Structural basis for the inhibitory effect of brefeldin A on guanine nucleotide-exchange proteins for ADP-ribosylation factors

Protein secretion through the endoplasmic reticulum and Golgi vesicular trafficking system is initiated by the binding of ADP-ribosylation factors (ARFs) to donor membranes, lending to recruitment of coatomer, bud formation, and eventual vesicle release. ARFs are approximate to 20-kDa GTPases that are active with bound GTP and inactive with GDP bound. Conversion of ARF-GDP to ARF-GTP is regulated by guanine nucleotide-exchange proteins. All known ARF guanine nucleotide exchange proteins contain a Sec7 domain of approximate to 200 amino acids that includes the active site and fall into two classes that differ in molecular size and susceptibility to inhibition by the fungal metabolite brefeldin A (BFA). To determine the structural basis of BFA sensitivity, chimeric molecules were constructed by using sequences from the Sec7 domains of BFA-sensitive yeast Sec7 protein (vSec7d) and the insensitive human cytohesin-1 (C-1Sec7). Biased on BFA inhibition of the activities of these molecules with recombinant yeast ARF2 as substrate, the Asp(965)-Met(975) sequence in ySec7d was shown to be responsible for BFA sensitivity. A C-1Sec7 mutant in which Ser(199), Asn(204), and Pro(209) were replaced with the corresponding ySec7d amino acids, Asp(965), Gln(970), and Met(975), exhibited BFA sensitivity similar to that of recombinant ySec7d (rySec7d). Single replacement in C-1Sec7 of Ser(199) or Pro(209) resulted in partial inhibition by BFA, whereas replacement of Gln(970) in ySec7d with Asn (as found in C-1Sec7) had no effect. ils predicted, the double C-1Sec7 mutant with S199D and k209M was BFA-sensitive, demonstrating that Asp(965) and Met(975) in ySec7d are major molecular determinants of BFA sensitivity.