REGULATION OF KINESIN MOTORS

这个项目的主要目的是阐明细胞如何将细胞器和分子复合体沿着微管(MT)运输到特定的目的地。为了解决这个问题，我们必须解释不同的动蛋白马达如何被动员来运载特定货物的分子基础和总体逻辑。为此，我们将证实我们的假设，即主要可溶于细胞的前进运动蛋白马达处于抑制的基态，直到它们被它们的货物结合并激活。运动蛋白的非运动区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。我们将在一些激动素的非运动区中确定结合货物和调节运动活动所需的结构域。这样的结构域映射研究将揭示货物结合和马达调节的合理机制，最重要的是，精确定位执行这些活动的蛋白质的候选结合位点。沿着这些思路对传统动蛋白的初步研究证实了这一策略--我们的结构域映射研究表明，传统动蛋白的抑制状态依赖于动蛋白轻链(KLC)，并涉及到一种折叠构象，在折叠构象中，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方法分离这些KLC TPR伙伴蛋白，并鉴定它们的功能。以类似的方式，第二个和第三个特定目标建议调查其他一些已知货物的动蛋白(异三聚体动蛋白II和单体KIF1A、B和C)的非运动区。通过在这些激动素的非运动区中确定与货物结合和马达激活有关的位点，我们将再次建立分离实现这些活动的蛋白质因子的理论基础。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，我们将在这些动蛋白的非运动区引入突变，破坏运动调节或货物结合。这些激动素突变体是我们建议的重点--与野生型而不是突变型结合的蛋白质很可能是体内真正的合作伙伴。这种负性对照的产生和使用从根本上区分了这一提议与之前识别与动蛋白相互作用的蛋白质的尝试。

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.