REGULATION OF KINESIN MOTORS

这个项目的主要目的是阐明细胞如何运输细胞器和分子复合物沿着微管（MTS）到特定的目的地。 为了解决这个问题，我们必须解释不同的驱动蛋白马达是如何被动员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，即进行性驱动蛋白马达，这是主要可溶于细胞中，是在一个抑制的基态，直到他们被绑定和激活的货物。 驱动蛋白的非运动区域如何指导运动活动和货物结合是研究计划的主要关注点。 我们将确定，在一些驱动蛋白的非运动区域，结合货物和调节运动活动所需的域。 这种结构域定位研究将揭示货物结合和运动调节的合理机制，最重要的是，确定实施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沿着这些路线验证了这一策略-我们的结构域作图研究表明，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域相互作用。此外，这些研究已经确定了KLC串联tetratricopeptide repeats（KLC TPR）作为主要候选人，以结合连接驱动蛋白与其货物或激活驱动蛋白与Mts相互作用的因子。 在第一个具体目标中，我们现在将使用生物化学和遗传学方法分离这些KLC TPR伴侣蛋白，并表征其功能。 以类似的方式，第二个和第三个具体的目标提出了研究一些其他驱动蛋白（异源三聚体驱动蛋白II和单体KIF 1A，B，& C）的非运动区，其货物是已知的。 通过在这些驱动蛋白的非运动区域内鉴定参与货物结合和运动激活的位点，我们将再次开发用于分离实施这些活动的蛋白质因子的基本原理。识别这些相互作用的蛋白质是该计划的最终目标。 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引入突变，破坏运动调节或货物结合。 这种驱动蛋白突变体是我们的建议的基石-蛋白质特异性结合野生型，但不突变，网站很可能是真正的合作伙伴在体内。 这种阴性对照的产生和使用从根本上将该提议与先前鉴定与驱动蛋白相互作用的蛋白质的尝试区分开。

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.