Structure and Function of Cytoplasmic Dynein

描述(由申请人提供)：分子马达蛋白在多种细胞内运输过程中发挥作用，包括细胞器的组织和运输、染色体分离、轴突运输和信号传递。运动依赖的过程对细胞和组织的生长、增殖和分化至关重要。如何在发育过程中调节运动功能，以及运动功能障碍与包括神经退行性疾病、先天性染色体综合征和出生缺陷在内的众多医学问题的关系是当前研究活动的重点。我们的工作集中在微管运动胞浆动力蛋白，以及这个单一的运动异构体如何完成多个任务。动力蛋白是如何针对特定的货物和/或蜂窝位置的？我们的目标将解决三种可能有助于动力蛋白靶向的非排他性机制。(1)细胞质动力蛋白含有多个亚基。单独的亚基或亚单位结构域可以指定动力蛋白连接在哪里以及连接到什么货物上。为了验证这一假设，我们将询问轻质中间体和中间链多肽中的结构域是否具有特定的功能。突变和分子遗传学方法将被用来破坏结构域的功能，并将表征突变的表型。(2)动力蛋白亚基的翻译后修饰可能控制亚基是否具有高亲和力结合货物的能力。与约翰·耶茨博士(斯克里普斯研究所)的合作将使用质谱学方法确定动力蛋白复合体内亚单位上的磷酸化位点。随后，确定的磷酸化位点将被突变，以模拟各自亚基的磷酸化或非磷酸化状态。将对表达突变亚基的转基因产生的表型进行分析，以揭示动力蛋白光调节的功能意义。(3)特定的结合伙伴或“效应器”蛋白可能介导动力蛋白马达靶向特定的货物或位置。我们将继续对前一阶段确定的候选相互作用蛋白进行功能分析，并将继续对其他相互作用基因座进行二次测试。

DESCRIPTION (provided by applicant): Molecular motor proteins function in a multitude of intracellular transport processes that include the organization and transport of organelles, chromosome segregation, axonal transport, and signaling. Motor dependent processes are critical for the growth, proliferation, and differentiation of cells and tissues. How motor function is regulated during development, and the relationship of motor dysfunction to numerous medical problems including neurodegenerative disease, congenital chromosomal syndromes, and birth defects is a current focus of research activity. Our work is focused on the microtubule motor cytoplasmic dynein, and how this single motor isoform accomplishes multiple tasks. How is dynein targeted to specific cargoes and/or cellular locations? Our aims will address three non-exclusive mechanisms that potentially contribute to dynein targeting. (1) Cytoplasmic dynein contains multiple subunits. The individual subunits or subunit domains could specify where, and to what cargo, dynein is attached. To test this hypothesis we will ask whether domains within the light intermediate and the intermediate chain polypeptides confer specific functions. Mutagenesis and molecular genetic approaches will be used to disrupt domain function and the mutant phenotypes will be characterized. (2) The posttranslational modification of dynein subunits might control whether subunits are competent to bind a cargo with high affinity. A collaboration with Dr. John Yates (Scripps Research Institute) will define the sites of phosphorylation on subunits within the dynein complex using a mass spectrometry approach. Subsequently, the phosphorylation sites identified will be mutated to mimic the phosphorylated or unphosphorylated state of the respective subunit. The phenotypes produced by transgenes that express the mutant subunits will be analyzed to reveal the functional significance of dynein phosphoregulation. (3) Specific binding partners or "effector" proteins might mediate the targeting of the dynein motor to specific cargoes or locations. We will pursue the functional analysis of candidate interacting proteins identified in the previous period and will continue with secondary tests on other interacting loci.