| Budget Amount *help |
¥8,900,000 (Direct Cost: ¥8,900,000)
Fiscal Year 1998: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 1997: ¥2,300,000 (Direct Cost: ¥2,300,000)
Fiscal Year 1996: ¥5,200,000 (Direct Cost: ¥5,200,000)
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| Research Abstract |
We study functions of calmodulin and rho-type GTPase. Recent genetic studies of yeast calmodulin (yCaM) have shown that alternations of different sets of Phe residues result in distinct functional defects (Ohya, Y., and Botstein, D.(1994) Science 263, 963-966). To examine the importance of Phe residues for target binding and activation, we purified mutant yCaMs containing single or double Phe to Ala substitutions and determined their ability to bind and activate two target proteins, calcineurin and CaM-dependent protein kinase (CaMK). Our results indicated that each target protein requires a specific subset of Phe residues of yCaM for target binding and activation, and that the subsets of Phe residues are required differently among various target proteins. One of four intragenic complementing groups of temperature-sensitive yeast calmodulin mutations, cmd1A, results in a characteristic functional defects in actin organization. We report that among the complementing mutations, a represe
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ntative mutant of cmd1A (cmd1-226 : F92A) is synthetically-lethal with a mutation in MYO2, which 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.1,3-beta-glucan synthase is a multi-enzyme complex that catalyzes the synthesis of 1,3-beta-linked glucan, a major structural component of the yeast cell wall. We found that Rho1 is a regulatory subunit of 1,3-beta-glucan synthase. Temperature-sensitive mutants in the essential Rho-type guanosine triphosphatase, Rho1, displayed thermolabile glucan synthase activity, which was restored by the addition of recombinant Rho1. Glucan synthase from mutants expressing constitutively active Rho1 did not require exogenous guanosine triphosphate for activity. Rho1 copurified with 1,3-beta-glucan synthase and associated with the Fks1 subunit of this complex in vivo. Both proteins were localized predominantly at sites of cell wall remodeling. Less
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