MUTAGENIC ANALYSIS OF YEAST ACTIN IN VIVO AND IN VITRO

我们的总体目标是获得分子水平的因子知识 有助于肌动蛋白的稳定性，聚合性，以及 肌动蛋白与控制其功能的蛋白质相互作用， cell.除了是收缩的两个主要组成部分之一 肌动蛋白在肌肉中产生机械，参与许多运动和 非肌肉细胞中细胞功能所需的收缩过程 包括胞质分裂、细胞沿着基质运动和细胞形状 保持战略定力肌球蛋白、原肌球蛋白和肌钙蛋白缺陷，所有蛋白质 与肌肉肌节中的肌动蛋白相互作用， 导致某种心肌病了解 这些疾病中肌节功能障碍的潜在机制以及 了解细胞如何执行和控制运动过程 它所涉及的，需要了解肌动蛋白的工作， 分子水平。最近解决的肌动蛋白的X射线晶体结构， 产生了两个未经检验的假设有关肌动蛋白的功能：1）一个10 在一个细胞中，肌动蛋白单体中肌动蛋白亚结构域3和4之间的残基环 螺旋链插入相对链中的疏水袋中， 产生F-肌动蛋白螺旋的稳定; 2）基于相似性 在肌动蛋白的三级结构中，HSC 7 O、H161和Q137参与了 肌动蛋白催化ATP水解，这是肌动蛋白的重要反应 稳定性和灯丝动力学。我们将测试这些假设使用网站- 酵母肌动蛋白偶联酵母表达系统的定向诱变 为了在体内和体外测试疏水性突变的影响， 环和H161和Q137对肌动蛋白结构和功能的影响。活的单倍体 只含有突变肌动蛋白的细胞将在 不同的温度，芽极性将被评估，肌动蛋白和 通过荧光显微镜分析几丁质沉积。突变体 将使用DNA酶I亲和性以活性形式分离肌动蛋白 色谱，并与野生型酵母肌动蛋白在其 热稳定性、可聚合性、与ATP的结合和水解，以及 与肌球蛋白的相互作用。使用突变肌动蛋白和工程化的反应性 在疏水环附近的半胱氨酸，我们将用荧光标记-SH， 自旋标记探针，以监测环区的构象变化， G和F-肌动蛋白使用荧光和EPR光谱。我们将测试 环突变对丝稳定性的影响， 扫描微量热法使用反应性硫醇亲和层析， 我们将建立一个分离突变体和野生型肌动蛋白的程序， 在同一个细胞中表达。这将使我们能够研究突变肌动蛋白 其本身与酵母活力不相容。这些 实验将提供有关稳定的新信息， F-肌动蛋白和ATP在肌动蛋白丝动力学中的作用， 肌动蛋白功能异常与表型的关系 由这些突变在体内产生。

Our overall goal is to gain knowledge at the molecular level of factors contributing to actin stability, polymerizability, and the ability of actin to interact with the proteins that control its function within the cell. Besides being one of the two major components of the contraction producing machinery in muscle, actin is involved in a number of motile and contractile processes needed for cell function in non-muscle cells including cytokinesis, cell movement along a substratum, and cell shape determination. Defects in myosin, tropomyosin, and troponin, all proteins that interact with actin in the muscle sarcomere, have recently been shown to be responsible for some forms of cardiomyopathy. Understanding the mechanism underlying sarcomere malfunction in these disorders as well as understanding how the cell carries out and controls the motile processes with which it is involved requires an understanding of actin works at the molecular level. The recently solved X-ray crystal structure of actin has generated two untested hypotheses concerning actin function: 1) a 10 residue loop between actin subdomains 3 and 4 in an actin monomer in one helical strand inserts into a hydrophobic pocket in the opposite strand to produce a stabilization of the F-actin helix; 2) based on the similarity in tertiary structures of actin and HSC7O, H161, and Q137 are involved in the actin catalyzed hydrolysis of ATP, a reaction important for actin stability and filament dynamics. We will test these hypotheses using site- directed mutagenesis of yeast actin coupled with a yeast expression system to test in vivo and in vitro the effects of mutations in the hydrophobic loop and in H161 and Q137 on actin structure and function. Viable haploid cells containing only the mutant actin will be tested for growth at different temperatures, budding polarity will be assessed, and actin and chitin deposition will be analyzed by fluorescence microscopy. Mutant actins will be isolated in active form using DNAse I affinity chromatography and compared with wild-type yeast actin in terms of their thermostability, polymerizability, binding to and hydrolysis of ATP, and interaction with myosin. Using a mutant actin with an engineered reactive Cys near the hydrophobic loop we will label the -SH with fluorescent and spin label probes to monitor conformation changes in the loop region of both G and F-actin using fluorescence and EPR spectroscopy. We will test the effects of the loop mutations on filament stability using differential scanning microcalorimetry. Using reactive thiol affinity chromatography, we will establish a procedure for separating mutant from wild-type actin when expressed in the same cell. This will allow us to study mutant actins which by themselves are not compatible with yeast viability. These experiments will provide new information concerning the stabilization of F-actin and the role that ATP plays in actin filament dynamics and allow correlation of abnormal actin function in vitro with the phenotypes produced by these mutations in vivo.