FUNCTIONAL SIGNIFICANCE OF THE ACTIN N-TERMINAL REGION

我们工作的总体目标是了解 控制肌动蛋白与肌球蛋白和 调节细胞内肌动蛋白行为的各种蛋白质。 我们 特别感兴趣的是肌动蛋白的N-末端区域， 不寻常的加工反应，涉及翻译后去除 乙酰基氨基酸通过特定的加工酶。 的这个区域 肌动蛋白还与蛋白质如肌球蛋白，凝溶胶蛋白， 因此，可能是重要的 控制细胞肌动蛋白功能。 这项拨款强调了 18个氨基酸的肌动蛋白N-末端区域的三个保守元件： 在成熟的加工过的肌动蛋白的N-末端的酸性氨基酸; 从残基6开始的保守的Ala-Leu-瓦尔;和保守的Asp-Asn- Gly-Ser-Gly起始于位置10，否则在结构上是 肌动蛋白多肽的高变部分。 我们将使用酵母 转化系统，其中携带突变的 将无内含子酵母肌动蛋白基因导入二倍体S. 酿酒酵母含有一个野生型肌动蛋白基因和一个失活肌动蛋白基因。 使用定点诱变，将在三个基因中的一个中进行突变。 刚刚描述的或将插入Met-1和Met-2之间的结构元件， Asp 1防止N-末端加工。 将对单倍体细胞进行检测， 单独使用突变的肌动蛋白保持活力的能力。 经变换的 二倍体将测试突变肌动蛋白对 牢房 我们将检查受影响的细胞中肌动蛋白沉积的变化 免疫荧光染色技术。 我们亦会研究 突变对几丁质沉积和转化酶分泌的影响， 这两个过程依赖于肌动蛋白的正常功能。 肽图谱 分离的突变肌动蛋白将表明是否改变影响 通过加工酶去除Met-1。 我们会隔离变种人 活性形式的肌动蛋白，并确定突变对肌动蛋白的影响 聚合，阳离子和核苷酸结合，以及肌动蛋白的相互作用 肌球蛋白和凝溶胶蛋白。 这些实验将为 N-末端加工和三个保守结构元件的作用 N-末端区域在体内和体外的肌动蛋白功能， 将使我们能够制备大量的突变肌动蛋白， 分析.

The overall goal of our work is to understand the nature of the interactions controlling the association of actin with myosin and with various proteins that modulate actin's behavior within the cell. We are particularly interested in actin's N-terminal region, the site of an unusual processing reaction involving the posttranslational removal of acetyl amino acids by a specific processing enzyme. This region of the actin also forms close contacts with proteins such as myosin, gelsolin, fragmin, depactin, and troponin I and therefore may be important in controlling cellular actin function. This grant addresses the importance of three conserved elements of the 18 amino acid actin N-terminal region: the acidic amino acids at the N-terminus of mature processed actin; the conserved Ala-Leu-Val beginning at residue 6; and the conserved Asp-Asn- Gly-Ser-Gly beginning at position 10 in what otherwise structurally is a hypervariable part of the actin polypeptide. We will use a yeast transformation system in which a centromeric plasmid carrying a mutated intronless yeast actin gene will be introduced into a diploid strain of S. cerevisiae containing one wild type actin gene and one inactive actin gene. Using site-directed mutagenesis, mutations will be made in one of the three structural elements just described or will be inserted between Met-1 and Asp1 to prevent N-terminal processing. Haploid cells will be tested for the ability to remain viable with the mutated actin alone. The transformed diploid will be tested for dominant effects exerted by the mutant actin on the cell. We will examine affected cells for changes in actin deposition patterns by immunofluorescent staining techniques. We will also examine the effects of the mutations on chitin deposition and invertase secretion, two processes that are dependent on proper actin function. Peptide mapping of the isolated mutant actin will indicate whether alterations affect removal of the Met-i by the processing enzyme. We will isolate the mutant actins in active form and determine the effects of the mutations on actin polymerization, cation and nucleotide binding, and the interaction of actin with myosin and gelsolin. These experiments will provide answers for the role of N-terminal processing and the three conserved structural elements of the N-terminal region both in vivo and in vitro in actin function and will allow us to prepare large quantities of mutant actins for biochemical analysis.