Brefeldin A inhibited activity of the Sec7 domain of p200, a mammalian guanine nucleotide-exchange protein for ADP-ribosylation factors

A brefeldin A (BFA)-inhibited guanine nucleotide-exchange protein (GEP) for ADP-ribosylation factors (ARF) was purified earlier from bovine brain cyto sol. Cloning and expression of the cDNA confirmed that the recombinant prot ein (p200) is a PEA-sensitive ARF GEP. p200 contains a domain that is 50% i dentical in amino acid sequence to a region in yeast Sec7, termed the Sec7 domain. Sec7 domains have been identified also in other proteins with ARF G EP activity, some of which are not inhibited by BFA. To identify structural elements that influence GEP activity and its BFA sensitivity, several trun cated mutants of p200 were made. Deletion of sequence C-terminal to the Sec 7 domain did not affect GEP activity. A protein lacking 594 amino acids at the N terminus, as well as sequence following the Sec7 domain, also had hig h activity, The mutant lacking 630 N-terminal amino acids was, however, onl y 1% as active, as was the Sec7 domain itself (mutant lacking 697 N-termina l residues). It appears that the Sec7 domain of p200 contains the catalytic site but additional sequence (perhaps especially that between positions 59 5 and 630) modifies activity dramatically. Myristoylated recombinant ARFs w ere better than non-myristoylated as substrates; ARFs 1 and 3 were better t han ARF5, and no activity was detected with ARF6. Physical interaction of t he Sec7 domain with an ARF1 mutant was demonstrated, but it was much weaker than that of the cytohesin-1 Sec7 domain with the same ARF protein. Effect s of BFA on p200 and all mutants with high activity were similar with simil ar to 50% inhibition at less than or equal to 50 mu M. The inactive BFA ana logue B36 did not inhibit the Sec7 domain or p200. Thus, the Sec7 domain of p200, like that of Sec7 itself (Sata, M., Donaldson, J. G., Moss, J., and Vaughan, M. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 4204-4208), plays a role in BFA inhibition as well as in GEP activity, although the latter is m arkedly modified by other structural elements.