REGULATION OF KINESIN MOTORS

驱动蛋白马达的调节

• 批准号: 6520103
• 负责人: BRUCE Jeffrey SCHNAPP
• 金额: $ 26.43万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-04-01 至 2004-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6520103
• 关键词: acid base balance; binding sites; conformation; intracellular transport; kinesin; laboratory rat; microtubules; mutant; protein sequence; recombinant proteins; tissue /cell culture; transfection

The broad aim of this project is to elucidate how cells transport organelles and molecular complexes along microtubules (Mts) to specific destinations. To address this problem, we must explain the molecular basis and the overall logic for how different kinesin motors are mobilized to carry specific cargoes. To his end, we will substantiate our hypothesis that processive kinesin motors, which are primarily soluble in the cell, are in an inhibited ground state until they are bound and activated by their cargoes. How the non-motor regions of kinesins direct motor activity and cargo binding is the research plan's principle concern. We will identify, within the non-motor regions of a few kinesins, the domains required for binding cargo and regulating motor activity. Such domain-mapping studies will bring to light plausible mechanisms for cargo binding and motor regulation, and most importantly, pinpoint candidate-binding sites for proteins that implement these activities. Preliminary studies along these lines on conventional kinesin validate this strategy - our domain-mapping studies indicate that the inhibited state of conventional kinesin depends on kinesin light chain (KLC) and involves a folded conformation, in which the C-terminus of kinesin heavy chain (KHC) interacts with its own motor domain. Moreover, these studies have identified the KLC tandem tetratricopeptide repeats (KLC TPRs) as prime candidates to bind factors that either link kinesin to its cargo or activate kinesin's interaction with Mts. In the first specific aim, we will now isolate these KLC TPR partner proteins, using biochemical and genetic approaches, and characterize their functions. In a similar fashion, the second and third specific aims propose to investigate the non-motor regions of a number of other kinesins (heterotrimeric kinesin II and monomeric KIF1A,B, & C) whose cargoes are known. By identifying, within the non- motor regions of these kinesins, sites involved in cargo binding and motor activation, we will again develop a rationale for isolating the protein factors that implement these activities. It is the identification of these interacting proteins that is this plan's ultimate goal. To ensure that we isolate proteins that are functionally relevant, we will introduce, into the nonmotor regions of these kinesins, mutations that disrupt motor regulation or cargo binding. Such kinesin mutants are the keystone of our proposal - proteins that bind specifically to wild-type, but not mutant, sites are likely to be the bona fide partners in vivo. The generation and use of such negative controls fundamentally distinguishes this proposal from previous attempts to identify proteins that interact with kinesins.

该项目的主要目标是阐明细胞如何沿着微管 (Mts) 将细胞器和分子复合物运输到特定目的地。 为了解决这个问题，我们必须解释如何动员不同的驱动蛋白马达来携带特定货物的分子基础和整体逻辑。 为此，我们将证实我们的假设，即主要可溶于细胞的进行性驱动蛋白马达处于受抑制的基态，直到它们被其货物结合并激活。 驱动蛋白的非运动区域如何指导运动活动和货物结合是该研究计划的主要关注点。 我们将在一些驱动蛋白的非运动区域内确定结合货物和调节运动活动所需的结构域。 这种结构域映射研究将揭示货物结合和运动调节的合理机制，最重要的是，查明实现这些活动的蛋白质的候选结合位点。 对传统驱动蛋白的初步研究验证了这一策略——我们的结构域映射研究表明，传统驱动蛋白的抑制状态取决于驱动蛋白轻链 (KLC)，并涉及折叠构象，其中驱动蛋白重链 (KHC) 的 C 末端与其自身的运动结构域相互作用。此外，这些研究已确定 KLC 串联四肽重复序列 (KLC TPR) 是结合驱动蛋白与其货物或激活驱动蛋白与 Mts 相互作用的因子的主要候选者。 在第一个具体目标中，我们现在将使用生化和遗传学方法分离这些 KLC TPR 伴侣蛋白，并表征它们的功能。 以类似的方式，第二个和第三个具体目标建议研究其货物已知的许多其他驱动蛋白（异三聚驱动蛋白 II 和单体 KIF1A、B 和 C）的非运动区域。 通过识别这些驱动蛋白的非运动区域内涉及货物结合和运动激活的位点，我们将再次开发分离执行这些活动的蛋白质因子的基本原理。该计划的最终目标是识别这些相互作用的蛋白质。 为了确保我们分离出功能相关的蛋白质，我们将在这些驱动蛋白的非运动区域中引入破坏运动调节或货物结合的突变。 这种驱动蛋白突变体是我们提议的基石——与野生型而非突变体位点特异性结合的蛋白质可能是体内真正的伙伴。 这种阴性对照的产生和使用从根本上区别了该提议与之前鉴定与驱动蛋白相互作用的蛋白质的尝试。