Vibrio effector protein VopQ inhibits fusion of V-ATPase-containing membranes

Abstract

Vesicle fusion governs many important biological processes, and imbalances in the regulation of membrane fusion can lead to a variety of diseases such as diabetes and neurological disorders. Here we show that the Vibrio parahaemolyticus effector protein VopQ is a potent inhibitor of membrane fusion based on an in vitro yeast vacuole fusion model. Previously, we demonstrated that VopQ binds to the V(o) domain of the conserved V-type H(+)-ATPase (V-ATPase) found on acidic compartments such as the yeast vacuole. VopQ forms a nonspecific, voltage-gated membrane channel of 18 Å resulting in neutralization of these compartments. We now present data showing that VopQ inhibits yeast vacuole fusion. Furthermore, we identified a unique mutation in VopQ that delineates its two functions, deacidification and inhibition of membrane fusion. The use of VopQ as a membrane fusion inhibitor in this manner now provides convincing evidence that vacuole fusion occurs independently of luminal acidification in vitro.

Keywords: SNARE; Vibrio parahaemolyticus; vesicle fusion; vp1680; yeast vacuole.

Fig. 1.

VopQ inhibits yeast homotypic vacuole fusion. (A) ALP vacuole fusion reactions in the absence or presence of increasing concentrations of recombinant His₆-VopQ. Standard fusion inhibitors were α-Vam3p, α-Sec17p, 1 µM Gdi1p, Gyp1-46, or ice incubation. Error bars indicate SD from the mean; n = 4. (B and C) The β-lactamase content-mixing assay (B) was performed in parallel with the Rh-PE dequenching lipid-mixing assay (C) in the absence of ATP or including α-Vam3p, MBP-VopQ, or MBP. Data for the content-mixing assay are reported as mean ± SD, n = 3. The Rh-PE dequenching plot is a representative curve measured from the same three experiments used for the content-mixing assay.

Fig. 2.

VopQ inhibits trans-SNARE complex formation. (A) At 0-, 10-, 20-, and 45-min intervals, ALP vacuole fusion reaction was incubated on ice, or one of the following fusion inhibitors was added: α-Sec17p, α-Ypt7p, α-Vam3p, or 200 nM VopQ. After 90 min, fusion was measured and compared with the fusion of an uninhibited fusion reaction. Error bars indicate SD from the mean; n = 3. (B) The quantitative docking assay was performed in the presence of 500 nM MBP, 2.8 µM Gdi1p and 11.4 µM Gyp1-46, or 500 nM MBP-VopQ. At least 130 clusters from 10 random fields were scored for each condition. (C) Trans-SNARE assays were performed without or with the following inhibitors: ice incubation, 1 µM Gdi1p/1 µM Gyp1-46, or 200 nM VopQ. (D) ALP vacuole fusion reactions were performed in the absence or presence of 200 nM MBP-VopQ. Recombinant 50 nM Vam7p SNARE, 20 nM HOPS complex, or α-Vam3p was added where indicated.

Fig. 3.

A pH gradient is not required for vacuole fusion. (A) ALP and β-lactamase fusion reactions were performed in parallel, using standard inhibitors or 200 nM MBP, 200 nM MBP-VopQ, or 500 nM bafilomycin. Maximal fusion was defined as the extent of fusion of the no-inhibitor reaction; error bars indicate SD from the mean; n = 3. (B) Schematic diagram of the mechanism of action of the ionophores used. (C) Proton translocation activity of vacuoles was assayed in the absence or presence of ATP, α-Vam3p, 500 nM bafilomycin, 1 µM valinomycin, 1 µM nigericin, or combinations of ionophores and bafilomycin. Curves are representative of three independent experiments. (D) ALP and β-lactamase fusion reactions were performed in parallel, using a combination of ionophores at the concentrations used in C. Maximal fusion was defined as the no-inhibitor reaction. Error bars indicate SD from the mean; n = 3.

Fig. 4.

VopQ^S200P does not induce vacuole fragmentation. (A) Serial dilutions of yeast strain BY4742 harboring pRS413-Gal1, pRS413-Gal1-VopQ, or pRS413-Gal1-VopQ^S200P in Complete Supplement Mixture (CSM) medium lacking histidine, supplemented with either 2% glucose or 2% galactose. (B) Vacuolar morphology of the BY4742 strain harboring pRS413-Gal1, pRS413-Gal1-VopQ, or pRS413-Gal1-VopQ^S200P was visualized via FM4-64 staining. (Scale bar, 5 μm.)

Fig. 5.

VopQ^S200P deacidifies the vacuole but does not inhibit fusion. (A) ALP vacuole fusion reactions or (B) β-lactamase fusion reactions were performed using standard inhibitors, MBP-VopQ, or MBP-VopQ^S200P. (C) Proton translocation activity was measured in the presence of MBP-VopQ or MBP-VopQ^S200P when proteins were added before the addition of ATP.