Molecular Dissection of Cytoplasmic Dynein

描述（由申请人提供）： 本研究的目的是利用结构-功能的方法，在模型系统S。啤酒。这项工作与人类疾病有关，因为缺乏功能性轴丝动力蛋白的人类发展为Kartagner综合征，这是一种导致慢性呼吸问题、不育和镜像器官定位的疾病。细胞质动力蛋白被认为存在于所有真核细胞中，并且在细胞生长和分裂中具有多种重要作用。由于缺乏可以在体外制造和分析突变蛋白的系统，动力蛋白领域的研究受到阻碍。本项目的第一个目的是建立体外实验来研究亲和纯化的酵母细胞质动力蛋白的运动特性。淡水河谷实验室和其他实验室率先采用了这种生物物理分析方法。这些试验将用于检验以下两个目标中描述的假设。与其他细胞骨架分子马达不同，动力蛋白含有多个核苷酸结合位点，可以结合和水解ATP以产生驱动运动的力。该项目的第二个目的是通过突变预测参与ATP水解的保守残基来确定每个ATP结合位点在动力蛋白功能中的相对贡献。最后，动力蛋白相关蛋白在动力蛋白介导的运动中的作用将被分析。一种这样的相关蛋白是Liscencephaly 1蛋白，当其有缺陷时，会导致人类严重的脑发育疾病。由于动力蛋白在进化上与分子马达驱动蛋白和肌球蛋白不同，这些研究代表了马达领域的前沿，并可能揭示马达蛋白功能的新机制。

DESCRIPTION (provided by applicant): The goal of this project is to characterize the mechanochemical properties of the molecular motor dynein using a structure- function approach in the model system, S. cerevisiae. This work is relevant to human disease because humans lacking functional axonemal dynein develop Kartagner syndrome, a disease resulting in chronic respiratory problems, infertility and mirror- image organ positioning. Cytoplasmic dynein is thought to be present in all eukaryotic cells and has multiple, essential roles in cell growth and division. Research in the dynein field has been hampered by the lack of a system in which mutant proteins can be made and analyzed in vitro. The first aim of this project is to develop in vitro assays to study the motile properties of affinity purified yeast cytoplasmic dynein. Such biophysical assays have been pioneered by the Vale lab and others. These assays will be used to test hypotheses described in the following two aims. Unlike other cytoskeletal molecular motors, dynein contains multiple nucleotide binding sites that may bind and hydrolyze ATP to produce the force that drives movement. The second aim of this project is to determine the relative contribution of each ATP binding site in dynein function by mutating conserved residues predicted to be involved in ATP hydrolysis. Finally, the role of dynein-associated proteins in dynein-mediated motility will be analyzed. One such associated protein is the Liscencephaly 1 protein, which, when defective causes a severe brain developmental disease in humans. As dyneins are evolutionarily distinct from the molecular motors kinesin and myosin, these studies represent a frontier in the motor field and may reveal novel mechanisms for motor protein function.