ACTIN CYTOSKELETON OF YEAST

我们对肌动蛋白细胞骨架的生物学特别感兴趣。 肌动蛋白结合蛋白如何调控其组装和组织。我们 建议在酿酒酵母中研究这些问题 因为这个系统有很好的遗传学、分子生物学、生物化学 和细胞生物学。首先，我们已经鉴定了酵母同源物 帽蛋白，一种普遍存在的肌动蛋白结合蛋白，调节肌动蛋白 并建议研究其在肌动蛋白细胞骨架和细胞骨架中的作用。 确定相互作用的基因。这种蛋白质有α和β两种成分 亚基，进行了纯化。相应的基因cap1和cap2分别是 克隆、测序和删除。抗体定位显示封顶 与皮质肌动蛋白斑块相关的蛋白质，但不与电缆或 胞质分裂环。零突变体单独存在是可行的，但在 与纤毛蛋白SAC6零突变结合。 为了分析覆盖蛋白在体内的作用，我们将研究 缺乏封闭性蛋白或具有过度封闭性的细胞的表型 蛋白质利用基因缺失和过表达菌株。我们将专注于 肌动蛋白细胞骨架可能存在的细胞生理学方面 涉及肌动蛋白的分布、芽位选择、细胞 形态、生长速度、交配形态发生和分泌。我们会 观察Null表型随时间的发展情况 变种人。 为了了解覆盖蛋白在体内的功能，我们将确定 细胞表型是否受改变的点突变的影响 已经在体外确定的封闭性蛋白的功能， 特异地限制蛋白质调节肌动蛋白聚合的能力 以及阴离子磷脂对其的抑制作用。此外，身份识别 表型异常但体外活性正常的突变体 表明封端蛋白具有不同于 这两种物质如结合到质膜上。其表型为 带有突变基因的转化子将被分析，并封顶 蛋白质将被部分纯化并进行体外活性分析， 从而将表型与体外活性联系起来。 我们将确定与封闭性蛋白结合的蛋白质的基因，并 具有相似或相关功能的蛋白质的基因 接近了。1.封顶蛋白缺失突变体的抑制分析。 空菌株将被诱变或转化为过表达 文库，并在限制性条件下选择生长。2. 封端蛋白点突变的抑制分析 上面，识别肌动蛋白和阴离子磷脂以外的分子 封端蛋白与其结合并分析阴离子的意义 磷脂抑制。3.因为肌动蛋白对生存能力和 帽蛋白不是，其他肌动蛋白调节蛋白的突变 结合帽蛋白的突变可能是致命的。我们 将通过诱变覆盖蛋白零的菌株来筛选这样的基因 在可逆选择的质粒上添加封端蛋白。

We are interested in the biology of the actin cytoskeleton, in particular how actin-binding proteins regulate its assembly and organization. We propose to study these questions in the yeast Saccharomyces cerevisae because this system has good genetics, molecular biology, biochemistry and cell biology. To begin, we have identified the yeast homologue of capping protein, a ubiquitous actin-binding protein that regulates actin assembly, and propose to study its role in the actin cytoskeleton and identify interacting genes. The protein, which has alpha and beta subunits, was purified. The corresponding genes, CAP1 and CAP2, were cloned, sequenced and deleted. Antibody localization shows capping protein in association with cortical patches of actin, but not cables or the cytokinesis ring. The null mutant is viable alone but lethal in combination with null mutations of fimbrin, SAC6. To analyze the role of capping protein in vivo, we will examine the phenotype of cells that lack capping protein or have an excess of capping protein using gene deletion and overexpression strains. We will focus on aspects of cell physiology in which the actin cytoskeleton may be involved, including actin distribution, bud site selection, cell morphology, growth rate, mating morphogenesis and secretion. We will observe the development of the null phenotype over time in conditional mutants. To understand the function of capping protein in vivo, we will determine whether cell phenotype is affected by point mutations that alter functions of capping protein that have been defined in vitro, specifically capping protein's ability to regulate actin polymerization and its inhibition by anionic phospholipids. In addition, identification of mutants with an abnormal phenotype but normal in vitro activities will indicate that capping protein has functions or interactions other than these two, such as binding to the plasma membrane. The phenotype of the transformants with mutant genes on plasmids will be analyzed, and capping protein will be partially purified and analyzed for in vitro activity, thus correlating phenotype with in vitro activity. We will identify genes for proteins that bind to capping protein and genes for proteins that have similar or related functions using several approaches. 1. Suppression analysis of the capping protein null mutants. The null strain will be mutagenized or transformed with an overexpression plasmid library and selected for growth at restrictive conditions. 2. Suppression analysis of the point mutants of capping protein, generated above, to identify molecules other than actin and anionic phospholipids to which capping protein binds and to analyze the significance of anionic phospholipid inhibition. 3. Because actin is essential for viability and capping protein is not, mutations in other actin regulatory proteins might be lethal in combination with mutations in capping protein. We will screen for such genes by mutagenizing a capping protein null strain with capping protein on a counter-selectable plasmid.