Characterization of the Saccharomyces cerevisiae cdc42-1ts allele and new temperature-conditional-lethal cdc42 alleles.
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 cdc42-1ts in Saccharomyces cerevisiae. We have determined the DNA and predicted amino-acid sequence of the cdc42-1ts 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 cdc42W97R temperature-sensitive allele in S. cerevisiae displayed the same cell-division-cycle arrest phenotype (large, round unbudded cells) as the cdc42-1ts mutant. However, it exhibited a bud-site selection defect and abnormal bud morphologies at the permissive temperature of 23 degrees 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 cdc42W97R mutant displayed a cold-sensitive, los-of-function phenotype when expressed from the thiamine-repressible nmt1 promoter under repressing conditions. In addition, cdc42T58A and cdc42S71P 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.
Miller
P J
PJ
Department of Microbiology and Molecular Genetics, Markey Center for Molecular Genetics, University of Vermont, Burlington 05405, USA.
Johnson
D I
DI
D000483:Alleles; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D018797:Cell Cycle Proteins / Q000235:genetics*; D005656:Fungal Proteins / Q000235:genetics*; D019204:GTP-Binding Proteins / Q000235:genetics*; D018628:Gene Dosage; D005804:Genes, Lethal; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D010641:Phenotype; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D012568:Schizosaccharomyces / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D013696:Temperature; D020846:cdc42 GTP-Binding Protein, Saccharomyces cerevisiae
D018797:Cell Cycle Proteins; D005656:Fungal Proteins; D019204:GTP-Binding Proteins; D020846:cdc42 GTP-Binding Protein, Saccharomyces cerevisiae