Study of multifunctional signalling proteins implicated in cell proliferation

我们研究了钙调素和rho型GTPase的功能。最近对酵母钙调蛋白（yCaM）的遗传研究表明，不同的Phe残基组的改变会导致不同的功能缺陷（Ohya， Y., and Botstein， D.(1994) Science 263, 963-966）。为了检验Phe残基对靶标结合和激活的重要性，我们纯化了含有单或双Phe到Ala取代的突变型ycam，并确定了它们结合和激活两个靶标蛋白——钙调磷酸酶和cam依赖性蛋白激酶（CaMK）的能力。我们的研究结果表明，每个靶蛋白都需要特定的yCaM的Phe残基亚群来结合和激活靶标，并且不同的靶蛋白对Phe残基亚群的需求不同。温度敏感酵母钙调蛋白突变的四个基因内互补组之一，cmd1A，导致肌动蛋白组织的特征功能缺陷。我们报道，在互补突变中，cmd1A的一个代表突变体（cmd1-226: F92A）与MYO2突变具有合成致死性，MYO2突变编码具有钙调蛋白结合域的V类非常规肌球蛋白。凝胶覆盖试验表明，F92A突变的钙调蛋白与GST-Myo2p融合蛋白的结合亲和力严重降低。钙调蛋白第92位的随机替换和定点突变表明，该位置允许存在疏水和芳香残基，这表明钙调蛋白和Myo2p之间疏水相互作用的重要性。1,3- β -葡聚糖合成酶是一种多酶复合物，催化1,3- β -连接葡聚糖的合成，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 -葡聚糖合成酶的调控亚基。必需的Rho1型鸟苷三磷酸酶Rho1的温度敏感突变体表现出耐热性葡聚糖合成酶活性，通过添加重组Rho1恢复了这种活性。来自表达组成型活性Rho1的突变体的葡聚糖合成酶不需要外源三磷酸鸟苷来维持活性。Rho1在体内与1,3- β -葡聚糖合成酶共化，并与该复合物的Fks1亚基相关。这两种蛋白主要定位于细胞壁重塑部位。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