Identification of functional connections between calmodulin and the yeast actin cytoskeleton.
One of four intragenic complementing groups of temperature-sensitive yeast calmodulin mutations, cmd1A, results in a characteristic functional defect in actin organization. We report here that among the complementing mutations, a representative cmd1A mutation (cmd1-226: F92A) is synthetically lethal with a mutation in MYO2 that encodes a class V unconventional myosin with calmodulin-binding domains. Gel overlay assay shows that a mutant calmodulin with the F92A alteration has severely reduced binding affinity to a GST-Myo2p fusion protein. Random replacement and site-directed mutagenesis at position 92 of calmodulin indicate that hydrophobic and aromatic residues are allowed at this position, suggesting an importance of hydrophobic interaction between calmodulin and Myo2p. To analyze other components involved in actin organization through calmodulin, we isolated and characterized mutations that show synthetic lethal interaction with cmd1-226; these "cax" mutants fell into five complementation groups. Interestingly, all the mutations themselves affect actin organization. Unlike cax2, cax3, cax4, and cax5 mutations, cax1 shows allele-specific synthetic lethality with the cmd1A allele. CAX1 is identical to ANP1/GEM3/MCD2, which is involved in protein glycosylation. CAX4 is identical to the ORF YGR036c, and CAX5 is identical to MNN10/SLC2/BED1. We discuss possible roles for Cax proteins in the regulation of the actin cytoskeleton.
Sekiya-Kawasaki
M
M
Department of Biological Sciences, Graduate School of Science, University of Tokyo, Tokyo 113-0033, Japan.
Botstein
D
D
Ohya
Y
Y
D000199:Actins / Q000378:metabolism*; D001483:Base Sequence; D002147:Calmodulin / Q000235:genetics / Q000378:metabolism*; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism*; D004271:DNA, Fungal; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D005804:Genes, Lethal; D005816:Genetic Complementation Test; D016296:Mutagenesis; D009154:Mutation; D018995:Myosin Heavy Chains; D024462:Myosin Type II; D024701:Myosin Type V; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D029702:Schizosaccharomyces pombe Proteins
D000199:Actins; D002147:Calmodulin; D002352:Carrier Proteins; D004271:DNA, Fungal; D005656:Fungal Proteins; C077512:MYO2 protein, S cerevisiae; C434211:MYO2 protein, S pombe; D029701:Saccharomyces cerevisiae Proteins; D029702:Schizosaccharomyces pombe Proteins; D024462:Myosin Type II; D024701:Myosin Type V; D018995:Myosin Heavy Chains