MOLECULAR ANALYSIS OF FLAGELLAR DYNEIN FUNCTION

动力蛋白是一种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、纤毛/鞭毛运动和细胞分裂。来自衣原体鞭毛的1.9MDa外动力蛋白臂为研究动力蛋白的结构和功能提供了一个很好的模型系统，因为它含有与胞质同工酶中的组分密切相关的组分，适合于经典/分子遗传学研究，并且可以大量纯化用于生物化学。主要的问题仍然是动力蛋白运动功能是如何控制的，以及酶与适当货物连接的机制。本申请提出了四个不同的轻链（LC），我们最近发现的动力蛋白颗粒内的研究。LC 4是一种与γ重链相关的Ca 2+结合EF-手蛋白，我们假设其参与Ca 2+介导的运动功能控制。所提出的实验将使用表现出不同Ca亲和力的LC 4的突变体形式在体外和体内直接测试这一点。第二个项目将分析与γ重链的马达结构域相关的LcI蛋白。这种LC直接与第二种蛋白质（p45）原位相互作用，从而靶向运动单位。我们建议鉴定p45并确定LC 1在重链上的结合位置。随后的实验将集中在定义这个新系统在动力蛋白功能中的作用。我们还将研究两个基本类的LC（Tctex 1/Tctex 2和LC 7/路障），位于动力蛋白颗粒的基础上，并在细胞质和鞭毛同工酶中发现发挥的一般作用。我们将在odal 2无效突变体背景中表达改变形式的LC 2（Tctex 2），其不能组装外臂以确定该LC类的功能。已在果蝇中鉴定了LC 7/roadblock蛋白的显性负突变。 因此，我们将在体内过表达该蛋白的突变形式，随后对菌株进行详细的生化/细胞生物学分析。 因此，本项目将提供关于这种复杂分子马达的组装、调节和活性的详细信息。

Dyneins are microtubule-based molecular motors involved in a wide variety of essential cellular functions including retrograde vesicular trafficking, 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 and function as it contains components closely related to those in the cytoplasmic isozyme, is amenable to classical/molecular genetic study and may be purified in large amount for biochemistry. Major questions remain about how dynein motor function is controlled and the mechanisms by which the enzyme is attached to the appropriate cargo. This application proposes study of four distinct light chains (LCs) that we have recently identified within the dynein particle. LC4 is a Ca2+-binding EF-hand protein associated with the gamma heavy chain that we hypothesize is involved in Ca2+-mediated control of motor function. The proposed experiments will test this directly both in vitro and in vivo using mutant versions of LC4 exhibiting different Ca affinities. The second project will analyze the LcI protein which is associated with the motor domain of the gamma heavy chain. This LC interacts directly in situ with a second protein (p45) that is thereby targeted to the motor unit. We propose to identify p45 and determine where LC1 binds on the heavy chain. Subsequent experiments will focus on defining the role of this novel system in dynein function. We also will examine the generic roles played by two essential classes of LC (Tctex1/Tctex2 and LC7/roadblock) that are located at the base of the dynein particle and are found in both cytoplasmic and flagellar isozymes. we will express altered versions of LC2 (Tctex2) in the odal2 null mutant background which is unable to assemble an outer arm to determine the function of this LC class. Dominant negative mutations for the LC7/roadblock proteins have been identified in Drosophila. Therefore, we will overexpress mutant forms of this protein in vivo and subsequently subject the strains to detailed biochemical/cell biological analysis. Thus, this project will provide detailed information on the assemble, regulation and activity of this complex molecular motor.