Molecular Analysis of Flagellar Dynein Function

描述(申请人提供)：动力蛋白是基于微管的分子马达，参与广泛的基本细胞功能，包括逆行运输囊泡、核膜破裂、纤毛/鞭毛运动和细胞分裂。衣藻鞭毛中的1.9个丙二醛动力蛋白外臂是研究动力蛋白结构、功能和调控的一个很好的模型系统，因为它含有与细胞质同工酶密切相关的成分，符合经典/分子遗传学，可以大量纯化用于生化分析。动力蛋白是ATPase AAA+家族中的一员，然而，ATP水解转化为机械运动的机制以及如何在分子水平上调节这一运动活性几乎完全不清楚。这份申请提出了四个具体的调查领域。1)我们将使用定点突变和体外生物化学来确定哪些结构域结合和/或水解核苷酸。我们还将测试结构域间相互作用是否调节ATP/ADP结合，以及微管结合茎是否在传递ATP驱动的构象变化中发挥积极作用。2)我们将使用电子显微镜和突变来测试我们的模型在动力蛋白运动单位内的亚区分配。此外，我们将在AAA1和N-末端结构域之间插入甘氨酸连接子，以测试最近提出的这两个片段之间是否发生了功率卒中。3)我们将研究硫氧还蛋白轻链和氧化还原敏感的对接复合体蛋白(DC)是否参与调节动力蛋白运动功能，以响应细胞氧化还原平衡的变化。这将包括分析表达DC3改变版本的突变菌株和硫氧还蛋白氧化还原活性部位的突变(从Cys到Ser)，以及对运动功能的体外和体内分析。4)我们将使用定点突变来检验(基于我们的核磁共振结构研究)的假设，即LC1蛋白的C-末端结构域控制伽马重链ATPase的方式类似于激活Ras/Rho GTP酶的间隙。最后，我们还将研究y重链相关的钙结合LC4蛋白是否作为钙感受器参与调节ATP依赖的动力微管相互作用。该项目将提供有关动力蛋白的基本运动机制的详细信息，并使我们能够定义控制运动功能的分子途径。

DESCRIPTION (provided by applicant): Dyneins are microtubule-based molecular motors involved in a wide variety of essential cellular functions including retrograde vesicle trafficking, nuclear envelope breakdown, ciliary/flagellar motility and cell division. The 1.9 MDa outer dynein arm from flagella of Chlamydomonas offers an excellent model system in which to study dynein structure, function and regulation as it contains components closely related to those in the cytoplasmic isozyme, is amenable to classical/molecular genetics and can be purified in large amounts for biochemical analysis. Dynein is a member of the AAA+ family of ATPases, however, the mechanisms by which ATP hydrolysis is converted to mechanical movement and how that motor activity is regulated at the molecular level remain almost completely unknown. This application proposes four specific areas of investigation. 1) We will use site-directed mutagenesis and in vitro biochemistry to address which domains bind and/or hydrolyze nucleotide. We will also test whether inter-domain interactions regulate ATP/ADP binding and whether the microtubule-binding stalk plays an active role in transmitting ATP-driven conformational change. 2) We will use electron microscopy and mutagenesis to test our model for subdomain assignments within the dynein motor unit. Further, we will insert a glycine linker between AAA1 and the N-terminal domain to test whether the power stroke occurs between these two segments as has been recently proposed. 3) We will examine whether thioredoxin light chains and the redox-sensitive docking complex protein (DCS) are involved in regulating dynein motor function in response to alterations in cellular redox poise. This will involve analysis of mutant strains expressing altered versions of DC3 and mutagenesis (Cys to Ser) of the thioredoxin redox-active sites combined with both in vitro and in vivo analysis of motor function. 4) We will use site-directed mutagenesis to test the hypothesis (based on our NMR structural studies) that the C-terminal domain of the LC1 protein controls gamma heavy chain ATPase in a manner similar to that observed with the GAPs that activate Ras/Rho GTPases. Finally, we will also examine whether the y heavy chain-associated Ca2+-binding LC4 protein acts as the Ca2+ sensor responsible for modulating ATP-dependent dyneinmicrotubule interactions. This project will provide detailed information on the fundamental motor mechanism of dynein and enable us to define the molecular pathways by which motor function is controlled.