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. 1999 Mar 16;96(6):2752-7.
doi: 10.1073/pnas.96.6.2752.

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

M Sata  1 , J Moss, M Vaughan
Affiliations

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

M Sata et al. Proc Natl Acad Sci U S A. .

Abstract

Protein secretion through the endoplasmic reticulum and Golgi vesicular trafficking system is initiated by the binding of ADP-ribosylation factors (ARFs) to donor membranes, leading to recruitment of coatomer, bud formation, and eventual vesicle release. ARFs are approximately 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 approximately 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 (ySec7d) and the insensitive human cytohesin-1 (C-1Sec7). Based on BFA inhibition of the activities of these molecules with recombinant yeast ARF2 as substrate, the Asp965-Met975 sequence in ySec7d was shown to be responsible for BFA sensitivity. A C-1Sec7 mutant in which Ser199, Asn204, and Pro209 were replaced with the corresponding ySec7d amino acids, Asp965, Gln970, and Met975, exhibited BFA sensitivity similar to that of recombinant ySec7d (rySec7d). Single replacement in C-1Sec7 of Ser199 or Pro209 resulted in partial inhibition by BFA, whereas replacement of Gln970 in ySec7d with Asn (as found in C-1Sec7) had no effect. As predicted, the double C-1Sec7 mutant with S199D and P209M was BFA-sensitive, demonstrating that Asp965 and Met975 in ySec7d are major molecular determinants of BFA sensitivity.

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Figures

Figure 1
Figure 1
Determination of ySec7d region required for BFA inhibition by using ySec7d/C-1Sec7 chimeras. (A) ryARF2 (50 pmol) and rySec7d (42 pmol), C-1Sec7 (45 pmol), or 50 pmol of each chimeric protein were incubated with 4 μM [35S]GTP[γS] in a total volume of 100 μl without (black bars) or with (gray bars) 6 μg of BFA (0.2 mM) for 3 h at 4°C before radioassay of protein-bound [35S]GTP[γS]. ryARF2 and GEPs were also incubated individually as controls (first six lanes). The experiment was repeated twice, with similar results. Data are means ± SEM of values from triplicate assays. Errors <0.03 pmol are not shown. (B) Diagram of ySec7d, C-1Sec7, and chimeric molecules.
Figure 2
Figure 2
Localization of determinants of BFA inhibition in the C-terminal one-third of ySec7d. (A) ryARF2 and 50 pmol of each chimeric GEP were incubated with 4 μM [35S]GTP[γS] without (black bars) or with (gray bars) 6 μg of BFA for 3 h at 4°C before collection of proteins for radioassay. ryARF2 and chimeric GEPs were also incubated individually as controls. Two independent experiments were performed with similar results. Errors <0.01 pmol are not shown. (B) Diagram of ySec7d/C-1Sec7 chimeras. Data are presented as in Fig. 1.
Figure 3
Figure 3
Alignment of Asp965–Met975 sequence of ySec7d with corresponding regions of other GEPs. Asp965, Gln970, and Met975 are highly conserved among BFA-sensitive ARF GEPs, whereas the corresponding Ser199, Asn204, and Pro209 in C-1Sec7 are conserved in BFA-insensitive ARF GEPs (boxed with solid lines). CYC(SNP) was made by replacing Asp965, Gln970, and Met975 in chimera CYC with Ser199, Asn204, and Pro209 as in C-1Sec7. In C-1Sec7, Ser199, Asn204, and Pro209 were replaced with the corresponding amino acids in ySec7d to yield C(DQM). The double mutant C(DM) contains Asp replacing Ser199 and Met replacing Pro209 in C-1Sec7. Mutated residues are in boldface. Numbers above the sequence correspond to positions in ySec7p. Hyphen indicates amino acid identical to that in the sequence at the top of each group.
Figure 4
Figure 4
Effect on BFA sensitivity of three residue replacements in C-1Sec7 and CYC. ryARF2 (50 pmol) and 50 pmol of each GEP mutant were incubated with 4 μM [35S]GTP[γS] without (black bars) or with (gray bars) 6 μg of BFA (0.2 mM) for 3 h at 4°C before assay of protein-bound [35S]GTP[γS]. ryARF2 and mutants were also incubated individually as controls. Data are presented as in Fig. 1. The experiment was repeated twice with similar results. Errors <0.02 pmol are not shown.
Figure 5
Figure 5
Specific amino acids in the ySec7d Asp965–Met975 sequence required for BFA inhibition. Ser199, Asn204, and Pro209 in C-1Sec7 were replaced by the corresponding amino acids in ySec7d (Asp965, Gln970, and Met975, respectively) to produce C(S199D), C(D204Q), and C(P209M). Gln970 in ySec7d was changed to Asn in Y(Q970N). ryARF2 (50 pmol), 50 pmol of each mutant protein, and 4 μM [35S]GTP[γS] were incubated for 3 h at 4°C without (black bars) or with (gray bars) 6 μg of BFA (0.2 mM) before assay of protein-bound [35S]GTP[γS]. ryARF2 and mutants were also incubated individually as controls. Data are presented as in Fig. 1. The experiment was repeated twice with similar results. Errors <0.05 pmol are not shown.
Figure 6
Figure 6
Effect of rySec7d and C(DM) on [35S]GTP[γS] binding and its inhibition by BFA or its analogue. ryARF2 and 1 μg of rySec7d (42 pmol) or the double mutant, C(DM) (50 pmol) were incubated for 3 h at 4°C with 4 μM [35S]GTP[γS] without (black bars) or with (gray bars) 9 μg of BFA or the inactive analogue B36 (0.3 mM) before radioassay of protein bound [35S]GTP[γS]. ryARF2, rySec7d, and C(DM) were also incubated individually as controls. Data are presented as in Fig. 1. The experiment was repeated twice with similar results. Errors <0.05 pmol are not shown.
Figure 7
Figure 7
Inhibition of rySec7d- or C(DM)-stimulated [35S]GTP[γS] binding to ryARF2 by BFA. ryARF2 and 1 μg of rySec7d (□) or C(DM) (■) and 4 μM [35S]GTP[γS] were incubated with the indicated amounts of BFA or MeOH (▴) for 3 h at 4°C before radioassay of protein-bound [35S]GTP[γS]. The decrease in binding caused by BFA is reported as percentage inhibition of binding in its absence. All assays contained 1.2% (vol/vol) CH3OH. Data are means ± SEM of values from triplicate determinations. Error bars smaller than symbols are not shown. The experiment was repeated twice.
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