Dissecting the functions of yeast COPI - Resubmission 01

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DESCRIPTION (provided by applicant): The coat protein I (COPI) complex is important for membrane traffic and compartmental organization in the secretory pathway, but the biological roles of COPI are still remarkably uncertain. Even though COPI is postulated to be essential for secretion and for Golgi cisternal maturation, yeast COPI temperature- sensitive (ts) mutants show surprisingly mild defects: secretory traffic is blocked only for some cargo proteins, and cisternal maturation is slowed but not arrested. In other studies, displacement of COPI from membranes alters Golgi architecture, but the underlying mechanism is unknown. We propose to clarify the action of COPI in Saccharomyces cerevisiae. Our main approach is a refined version of the "anchor-away" method. Addition of rapamycin causes a tagged protein to be sequestered at an anchor site, thereby functionally inactivating the tagged protein. Preliminary data confirm that COPI can be sequestered and inactivated by rapamycin within minutes. In addition, we are optimizing a method for correlative fluorescence and electron microscopy of yeast. The working hypothesis is that (a) COPI is essential for intra-Golgi traffic because COPI vesicles drive cisternal maturation, and (b) COPI negatively regulates homotypic fusion at the early Golgi. Specific Aim #1 is to test the role of COPI in Golgi cisternal maturation. The hypothesis is that COPI vesicles recycle resident Golgi proteins from older to younger cisternae, thereby driving cisternal maturation. By combining the anchor-away method with our established video microscopy technique for visualizing yeast Golgi dynamics, we will test whether cisternal maturation requires COPI. The prediction is that when COPI is inactivated, cisternal maturation will "freeze". Specific Aim #2 is to test the role of COPI in traffic through the secretory pathwa. The hypothesis is that all protein traffic through the secretory pathway requires COPI. Secretory traffic will be assayed by pulse-chase analysis. The prediction is that inactivation of COPI will completely block secretory traffic by preventing the recycling of key trafficking components such as SNAREs and ER export receptors. Specific Aim #3 is to test the role of COPI in Golgi homotypic fusion. The hypothesis is that in addition to promoting vesicle formation, COPI restrains the homotypic fusion of early Golgi membranes. In this context, COPI may negatively regulate Golgi cisternal size and Golgi fenestration. We will test these ideas using fluorescence and electron microscopy. The prediction is that a controlled reduction of Golgi-associated COPI will enhance homotypic fusion. For over a decade, the function of COPI has been one of the central mysteries in the secretion field. New yeast tools will enable us to make major inroads into this problem.