ARF function requires the regulated alternation between GTP-bound active and GDP-bound inactive forms. GTP binding is catalyzed by guanine nucleotide-exchange proteins (GEPs) and inactivation by GTPase- activating proteins (GAPs). Two general types of GEPs have been recognized, a family of ~200-kDa proteins that are inhibited by BFA (a drug that interferes with protein secretion and causes reversible disintegration of Golgi cisternae) and smaller ~55-kDa GEPs that are BFA-resistant. All GEPs have so-called Sec7 domains of ~200 amino acids that are responsible for the GEP activity, as well as its BFA sensitivity. We had earlier purified two BFA-inhibited GEPs and cloned one cDNA. This year, detailed characterization of the recombinant protein was completed. Removal of sequence C-terminal to the Sec7 domain plus up to 596 residues from the N-terminus had little effect on activity, but further deletion of 36 N-terminal amino acids decreased activity ~200-fold to the level of the Sec7 domain itself. Ongoing work will identify the structural elements responsible for the dramatic enhancement of Sec7 domain catalytic activity. The second of the BFA- inhibited GEPs, termed BIG2, was cloned, characterized, and reported this year. Deduced amino acid sequences of the bovine and human proteins are 99.5% identical and ~74% identical to that of the previously characterized BIG1. By comparison of their functions, expression and subcellular distributions, we should obtain clues to the biological roles of these two apparently very similar BFA-sensitive GEPs. - ARF, brefeldin A, guanine nucleotide-exchange protein, Sec7, arfaptin
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