Molecular Analysis of Flagellar Dynein Function

描述（由申请人提供）：动力蛋白是基于微管的分子马达，参与多种基本细胞功能，包括逆行囊泡运输、核膜破裂、纤毛/鞭毛运动和细胞分裂。 来自衣原体鞭毛的1.9MDa外动力蛋白臂提供了一个很好的模型系统，其中研究动力蛋白的结构，功能和调节，因为它包含与细胞质同工酶中的那些密切相关的组分，适合于经典/分子遗传学，并且可以大量纯化用于生化分析。动力蛋白是ATP酶AAA+家族的成员，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运动活性仍然几乎完全未知。本申请提出了四个具体的调查领域。1)我们将使用定点诱变和体外生物化学来解决哪些结构域结合和/或水解核苷酸。我们还将测试是否域间相互作用调节ATP/ADP结合，以及微管结合柄是否在传输ATP驱动的构象变化中起着积极的作用。 2)我们将使用电子显微镜和诱变来测试我们的动力蛋白运动单元内的子域分配模型。此外，我们将在AAA 1和N-末端结构域之间插入甘氨酸接头，以测试是否如最近提出的那样在这两个片段之间发生动力冲程。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物蛋白（DCS）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改变。这将涉及突变株表达的DC 3和突变形式（半胱氨酸丝氨酸）的硫氧还蛋白氧化还原活性位点结合在体外和体内分析的运动功能的突变株的分析。4)我们将使用定点诱变来测试假设（基于我们的NMR结构研究），即LC 1蛋白的C-末端结构域以与激活Ras/Rho GTP酶的GAP所观察到的类似的方式控制γ重链ATP酶。最后，我们还将研究是否y重链相关的Ca 2+结合LC 4蛋白作为Ca 2+传感器负责调节ATP依赖的动力蛋白微管相互作用。本研究将提供动力蛋白基本运动机制的详细信息，并使我们能够确定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.