Regulation of Coat Proteins in the ER-Golgi System

衣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覆的坑，积聚特定的货物，然后发芽。在高尔基体上，COPI被用于高尔基体内部的运输，也用于将货物受体和其他循环蛋白返回内质网。许多蛋白质从高尔基体进入细胞表面或核内体都需要高尔基定位的网格蛋白外壳。在GTP状态下，这两种外壳都被小的Arf家族GTPase （Arf1, Arf3， Arf4或Arf5）招募到高尔基膜上，并通过GTPase的水解从脱膜囊泡中释放出来。高尔基蛋白通过交换因子（主要是早期高尔基膜上的GBF1）募集，并由GAP蛋白（主要是早期高尔基膜上的ArfGAP1和ArfGAP2）释放。特别是GAP蛋白的作用，人们知之甚少。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（Arfs， GEFs和gap）在货物招募和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介导的转运中发挥着不同的作用，其中Arf4在高尔基体到内质网逆行转运中起着特定的作用，这是以前没有认识到的。我们进一步假设，ArfGAP1刺激Arf4的GTP水解，而不是ArfGAP2。在我们的第一个和第二个目标中，我们提出了实验来阐明这些不同蛋白质在不同逆行货物的逆行运输中的作用，包括ERGIC53和高尔基酶。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Arfs， arfgap和货物的位置。由于我们的初步数据表明，单个COPI小泡可以很容易地可视化，这项工作应该明确地解决与不同Arfs的作用相关的问题，以及是否存在不同类型的COPI小泡。明确回答这些问题的强大技术以前并不存在，我们的工作可以在理解高尔基体的功能方面取得重大进展。在之前的工作中，我们已经发现证据表明，晚期高尔基体（调节网格蛋白囊泡形成）上的Arfs和arfgap与调节COPI囊泡形成的Arfs和arfgap不同。这些网格蛋白囊泡对于甘露糖6-磷酸受体的循环和其他多种功能至关重要。我们假设反式高尔基网络中网格蛋白囊泡形成的一个子集是由Arf3和ArfGAP3调控的，并在我们的第三个目标中提出了实验来完善这一假设，包括使用超分辨率成像。对于这些研究，我们将使用光学显微镜，siRNA，生物物理和生化技术的组合。这项研究的结果将对研究分泌途径的细胞生物学家有很大的兴趣。

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.