Mot Gen Genet (1992) 235:285-~291 © Springer-Vertag 1992 SCM4, a gene that suppresses mutant cdc4 function in budding yeast Simon A. Smith ~, Parveen Kumar ~, Irving Johnston 2, and John Rosamond ~ ~ Department of Biochemistryand Molecular Biology,Universityof Manchester, Oxford Road, Manchester M13 9PT, UK a Department of Biochemistry, University College, London, Gower Street, London UK ReceivedApril 13, 1992 / Accepted May 25, 1992 Summary. The gene SCM4 encodes a protein which sup- presses a temperature-sensitive allele of the cell division cycle gene CDC4 in Saecharomyces cerevisiae. SCM4 was cloned on a 1.8 kb BamHI fragment of yeast geno- mic DNA in the high copy-number vector pJDB207, which results in a 50- to 100-fold increase in the level of the 700 nucleotide SCM4 transcript in vivo. The SCM4 gene encodes a 20.2 kDa protein of 187 amino- acids with a clear tripartite domain structure in which a region rich in charged residues separates two domains of largely uncharged amino acids. Although the appar- ent allele specificity of cdc4 suppression suggests that the CDC4 and SCM4 proteins interact, disruption of SCM4 demonstrates that the gene product is not essen- tial for mitosis or meiosis; however, it may be a member of a family of related, functionally redundant proteins. Key words: Saccharomyces cet~visiae .... Cell cycle - CDC4- Suppression Introduction Classical and molecular genetic analysis of mitotic cell division in the budding yeast Saccharomyces cerevisiae has identified a major control point, termed ' Start', that operates late in the G1 phase of the cell cycle (revie~ved in Wheals 1987). Progress from "Start" to S phase re- quires the function of a number of cell division cycle (CDC) genes including CDC4 (Byers and Goetsch 1974, 1975). Cells carrying a conditional-lethal, temperature- sensitive mutation in CDC4 arrest division at the restric- tive temperature with duplicated spindle pole bodies ly- ing adjacent to each other in the nuclear membrane and connected by a bridge structure (Byers and Goetsch 1974). DNA synthesis does not occur in these mutants under restrictive conditions, although the cells continue to undergo periodic budding after arrest, leading to a Offprint requests to: J. Rosamond characteristic terminal phenotype in which multiple, long, thin anucleate buds extend from a single mother cell. In addition, CDC4 function is also needed for kar- yogamy after conjugation (Dutcher and Hartwell 1982) and at various times during sporulation (Simchen and Hirschberg 1977). The CDC4 gene has been cloned and sequenced (Yo- chem and Byers 1987); the gene encodes a 779 amino acid protein, the predicted sequence of which has homol- ogy both to the protein product of the v-ets and to the /%subunit of transducin (Peterson et al. 1984; Fong et al. 1986). However, interpretation of the functional signifi- cance of these homologies with respect to the activity of the CDC4 protein is complicated by the demonstra- tion that the CDC4 gene product is localised in the yeast nucleus in apparent association with the nucleoskeleton (Choi et al. 1990). In order to understand more fully the role of CDC4 in the mitotic cell division cycle, we have attempted to identify gene products with which the CDC4 protein interacts. The approach that we took was to screen for genes that could suppress mutant cdc4 function when overexpressed from a high copy-number plasmid. Using this strategy, we have isolated a gene, SCM4, which can suppress the mitotic effects of the cdc4-3 mutation when cloned in the vector pJDB207, a derivative of pJDB219 (Beggs 1978). Here we describe the isolation and characterisation of SCM4. Materials and methods Strains and media. Escherichia coli strain HW87 (F- A (araD139-leu) lacX74 galK hsdR rpsL srb recA) was used as host for the routine maintenance and propagation of plasmids (Patterson et al. 1986); E. coli strain JM103 (Alac-pro thi strA supE endA sbcBt5 hsdR4 )UtraD36 proAB laclqZAM15) was used for the maintenance and propagation of MI 3 recombinant phage (Messing et al, 1981). Bacterial cultures were grown in L broth or sup- plemented M9 media (Miller 1972); when necessary, am- picillin was added to a final concentration of 50 ~g/ml.