Figure 4. Sey1p-mediated membrane fusion... | Rockefeller University Press

Figure 4.

$Figure 4. Sey1p-mediated membrane fusion in vitro. (a) Wild-type (WT) Sey1p or the indicated mutants were purified (left blot shows a Coomassie-stained SDS gel) and reconstituted into proteoliposomes. Flotation in a sucrose gradient (right) shows efficient reconstitution of the proteins (T, top fraction; B, bottom fraction). The black line indicates that intervening lanes have been spliced out. (b) Donor (D) and acceptor (A) proteoliposomes containing WT Sey1p or the indicated mutants were analyzed by SDS-PAGE and Coomassie staining. (c) WT Sey1p was reconstituted at the indicated protein to lipid ratios into proteoliposomes containing fluorescent lipids at quenching concentrations or unlabeled lipids (donor and acceptor vesicles, respectively). Donor and acceptor proteoliposomes were mixed at a ratio of 1:3 (600 µM of total lipid) and incubated for 10 min at 37°C before the addition of 1 mM GTP. The increase in fluorescence caused by lipid mixing was followed at 1-min intervals in a fluorescence plate reader. Control reactions were performed in the absence of Mg2+ or in the presence of GDP or GTPγS instead of GTP. (d) Fusion with WT Sey1p was compared with that of the indicated Sey1p mutants. Mutant Sey1K50A has a mutation in the phosphate-binding loop (P-loop) of the active site, and mutant A592V is analogous to one in a plant homologue that causes ER morphology defects. (e) GTPase activity of WT and the indicated Sey1p mutants. Error bars show means ± SD. (f) A sey1Δ yop1Δ strain expressing Sec63-GFP and plasmids encoding the indicated Sey1 mutants were visualized as in Fig. 1. Bar, 1 µm.$

Sey1p-mediated membrane fusion in vitro. (a) Wild-type (WT) Sey1p or the indicated mutants were purified (left blot shows a Coomassie-stained SDS gel) and reconstituted into proteoliposomes. Flotation in a sucrose gradient (right) shows efficient reconstitution of the proteins (T, top fraction; B, bottom fraction). The black line indicates that intervening lanes have been spliced out. (b) Donor (D) and acceptor (A) proteoliposomes containing WT Sey1p or the indicated mutants were analyzed by SDS-PAGE and Coomassie staining. (c) WT Sey1p was reconstituted at the indicated protein to lipid ratios into proteoliposomes containing fluorescent lipids at quenching concentrations or unlabeled lipids (donor and acceptor vesicles, respectively). Donor and acceptor proteoliposomes were mixed at a ratio of 1:3 (600 µM of total lipid) and incubated for 10 min at 37°C before the addition of 1 mM GTP. The increase in fluorescence caused by lipid mixing was followed at 1-min intervals in a fluorescence plate reader. Control reactions were performed in the absence of Mg²⁺ or in the presence of GDP or GTPγS instead of GTP. (d) Fusion with WT Sey1p was compared with that of the indicated Sey1p mutants. Mutant Sey1K50A has a mutation in the phosphate-binding loop (P-loop) of the active site, and mutant A592V is analogous to one in a plant homologue that causes ER morphology defects. (e) GTPase activity of WT and the indicated Sey1p mutants. Error bars show means ± SD. (f) A sey1Δ yop1Δ strain expressing Sec63-GFP and plasmids encoding the indicated Sey1 mutants were visualized as in Fig. 1. Bar, 1 µm.