THE REGULATION OF GOLGI COAT PROTEIN BY ARF AND BREFELDIN A

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The main objectives of this project are the elucidation of the cellular functions of the family of ADP-ribosylation factors (ARFs) and the mechanism of their regulation. As Brefeldin A (BFA) has recently been shown to affect ARF functions and cellular distribution, we have recently expanded our efforts to include a better understanding of the mechanism of BFA action. Specific inhibitors of ARF activities have been exploited to provide evidence for the role of ARF proteins in a variety of intracellular membrane events. Anti-ARF peptides were found to be potent inhibitors of ER-Golgi transport, intra-Golgi transport, and endosome-endosome fusion. In addition, sensitivity of each of these assays, as well as nuclear membrane fusion, to GTPgammaS was shown to be mediated by ARF proteins. A search for regulators of ARF activity has identified an activity on Golgi membranes which appears to activate ARF. This activity is being investigated as a potential site of action of Brefeldin A and for its potential role as regulator of the protein secretory pathway. The ability of Brefeldin A to alter the structure of the Golgi and location of specific coat proteins (ARF and beta-COP) is being investigated in concert with the development of Brefeldin A as a potential anticancer agent. We have used inducible mammalian expression vectors to control the expression of ARF genes. These cell lines will be used to analyze the effects of varied expression of wild-type and mutant ARF proteins on the secretory pathway and on the integrity of intracellular organelles, e.g. Golgi complex. The three dimensional crystal structure of ARF is also being solved to aid in the determination of critical functional domains and ultimate design of inhibitors.