Chaperone Mechanisms in Clathrin Mediated Neuronal Vesicle Trafficking

 描述(由申请人提供)：蛋白质和膜的运输依赖于精心设计的反应，这些反应使膜变形，并顺序地组装和分解蛋白质复合体。Hsp70--参与蛋白质折叠和许多细胞蛋白质加工反应的普遍存在的伴侣蛋白--在运输过程中发挥马达的作用，例如蛋白质转位到内质网和线粒体，以及分解新生内吞小泡周围形成的网状蛋白外壳。Hsp70的普遍作用表明，在这些看似不同的反应中，有一种共同的机制被利用，但是虽然我们了解Hsp70是如何结合和释放蛋白质底物的，但我们对Hsp70如何产生移动蛋白质或分解蛋白质复合体的力的理解是有限的。与Hsp70介导的蛋白质移位或异质聚集体解离等过程不同，可以实时准确地监测外壳拆解，知道所有参与者的结构和反应快照，涂层稳定性可以很容易地控制，并且拆卸所需的单一Hsc70结合位点是已知的。通过利用这些特征和我们开发的在细菌中生产功能网状蛋白的协议，我们获得了证据，表明之前提出的功率冲程模型和布朗棘轮/空间楔形模型都不能解释Hsc70如何分解外套。相反，我们的数据表明，涂层是通过涂层墙和Hsc70之间的碰撞产生的压力来分解的，Hsc70与柔性系绳紧密相对。我们还发现，所有HSP70的特征--自我关联--放大了这种力量，从而为一种从未出现过的普遍现象提供了生物功能。在我们提议的工作中，我们将测试和提炼这种Hsp70力产生的碰撞压力机制(目标1)。Hsc70与其他伴侣相互作用，其中Hsp110起Hsp70核苷酸交换因子(NEF)的作用。我们确定了Hsc70：Hsp110复合体的结构，并利用这些结构信息设计了实验，揭示了Hsp110在突触小泡循环过程中调节Hsc70分子伴侣的作用。后一项实验使用了巨大的七鳃鳗网状脊髓突触，它代表了我们体外clathrin/Hsc70系统的体内实验补充。由于神经元突触是主要用于膜运输的隔室，它们为解决运输中的基本生物学问题以及伴侣在这些过程中的作用提供了一个特殊的系统。在我们提出的研究中，我们将利用体外的clathrin/Hsc70系统和体内的Lamprey RS突触来进一步确定Hsp110的核苷酸交换机制(目标2)，以及它在膜运输过程中调节Hsc70和笼蛋白的有效性的机制(目标3)。这些研究将增进我们对Hsp70如何产生移位蛋白质和重塑蛋白质复合体的力量，以及它们的活动是如何受到调控的理解。他们还将阐明突触传递和网状蛋白介导的囊泡运输的基本机制。

 DESCRIPTION (provided by applicant): Protein and membrane trafficking depends on choreographed reactions which deform membranes and sequentially assemble and disassemble protein complexes. Hsp70s--the ubiquitous chaperones involved in protein folding and many cellular protein processing reactions--function as motors in trafficking processes such as translocation of proteins into ER and mitochondria, and disassembly of the clathrin coats that form around nascent endocytic vesicles. The ubiquitous role of Hsp70s suggests a common mechanism is being harnessed in these seemingly disparate reactions, but while we understand how Hsp70s bind and release protein substrates, our understanding of how Hsp70s generate force to move proteins or take apart protein complexes is limited. Clathrin coat disassembly provides an exceptional system to study these force generation mechanisms: unlike Hsp70 mediated processes such as protein translocation or dissociation of heterogeneous aggregates, coat disassembly can be precisely monitored in real time, structures of all players and reaction snapshots are known, coat stability can be easily controlled, and the single Hsc70 binding site in clathrin required for disassembly is known. By exploiting these features and our development of protocols for producing functional clathrin in bacteria, we have obtained evidence that neither previously proposed power- stroke nor Brownian ratchet/steric wedge models can explain how Hsc70 disassembles coats. Instead, our data indicate that coats are disassembled through pressure generated by collisions between coat walls and Hsc70s bound to flexible tethers in close apposition to these walls. We also discovered that self-association, characteristic of all Hsp70s, amplifies this force, thus providing a biological function for a ubiquitous phenomenon that has never had one. In our proposed work we will test and refine this collision pressure mechanism of Hsp70 force generation (Aim 1). Hsc70 cooperates with other chaperones, among them Hsp110, which acts as an Hsp70 nucleotide exchange factor (NEF). We determined the structure of the Hsc70:Hsp110 complex, and used the structural information to design experiments which revealed that Hsp110 regulates Hsc70 chaperoning of clathrin during synaptic vesicle recycling. The latter experiments used the giant lamprey reticulospinal synapse, which represents the in vivo experimental complement to our in vitro clathrin/Hsc70 system. Because neuronal synapses are compartments devoted primarily to membrane trafficking, they provide an exceptional system for addressing fundamental biological questions in trafficking and the role of chaperones in these processes. In our proposed studies we will exploit both the in vitro clathrin/Hsc70 system and the in vivo, lamprey RS synapse to further define the nucleotide exchange mechanism of Hsp110 (Aim 2), and the mechanism by which it regulates Hsc70 and clathrin availability during membrane trafficking (Aim 3). These studies will advance our understanding of how Hsp70s generate the forces by which they translocate proteins and remodel protein complexes, and of how their activities are regulated. They will also elucidate fundamental mechanisms underlying synaptic transmission and clathrin mediated vesicular trafficking.