Regulation of cytoplasmic dynein in vivo

摘要 基于微管的细胞内转运对于所有真核细胞的功能至关重要。细胞质 动力蛋白是一种负末端导向的微管马达，可运输多种货物，动力蛋白-货物 相互作用需要动力蛋白复合物和特定的货物衔接子。动力蛋白与货物的相互作用 在体内调节以及动力蛋白活性如何与货物结合协调是主要的开放性问题， 外地最近，结构研究和体外运动性测定得出了这样的假设： 运输到动力蛋白Arp 1细丝尖端的货物可以通过改变 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构象变化中蛋白质的缺失和身份仍有待确定。我们 先前发现，在构巢曲霉中，动力蛋白的尖端蛋白p25对动力蛋白很重要， 早期内体相互作用最近，我们发现，p25是必需的相互作用， 动力蛋白-动力蛋白和早期核内体动力蛋白衔接子HookA。根据最近的数据，我们 假设p25是调节动力蛋白构象关键蛋白。具体目标1是揭示 dynactin p25和货物适配器HookA在调节dynactin中的作用。基于敏化发射 FRET成像，微管沉淀和体外运动试验将用于实现这一目标。在 此外，我们最近的遗传学工作已经发现了一种类似vezatin的蛋白质，VezA，作为一种新的调节剂， 动力蛋白-货物相互作用VezA不像HookA那样是货物适配器，但会影响动力蛋白的积累 在货物装载点，微管正端。VezA如何调节动力蛋白仍然是一个谜。 具体目标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识别VezA动力蛋白途径中的新参与者。

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.