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CDC42 and Three Newly Identified Genes Including the Ras-Related

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CDC42 and Three Newly Identified Genes Including the Ras-Related 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Communicated by Leland Hartwell, July 31, 1989 ABSTRACT Genes CDC24, CDC42, and CDC43 are re- Johnson, and J.R.P., unpublished data), and overproduction quired for the establishment ofcell polarity and the localization of the CDC42 product can produce a mislocalization of of secretion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seen in some cdc24 mutants (D. these genes fail to form buds and display isotropic expansion of Johnson and J.R.P., unpublished data).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 To identify other genes that may be involved CDC42 (D. Johnson and J.R.P., unpublished data) revealed in these processes, we screened yeast genomic DNA libraries for that it is a member of the rho family (11) of ras oncogene- heterologous genes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 complemen- tation. 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).
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