CHARACTERIZATION OF THE ARF FAMILY OF REGULATORY GTP-BINDING PROTEINS

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The ARF proteins are a sub-family of structurally and functionally conserved 21 kDa GTP binding proteins. ARF has now been shown to have essential functions in the maintenance of the integrity and proper functioning of the endoplasmic reticulum, Golgi complex, and endosome compartments. These activities are the targets for the actions of Brefeldin A, a fungal metabolite under development in the the Developmental Therapeutics Program as a potential anti-neoplastic agent in man. Work described in this project focuses on characterizing the members of the ARF family with regard to sequence, cellular location, and functions. We have discovered that in humans this family consists not only of the five previously identified gene products, but of more than 10 structurally related genes and their products. We are engaged in cloning and sequencing this newly enlarged family to begin to document its size, functions and importance to cell physiology and pathophysiology. In addition, as sequence information becomes available we continue to produce more specific antisera for diagnostic purposes and immunolocalization. We have continued to exploit simpler eukaryotic organisms, particularly yeast, to identify new functions for ARF proteins. We will perform functional screens to identify extragenic suppressors of ARF mutations in efforts to identify other components of the ARF pathway and potential regulators of ARF activities. Expression of a number of mutants in yeast has also served as background for similar studies now underway to express wild-type and mutant ARF proteins in mammalian cells in culture. An exhaustive screen has now identified at least one temperature sensitive allele of ARF1 which will be used to assist in defining functions of ARF, consequences of an arf null cell, and functional screens for extragenic suppressors of the arf null cell. The three dimensional crystal structure of ARF is also being solved to aid in the determination of critical functional domains and ultimate design of specific inhibitors.