FUNCTIONAL CHARACTERIZATION OF ARF REGULATORY FACTORS

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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. Increasing evidence has accumulated in the past year that supports our hypothesis that membrane phospholipids play a critical and dynamic role in the actions of ARF proteins. As Brefeldin A (BFA) has recently been shown to affect ARF functions and cellular distribution, we have expanded our efforts to include a better understanding of the mechanism of BFA action. ARF proteins are unique among the approximately 100 known GTP binding proteins in having a strict dependence on phospholipids to allow nucleotide exchange. Recent work suggests that in addition to a general lipid dependence there are also distinct effects of specific phospholipids that dramatically effect ARF activities. Specifically, ARF binds PIP2 with high affinity, resulting in a protein with reduced affinity for guanine nucleotides. Thus, we are testing the hypothesis that lipids may be the source of the guanine nucleotide exchange factor, observed for many other GTP binding proteins. The N-myristoylation of ARF proteins is a normal co-translation modification occurring in eukaryotic cells. The role of the myristate moiety is also under investigation and may serve as a model for other regulatory myristoylated proteins; e.g. p60 src, cAMP dependent protein kinase, etc. A protein with the ability to specifically bind ARF and promote the GTPase activity by ARF1 has been identified and partially purified. This ARF-GAP (GTPase activating protein) is under investigation as both potential effector and down-regulator of ARF actions.