Regulation of Coat Proteins in the ER-Golgi System

外壳蛋白调节货物招募和囊泡形成。它们的主要作用是形成包被的坑，积累特定的货物，然后发芽。在高尔基体上，COPI 涂层是在高尔基体内部运输以及将货物受体和其他回收蛋白返回内质网所必需的。许多蛋白质从高尔基体进展到细胞表面或内体需要高尔基体局部网格蛋白外壳。两种涂层均由处于 GTP 状态的小型 Arf 家族 GTP 酶（Arf1、Arf3、Arf4 或 Arf5）招募至高尔基体膜，并通过 GTP 酶的水解从脱壳囊泡中释放。高尔基体 Arf 由交换因子（主要是早期高尔基体膜上的 GBF1）招募，并由 GAP 蛋白（主要是早期高尔基体膜上的 ArfGAP1 和 ArfGAP2）释放。人们对 GAP 蛋白的作用尤其了解甚少。我们实验室的一个主要长期目标是了解这些和其他调节因子（Arf、GEF 和 GAP）在货物招募以及 COPI 和网格蛋白囊泡形成中的独特作用。根据我们的初步数据，我们假设 Arf1 和 Arf4 在 COPI 介导的转运中发挥不同的作用，其中 Arf4 在高尔基体到 ER 逆行转运中发挥着以前未认识到的特定作用。我们进一步假设，Arf4 的 GTP 水解是由 ArfGAP1 刺激的，而不是由 ArfGAP2 刺激的。在我们的第一个和第二个目标中，我们提出实验来阐明这些不同蛋白质在不同逆行货物（包括 ERGIC53 和高尔基酶）的逆行运输中的作用。作为这项工作的一部分，我们将使用超分辨率双标签 PALM/STORM 直接绘制 COPI 涂层坑中不同 Arf、ArfGAP 和货物的位置。由于我们的初步数据表明单个 COPI 凹坑可以很容易地可视化，因此这项工作应该明确解决与不同 Arf 的作用以及是否存在不同类别的 COPI 囊泡相关的问题。以前并不存在明确回答这些问题的强大技术，我们的工作可以在理解高尔基体功能方面取得重大进展。在之前的工作中，我们发现证据表明晚期高尔基体上的 Arfs 和 ArfGAP 组（调节网格蛋白囊泡形成）与调节 COPI 囊泡形成的组不同。这些网格蛋白囊泡对于 6-磷酸甘露糖受体的循环和其他多种功能非常重要。我们假设反式高尔基体网络上网格蛋白囊泡形成的一个子集受到 Arf3 和 ArfGAP3 的调节，并提出实验来完善我们的第三个目标中的这一假设，包括使用超分辨率成像。对于这些研究，我们将结合使用光学显微镜、siRNA、生物物理和生化技术。  这项研究的结果将引起研究分泌途径的细胞生物学家的极大兴趣。

Coat proteins regulate cargo recruitment and vesicle formation. Their primary role is to form coated pits which accumulate specific cargoes and then bud. On the Golgi apparatus, the COPI coat is required for trafficking inside the Golgi and also to return cargo receptors and other recycling proteins to the endoplasmic reticulum. Golgi localized clathrin coats are required for many proteins to progress from the Golgi apparatus to the cell surface or endosome. Both coats are recruited to Golgi membranes by small Arf family GTPases (Arf1, Arf3, Arf4 or Arf5) in the GTP state, and released from uncoating vesicles by hydrolysis of the GTPase. Golgi Arfs are recruited by exchange factors (primarily GBF1 on early Golgi membranes) and released by GAP proteins (primarily ArfGAP1 and ArfGAP2 on early Golgi membranes). The roles of the GAP proteins in particular, are poorly understood. A major long term objective of our laboratory is to understand the distinct roles of these and other regulatory factors (Arfs, GEFs and GAPs) in cargo recruitment and in the formation of both COPI and clathrin vesicles. Based on our preliminary data we hypothesize that Arf1 and Arf4 play distinct roles in COPI mediated trafficking, with Arf4 playing a specific role in Golgi to ER retrograde trafficking not previously recognized. We further hypothesize, that GTP hydrolysis of Arf4 is stimulated by ArfGAP1, but not ArfGAP2. In our first and second aims we propose experiments to clarify the roles of these different proteins in the retrograde trafficking of different retrograde cargoes, including ERGIC53 and Golgi enzymes. As part of this work, we will use super-resolution double label PALM/STORM to directly map the locations of different Arfs, ArfGAPs and cargoes in COPI coated pits. As our preliminary data indicates that individual COPI pits can be easily visualized, this work should definitively resolve questions related to the roles of the different Arfs, and whether there are distinct classes of COPI vesicles. Powerful techniques for unambiguously answering these question have not previously existed, and our work can create major advances in understanding of the functioning of the Golgi apparatus. In previous work, we have found evidence that the set of Arfs and ArfGAPs on the late Golgi apparatus (regulating clathrin vesicle formation) is distinct from those regulating COPI vesicle formation. These clathrin vesicles are important for cycling of the mannose 6-phosphate receptors, and other diverse functions. We hypothesize that a subset of clathrin vesicle formation on the trans Golgi network is regulated by Arf3 and ArfGAP3 and propose experiments to refine this hypothesis in our third aim, including the use of super-resolution imaging. For these studies, we will use a combination of light microscopy, siRNA, biophysical and biochemical techniques.  The results of this study will be of great interest to cell biologists studying the secretory pathway.