The unconventional myosin, Myo2p, is a calmodulin target at sites of cell growth in Saccharomyces cerevisiae.
Myo2p is an unconventional myosin required for polarized growth in Saccharomyces cerevisiae. Four lines of evidence suggest that (a) Myo2p is a target of calmodulin at sites of cell growth, and (b) the interaction between Myo2p and calmodulin is Ca2+ independent. First, as assessed by indirect immunofluorescence, the distributions of Myo2p and calmodulin are nearly indistinguishable throughout the cell cycle. Second, a genetic analysis indicates that mutations in CMD1 show allele-specific synthetic lethality with the myo2-66 conditional mutation. Mutations that inactivate the Ca(2+)-binding sites of calmodulin have little or no effect on strains carrying myo2-66, whereas an allele with a mutation outside the Ca(2+)-binding sites dramatically increases the severity of the phenotype conferred by myo2-66. Third, Myo2p coimmunoprecipitates with calmodulin in the presence of Ca2+ or EGTA. Finally, we used a modified gel overlay assay to demonstrate direct interaction between calmodulin and fusion proteins containing portions of Myo2p. Calmodulin binds specifically to the region of Myo2p containing six tandem repeats of a motif called an IQ site. Binding occurs in either Ca2+ or EGTA, and only two sites are required to observe binding.
Brockerhoff
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Department of Biochemistry, University of Washington, Seattle 98195.
Stevens
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RC
Davis
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TN
D000483:Alleles; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism; D002147:Calmodulin / Q000235:genetics / Q000378:metabolism*; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002455:Cell Division; D003598:Cytoskeletal Proteins; D004533:Egtazic Acid / Q000494:pharmacology; D005455:Fluorescent Antibody Technique; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D018995:Myosin Heavy Chains; D024462:Myosin Type II; D024701:Myosin Type V; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D029702:Schizosaccharomyces pombe Proteins
D002147:Calmodulin; D002352:Carrier Proteins; D003598:Cytoskeletal Proteins; D005656:Fungal Proteins; C077512:MYO2 protein, S cerevisiae; C434211:MYO2 protein, S pombe; D011993:Recombinant Fusion Proteins; C065889:SPA2 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D029702:Schizosaccharomyces pombe Proteins; D004533:Egtazic Acid; D024462:Myosin Type II; D024701:Myosin Type V; D018995:Myosin Heavy Chains; D002118:Calcium