MUTAGENIC ANALYSIS OF YEAST ACTIN IN VIVO AND IN VITRO

我们的总体目标是在因子的分子水平上获得知识 有助于肌动蛋白的稳定性、聚合性和 肌动蛋白与控制其功能的蛋白质相互作用 手机。除了是收缩的两个主要组成部分之一 在肌肉中生产机械，肌动蛋白参与了许多运动和 非肌肉细胞中细胞功能所需的收缩过程 包括胞质分裂、细胞沿底物的运动和细胞形状 决心。肌球蛋白、原肌球蛋白和肌钙蛋白缺陷，都是蛋白质 与肌肉肌节中的肌动蛋白相互作用，最近被证明 对某些形式的心肌病负责。了解 这些疾病中肌节功能障碍的机制以及 了解细胞如何执行和控制运动过程 需要了解肌动蛋白在 分子水平。最近解决的肌动蛋白的X射线晶体结构 产生了两个关于肌动蛋白功能的未经测试的假设：1)A 10 肌动蛋白单体中肌动蛋白亚区3和4之间的残基环 螺旋链插入到相反链中的疏水口袋中以 产生F-肌动蛋白螺旋的稳定化；2)基于相似性 在肌动蛋白和HSC7O的三级结构中，H161和Q137参与了 肌动蛋白催化ATP的水解，这是一个对肌动蛋白很重要的反应 稳定性和细丝动力学。我们将使用Site-Site验证这些假设- 酵母肌动蛋白与酵母表达系统偶联的定向突变 在体内和体外测试突变对疏水性的影响 环以及在H161和Q137中对肌动蛋白结构和功能的影响。活单倍体 只含有突变肌动蛋白的细胞将在 不同的温度，发芽的极性将被评估，肌动蛋白和 甲壳素沉积将通过荧光显微镜进行分析。突变型 肌动蛋白将使用DNase I亲和力以活性形式分离 层析及与野生型酵母肌动蛋白的比较 热稳定性、聚合性、与三磷酸腺苷的结合和水解，以及 与肌球蛋白的相互作用。使用突变的肌动蛋白和工程反应 在疏水环附近我们将用荧光标记-SH和 用于监测环区构象变化的自旋标记探针 G-肌动蛋白和F-肌动蛋白的荧光和EPR光谱分析。我们将测试 用差动法研究环路突变对细丝稳定性的影响 扫描微量热法。使用反应性硫醇亲和层析， 我们将建立一种从野生型肌动蛋白中分离突变体的程序 当在同一细胞中表达时。这将使我们能够研究突变的肌动蛋白 它们本身与酵母的活力是不相容的。这些 实验将提供关于稳定的新信息 F-肌动蛋白和ATP在肌动蛋白细丝动力学中的作用 体外肌动蛋白功能异常与表型的关系 由体内的这些突变产生。

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.