Regulation of Coat Proteins in the ER-Golgi System

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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.