MECHANISMS OF AP-3 FUNCTION IN VESICLE FORMATION AND GOLGI MATURATION

项目概要 分泌途径折叠、组装和分类膜和分泌蛋白，并将它们靶向 细胞内外的不同位置。细胞的“大中央车站”是跨高尔基体网络 (TGN)，一种极其动态且复杂的结构，可将货物包装到单独的集装箱中并将其分派到终端目的地。 AP-3 运输复合物从内体或内体中对货物进行分类 高尔基体进入容器，绕过内体多泡体，进入内溶酶体区室 和 ESRT 途径。 AP-3 是 HIV 和其他病毒衣壳的高效组装所必需的。遗传性病变 AP-3 功能受损会导致多种疾病，包括 2 型赫曼斯基-普德拉克综合征和癫痫样综合征。尽管 AP-3 很重要，但人们对它的功能机制知之甚少。 在这个合作项目中，我们利用已发表和未发表的遗传、细胞生物学和生化进展，并采用最先进的成像方法来剖析 AP-3 出芽的机制和 AP-3 功能对模型生物酵母中 TGN 整体动态的影响 酿酒酵母。在目标 1 中，我们以蛋白质组学调查、功能基因组调查和后续分析为基础，其中 鉴定新的候选 AP-3 辅因子，包括泛素连接酶。我们将利用这些进展来测试有关 TGN 中 AP-3 囊泡的组装、成熟和调度的假设。在目标 2 中，我们建立在 初步研究结果表明，AP-3 活性的破坏会导致高尔基体结构发生惊人且意想不到的整体扰动。根据已发表和未发表的数据，我们假设不同的运营商 AP-3 航母以有计划的、连续的方式退出 TGN，提前离开。我们将对此进行进一步测试 建模并剖析事件序列被编程的分子机制。在一起， 这些研究应该为高尔基体-内溶酶体系统中的 AP-3 囊泡形成和细胞器成熟动力学建立新的范例。

PROJECT SUMMARY The secretory pathway folds, assembles, and sorts membrane and secreted proteins and targets them to a variety of locations inside and outside the cell. The `Grand Central Station' of the cell is the trans Golgi network (TGN), an incredibly dynamic and complex structure that packages cargo into separate containers and dispatches them to their terminal destinations. The AP-3 trafficking complex sorts cargo from endosomes or the Golgi into containers bound for endolysosomal compartments, bypassing the endosomal multivesicular body and ESCRT pathway. AP-3 is needed for productive assembly of HIV and other viral capsids. Genetic lesions impairing AP-3 function cause multiple diseases, including Hermansky-Pudlak Syndrome type 2 and an epilepsy-like syndrome. Despite its importance, remarkably little is known about the mechanisms of AP-3 function. In this collaborative Project, we exploit published and unpublished genetic, cell biological, and biochemical advances, and employ state of the art imaging approaches, to dissect the mechanisms of AP-3 budding and the consequences of AP-3 function for the overall dynamics of the TGN, in the model organism Saccharomyces cerevisiae. In Aim 1, we build on proteomic surveys, functional genomic surveys, and follow-up analyses, wich identify new candidate AP-3 cofactors including a ubiquitin ligase. We will use these advances to test hypotheses about the assembly, maturation, and dispatch of AP-3 vesicles from the TGN. In Aim 2, we build on preliminary findings showing that disruption of AP-3 activity causes striking and unexpected global perturbations of Golgi architecture. We hypothesize, based on published and unpublished data, that distinct carriers exit the TGN in a programmed, sequential manner, with AP-3 carriers departing early. We will further test this model and dissect the molecular mechanisms though which the sequence of events is programmed. Together, these studies should establish new paradigms for AP-3 vesicle formation and for organelle maturation dynamics in the Golgi-endolysosomal system.