Purification and cloning of a brefeldin A-inhibited guanine nucleotide- exchange protein for ADP-ribosylation factors

Abstract

Activation of ADP-ribosylation factors (ARFs), ~20kDa guanine nucleotide-binding proteins that play an important role in intracellular vesicular trafficking, depends on guanine nucleotide-exchange proteins (GEPs), which accelerate replacement of bound GDP with GTP. Two major families of ARF GEPs are known: ~200-kDa molecules that are inhibited by brefeldin A (BFA), a fungal metabolite that blocks protein secretion and causes apparent disintegration of Golgi structure, and ~50-kDa GEPs that are insensitive to BFA. We describe here two human brain cDNAs that encode BFA- inhibited GEPs. One is a ~209-kDa protein 99.5% identical in deduced amino acid sequence (1,849 residues) to a BFA-inhibited ARF GEP (p200) from bovine brain. The other smaller protein, which is ~74% identical (1,785 amino acids), represents a previously unknown gene. We propose that the former, p200, be named BIG1 for (brefeldin A-inhibited GEP1) and the second, which encodes a ~202-kDa protein, BIG2. A protein containing sequences found in BIG2 had been purified earlier from bovine brain. Human tissues contained a 7.5-kilobase BIG1 mRNA and a 9.4-kilobase BIG2 transcript. The BIG1 and BIG2 genes were localized, respectively, to chromosomes 8 and 20. BIG2, synthesized as a His6 fusion protein in Sf9 cells, accelerated guanosine 5'- 3-O-(thio)triphosphate binding by recombinant ARF1, ARF5, and ARF6. It activated native ARF (mixture of ARF1 and ARF3) more effectively than it did any of the nonmyristoylated recombinant ARFs. BIG2 activity was inhibited by BFA in a concentration-dependent manner but not by B17, a structural analog without effects on Golgi function. Although several clones for ~50-KDa BFA- insensitive ARF GEPs are known, these new clones for the ~200-kDa BIG1 and BIG2 should facilitate characterization of this rather different family of proteins as well as the elucidation of mechanisms of regulation of BFA- sensitive ARF function in Golgi transport.