Abstract
Activation of ADP-ribosylation factors (ARFs), approximately 20-kDa 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: approximately 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 approximately 50-kDa GEPs that are insensitive to BFA. We describe here two human brain cDNAs that encode BFA-inhibited GEPs. One is a approximately 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 approximately 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 approximately 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 approximately 50-kDa BFA-insensitive ARF GEPs are known, these new clones for the approximately 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.
Highlights
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AF084520 and AF084521
Its Sec7 domain was demonstrated relatively recently to function as a brefeldin A (BFA)-inhibited ADP-ribosylation factors (ARFs) guanine nucleotide-exchange proteins (GEPs) [16]
Human BIG1 and BIG2 cDNAs—BIG2 was initially purified as part of a macromolecular complex of ϳ670 kDa from bovine brain
Summary
Vol 274, No 18, Issue of April 30, pp. 12308 –12315, 1999 Printed in U.S.A. Purification and Cloning of a Brefeldin A-inhibited Guanine Nucleotide-exchange Protein for ADP-ribosylation Factors*. Replacement of GDP by GTP is accelerated by ARF GEPs or guanine nucleotide-exchange proteins, several of which have been identified [7] These include Gea and Gea from yeast [8], mammalian B2-1 or cytohesin-1 [9] and cytohesin-2 or ARNO [10], which are 83% identical in amino acid sequence, and GRP1 (general receptor for phosphoinositides), a third member of the cytohesin group [11]. BFA is a fungal fatty acid metabolite with a monocyclic lactone ring that blocks protein secretion reversibly and causes apparent collapse of Golgi membranes into the endoplasmic reticulum [17, 18] These effects result from BFA inhibition of GEP-catalyzed ARF activation (GTP binding) [19, 20]. We propose that because of their BFA sensitivity and size relative to the cytohesins, these mammalian ARF GEPs be named BIG1 (for BFA-inhibited GEP) and BIG2, respectively
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