNA and a 1.0-kb fragment from the also near appears not to be allelic rsr:: URA3 DNA. SMR3, which maps ADE9, to MSBI communication). Interestingly, Segregants containing the disruption were viable and grew (J. Golin, personal not a cdc42 mutation as well as those that contained the intact RSRJ gene. How- MSBJ (but MSB2) could also suppress ever, as judged by Calcofluor staining of chitin rings (3), (Fig. 1). In contrast to the suppression of cdc24, the sup- segregants containing the rsrl:: URA3 disruption displayed a pression of cdc42 was effective even on SC plates lacking randomized pattern of budding sites (Fig. 5), a phenotype sorbitol at 36°C. observed previously in cells carrying some mutant alleles of CDC24 (1) and in cells overexpressing CDC42 (D. Johnson DISCUSSION and J.R.P., unpublished data). The random budding pattern cosegregated with the URA3 marking the RSRJ disruption in In attempts to clone genes by complementation of mutations, seven tetrads from a cross between strains Y301 and Y124 it has often been observed that heterologous sequences are and was also evident in a/a rsrl :: URA3/rsrl:: URA3 dip- isolated in addition to the wild-type version of the mutant loids (data not shown). gene. In many cases, it seems clear that the heterologous genes are related in function to the gene originally marked by mutation (2, 12-19).. Thus, it appears that the "multicopy suppression" approach should be a generally useful tactic for the identification offunctionally related genes in appropriate organisms; this approach has the advantage that the identification and cloning of a new gene ofinterest occur in a single step. In the present study, we employed this approach to search for additional genes involved in bud emergence and the establishment of cell polarity in yeast by seeking heter- FIG. 5. Randomization of bud position due to deletion of RSRJ. ologous sequences that would suppress a Ts- cdc24 muta- An rsrl:: URA3 (A) and an RSR1 (B) segregant from a cross of strains tion. We identified four such sequences, including the known Y301 and Y124 were stained with Calcofluor (3) to reveal the pattern gene CDC42 and the three apparently new genes RSRl, of bud scars. (x 1600.) MSBI, and MSB2. Downloaded by guest on September 24, 2021 9980 Genetics: Bender and Pringle Proc. Nati. Acad. Sci. USA 86 (1989) It is ofcourse possible that any given gene identified in this 1. Sloat, B. F., Adams, A. & Pringle, J. R. (1981) J. Cell Biol. 89, way may suppress for some trivial reason (e.g., by altering 395-405. the intracellular milieu in such a way as to stabilize nonspe- 2. Pringle, J. R., Lillie, S. H., Adams, A. E. M., Jacobs, C. W., cifically a previously thermolabile gene product). Evidence Haarer, B. K., Coleman, K. G., Robinson, J. S., Bloom, L. & Preston, R. A. (1986) in Yeast Cell Biology, UCLA Symposia on against such trivial explanations in the present study is Molecular and Cellular Biology, ed. Hicks, J. B. (Liss, New York), provided by the observations (i) that mutations in CDC42 Vol. 33, pp. 47-80. produce phenotypes very similar to those of cdc24 mutants 3. Sloat, B. F. & Pringle, J. R. (1978) Science 200, 1171-1173. (refs. 1-3 and 5; A. Adams, D. Johnson, and J.R.P., unpub- 4. Field, C. & Schekman, R. (1980) J. Cell Biol. 86, 123-128. lished data); (ii) that RSRI is required for normal bud 5. Adams, A. E. M. & Pringle, J. R. (1984) J. Cell Biol. 98, 934-945. localization; and (iii) that MSBI suppresses both cdc24 and 6. Hartwell, L. H., Culotti, J., Pringle, J. R. & Reid, B. J. (1974) cdc42. Science 183, 46-51. We do not have a comparable argument for MSB2. 7. Ohya, Y., Miyamoto, S., Ohsumi, Y. & Anraku, Y. (1986) J. A noteworthy feature of our results is that, except in one Bacteriol. 165, 28-33. case, multicopy suppression at 360C was observed only in the 8. Miyamoto, S., Ohya, Y., Ohsumi, Y. & Anraku, Y. (1987) Gene 54, presence of 1 M sorbitol. As the cdc24 and cdc42 mutations 125-132. affect cell-wall biogenesis, it is possible that the sorbitol is 9. Kropf, D. L. & Quatrano, R. S. (1987) Planta 171, 158-170. required to stabilize malformed cell walls against lysis. How- 10. Schmid, J. & Harold, F. M. (1988) J. Gen. Microbiol. 134, 2623- ever, we think it is more likely that the presence of sorbitol 2631. a 11. Madaule, P., Axel, R. & Myers, A. M. (1987) Proc. Natl. Acad. Sci. produces partial stabilization of the Ts- cdc24 product, USA 84, 779-783. which is then more susceptible to suppression. This view is 12. Hinnebusch, A. G. & Fink, G. R. (1983) Proc. NatI. Acad. Sci. supported by the observations (i) that many Ts- mutations USA 80, 5374-5378. prove to be osmotic remedial (42); (ii) that a similar case of 13. MacKay, V. L. (1983) Methods Enzymol. 101, 325-343. high-osmolarity-dependent multicopy suppression has been 14. Ivy, J. M., Klar, A. J. S. & Hicks, J. B. (1986) Mol. Cell. Biol. 6, observed with the Schizosaccharomyces pombe DNA ligase 688-702. gene (43), where effects on cell wall stability are unlikely; and 15. Hayles, J., Aves, S. & Nurse, P. (1986) EMBO J. 5, 3373-3379. 16. Dietzel, C. & Kurdan, J. (1987) Cell 50, 1001-1010. (iii) that we also observed multicopy suppression in the 17. Salminen, A. & Novick, P. J. (1987) Cell 49, 527-538. absence of sorbitol at temperatures lower than 360C (unpub- 18. Booher, R. & Beach, D. (1987) EMBO J. 6, 3441-3447. lished data). We suggest that the use of partially ameliorating 19. Berg, C. M., Wang, M., Vartak, N. B. & Liu, L. (1988) Gene 65, conditions (high osmolarity or semipermissive temperatures) 195-202. may often facilitate searches for interacting genes by both 20. Broach, J. R., Strathern, J. N. & Hicks, J. B. (1979) Gene 8, multicopy suppression and conventional suppressor ap- 121-133. proaches. In at least some cases (see also ref. 17), multicopy 21. Bender, A. & Sprague, G. F., Jr. (1989) Genetics 121, 463-476. 22. Wilkinson, L. E. & Pringle, J. R. (1974) Exp. Cell Res. 89, 175-187. suppression was effected by merely a small increase in gene 23. Bedard, D. P., Johnston, G. C. & Singer, R. A. (1981) Curr. Genet. copy number. 4, 205-214. The predicted amino acid sequence and map position of 24. Mortimer, R. K. & Hawthorne, D. C. (1973) Genetics 74, 33-54. RSR1 indicate that the RSRJ product is a member of the 25. Carlson, M. & Botstein, D. (1982) Cell 28, 145-154. rapidly expanding superfamily of GTP-interactive proteins in 26. Rose, M. D., Novick, P., Thomas, J. H., Botstein, D. & Fink, yeast, which now includes at least a dozen known members. G. R. (1987) Gene 60, 237-243. The protein encoded by RSRJ is more similar to the para- 27. Coleman, K. G., Steensma, H. Y., Kaback, D. B. & Pringle, J. R. (1986) Mol. Cell. Biol. 6, 4516-4525. digmatic ras proteins than other known yeast proteins with 28. Sherman, F., Fink, G. R. & Hicks, J. B. (1986) Methods in Yeast the exception of the RAS] and RAS2 products. However, as Genetics (Cold Spring Harbor Lab., Cold Spring Harbor, NY). yet there is no reason to suspect that the physiological roles 29. Sanger, F., Nicklen, S. & Coulson, A. R. (1977) Proc. Natl. Acad. of RSRJ are related to those of RAS] and RAS2. Of known Sci. USA 74, 5463-5467. ras-related genes, RSR1 finds its closest homologue in the 30. Maniatis, T., Fritsch, E. F. & Sambrook, J. (1982) Molecular mammalian gene rapla (34), but the functional significance of Cloning: A Laboratory Manual (Cold Spring Harbor Lab., Cold this similarity is not clear. Interestingly, both RSRJ and Spring Harbor, NY). 31. Drabkin, H. J. & RajBhandary, U. L. (1985) J. Biol. Chem. 260, rapla lack the consensus glutamine at position 61; RSRl 5596-5602. encodes an isoleucine at this position, which in the Ha-ras 32. Capon, D. J., Chen, E. Y., Levinson, A. D., Seeburg, P. H. & protein is a moderately strong activating mutation (44). The Goeddel, D. V. (1983) Nature (London) 302, 33-37. nonlethality of the rsrl deletion suggests that this gene might 33. Powers, S., Kataoka, T., Fasano, O., Goldfarb, M., Strathern, J., be functionally redundant. Broach, J. & Wigler, M. (1984) Cell 36, 607-612. The finding of both CDC42 and RSRJ among the genes 34. Pizon, V., Chardin, P., Lerosey, I., Olofsson, B. & Tavitian, A. capable of suppressing a Ts- cdc24 mutation underscores the (1988) Oncogene 3, 201-204. 35. De Vos, A. M., Tong, L., Milburn, M. V., Matias, P. M., Jancarik, apparent interaction between a putative Ca2l-interactive J., Noguchi, S., Nishimura, S., Miura, K., Ohtsuka, E. & Kim, protein and putative GTP-interactive, membrane-associated S.-H. (1988) Science 239, 888-893. proteins in the development of cell polarity and the estab- 36. Willumsen, B. M., Papageorge, A. G., Kung, H.-F., Bekesi, E., lishment of the budding site. Although extensive speculation Robins, T., Johnsen, M., Vass, W. C. & Lowy, D. R. (1986) Mol. about the nature of these interactions is unwarranted until Cell. Biol. 6, 2646-2654. more is known about other gene products involved in these 37. Sigal, I. S., Gibbs, J. B., D'Alonzo, J. S. & Scolnick, E. M. (1986) processes, it is worth noting that the GTP-binding/hydrolysis Proc. Natl. Acad. Sci. USA 83, 4725-4729. 38. Clarke, S., Vogel, J. P., Deschenes, R. J. & Stock, J. (1988) Proc. motif appears to be a general mechanism for switching Natl. Acad. Sci. USA 85, 4643-4647. proteins between active and inactive forms and that Ca2+ 39. Rose, M., Grisafi, P. & Botstein, D. (1984) Gene 29, 113-124. gradients or currents have been associated with polarized cell 40. Mortimer, R. K. & Schild, D. (1980) Microbiol. Rev. 44, 519-571. growth in a variety of cell types (9, 10). 41. Mortimer, R. K., Schild, D., Contopoulou, C. R. & Kans, J. A. (1989) Yeast 5, 321-403. We thank D. Johnson for strains, plasmids, and valuable discus- 42. Hawthorne, D. C. & Friis, J. (1964) Genetics 50, 829-839. sions; L. Riles and J. Dutchik for mapping RSRJ to the right arm of 43. Johnston, L. H., Barker, D. G. & Nurse, P. (1986) Gene 41, chromosome VII; J. Golin for testing the allelism of SMR3 and 321-325. MSBI; and D. Jenness and G. Johnston for strains. This work was 44. Der, C. J., Finkel, T. & Cooper, G. M. (1986) Cell 44, 167-176. supported by Public Health Service Grant GM31006 and Postdoc- 45. Lawrence, C. B., Goldman, D. A. & Hood, R. H. (1986) Bull. toral Fellowship GM12276. Math. Biol. 48, 569-583. Downloaded by guest on September 24, 2021