Regulation of cytoplasmic dynein in vivo

摘要 基于微管的细胞内转运对所有真核细胞的功能至关重要。细胞质 动力蛋白是一种负端定向的微管马达，可以运输各种货物，以及动力蛋白货物 相互作用需要动力蛋白复合体和特定的货物适配器。动力蛋白与货物的相互作用如何 体内调节以及动力蛋白活性如何与货物结合相协调是目前尚未解决的主要问题 田野。最近，结构研究和体外运动分析导致了一种假设，即结合 连接到dynactin Arp1细丝尖端的货物可能通过改变 它的构象，导致动力蛋白激活。然而，支持这一假设的体内证据是 P150‘S构象变化所涉及的蛋白质缺失和同源性尚待确定。我们 此前发现，尼杜拉曲霉中动力蛋白的尖端蛋白p25对动力蛋白具有重要作用。 早期的内小体相互作用。最近，我们发现p25是相互作用所必需的。 动力蛋白-动力蛋白和Hooka，早期的内体动力蛋白适配器。根据我们最近的数据，我们 假设p25是调节动力蛋白构象的关键蛋白。具体目标1是揭示 动力蛋白p25和货物接头钩子在调节动力蛋白中的作用。基于敏化发射的 为了实现这一目标，将使用FRET成像、微管丸化和体外动力分析。在……里面 此外，我们最近的基因研究发现了一种维他丁样蛋白veza，作为一种新的调节因子。 动力蛋白-货物相互作用。Veza不是像Hooka那样的货物适配器，但会影响动力蛋白的积累 在装货现场，微管加端。Veza是如何调节dynein的，这仍然是个谜。 具体目的2是确定veza在动力蛋白介导的囊泡运输中的作用机制。我们 将使用蛋白质组学和遗传学方法来确定veza-dynein途径中的新成员。

Abstract Microtubule-based intracellular transport is critical for the function of all eukaryotic cells. Cytoplasmic dynein is a minus-end-directed microtubule motor that transports a variety of cargoes, and dynein-cargo interactions require the dynactin complex and specific cargo adapters. How dynein-cargo interactions are regulated in vivo and how dynein activity is coordinated with cargo binding are major open questions in the field. Recently, structural studies and in vitro motility assays led to the hypothesis that the binding of cargo to the pointed end of dynactin's Arp1 filament may unlock dynactin's p150 subunit via a change in its conformation, leading to dynein activation. However, in vivo evidence supporting this hypothesis is missing and the identity of proteins involved in p150's conformational change remain to be defined. We previously found that dynactin's pointed-end protein p25 in Aspergillus nidulans is important for dynein- early endosome interaction. Recently, we discovered that p25 is required for the interaction between dynein-dynactin and HookA, the early endosome dynein adapter. Based on our recent data, we hypothesize that p25 is a key protein regulating dynactin conformation. Specific Aim 1 is to reveal the roles of dynactin p25 and the cargo adapter HookA in regulating dynactin. Sensitized emission-based FRET imaging, microtubule pelleting and in vitro motility assays will be used to achieve this aim. In addition, our recent genetic work has uncovered a vezatin-like protein, VezA, as a novel regulator for dynein-cargo interaction. VezA is not a cargo adapter like HookA but affects the accumulation of dynein at the cargo-loading site, the microtubule plus end. How VezA regulates dynein remains a mystery. Specific Aim 2 is to determine the mechanism of VezA action in dynein-mediated vesicle transport. We will use proteomic and genetic approaches to identify new players in the VezA-dynein pathway.