Characterization of the Saccharomyces cerevisiae cdc42 ‐1 ts Allele and New Temperature‐Conditional‐Lethal cdc42 Alleles

Abstract Cdc42p is a highly conserved GTPase involved in controlling cell polarity and polarizing the actin cytoskeleton. The CDC42 gene was first identified by the temperature‐sensitive cell‐division‐cycle mutant cdc42 ‐1 ts in Saccharomyces cerevisiae . We have determined the DNA and predicted amino‐acid sequence of the cdc42 ‐1 ts allele and identified multiple mutations in the coding region and 5′ promoter region, thereby limiting its usefulness in genetic screens. Therefore, we generated additional temperature‐conditional‐lethal alleles in highly conserved amino‐acid residues of both S. cerevisiae and Schizosaccharomyces pombe Cdc42p. The cdc42 W97R temperature‐sensitive allele in S. cerevisiae displayed the same cell‐division‐cycle arrest phenotype (large, round unbudded cells) as the cdc42 ‐1 ts mutant. However, it exhibited a bud‐site selection defect and abnormal bud morphologies at the permissive temperature of 23°C. These phenotypes suggest that Cdc42p functions in bud‐site selection early in the morphogenetic process and also in polarizing growth patterns leading to proper bud morphogenesis later in the process. In S. pombe , the cdc42 W97R mutant displayed a cold‐sensitive, loss‐of‐function phenotype when expressed from the thiamine‐repressible nmt1 promoter under repressing conditions. In addition, cdc42 T58A and cdc42 S71P mutants showed a temperature‐sensitive loss‐of‐function phenotype when expressed in S. pombe ; these mutants did not display a conditional phenotype when expressed in S. cerevisiae . These new conditional‐lethal cdc42 alleles will be important reagents for the further dissection of the cell polarity pathway in both yeasts. © 1997 John Wiley & Sons, Ltd.