Study of multifunctional signalling proteins implicated in cell proliferation

我们研究了钙调蛋白和Rho型GTP酶的功能。最近对酵母钙调素(YCaM)的遗传学研究表明，不同的Phe残基的改变会导致不同的功能缺陷(Ohya，Y.和Botstein，D.(1994)科学263,963-966)。为了检测Phe残基对靶结合和激活的重要性，我们纯化了含有单个或双Phe到Ala取代的突变体yCaM，并测定了它们结合和激活两种靶蛋白的能力，即钙调神经磷酸酶和CaM依赖的蛋白激酶(CaMK)。我们的结果表明，每个靶蛋白需要yCaM的一个特定的Phe残基来结合和激活靶蛋白，并且不同的靶蛋白需要不同的Phe残基亚集。温度敏感型酵母钙调素突变的四个基因内互补组之一，cmd1A，导致肌动蛋白组织中特有的功能缺陷。我们报告了在互补突变中，一个代表…更多的cmd1A(cmd1-226：F92A)突变是人工致死的，MYO2突变编码带有钙调蛋白结合区的V类非常规肌球蛋白。凝胶覆盖分析表明，带有F92a突变的钙调蛋白严重降低了与GST-Myo2p融合蛋白的结合亲和力。钙调蛋白第92位的随机替换和定点突变表明，该位置允许疏水和芳香族残基，这表明钙调蛋白和Myo2p之间的疏水相互作用的重要性。1，3-β-葡聚糖合成酶是一个多酶复合体，催化1，3-β-连接葡聚糖的合成，1，3-β-连接葡聚糖是酵母细胞壁的主要结构成分。我们发现Rho1是1，3-β-葡聚糖合成酶的一个调节亚单位。Rho型鸟苷三磷酸酶Rho1的温度敏感突变体表现出不耐热的葡聚糖合成酶活性，通过添加重组Rho1可以恢复这种活性。来自表达具有固有活性的Rho1的突变体的葡聚糖合成酶不需要外源三磷酸鸟苷来激活。Rho1与1，3-β-葡聚糖合成酶结合，在体内与该复合体的Fks1亚单位结合。这两种蛋白主要定位在细胞壁重塑的部位。较少

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 … More 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