Sey1p at its physiological concentration is not sufficient to induce liposome fusion. (A) Proteoliposomes with the indicated Sey1p-to-lipid ratio were generated as described in the Materials and methods. Donor and acceptor proteoliposomes were mixed and incubated at 30°C for 10 min. After GTP and Mg²⁺ were added, and NBD fluorescence was measured at 30-s intervals for 30 min. β-Octylglucoside was then added to determine total fluorescence. Fusion is expressed as the percentage of total fluorescence. All experiments were performed multiple times with similar results, and the data shown are representative of all results. (B) Overexpression of Sey1p restores fusion to sec22Δ microsomes. The indicated microsomes were incubated on ice or at 27°C with GTP/ATP in the absence or presence of anti-Sey1p antibodies for 90 min. SEY1-OE, SEY1 overexpressor. Data represent the means ± SEM (error bars; n = 3). ***, P < 0.001, Tukey’s test between the no inhibitor groups of the indicated genotypes. (C) Protein profiles of wild-type, sec22Δ, and sec22Δ SEY1-OE microsomes. (D) The fusion of sec22Δ SEY1-OE microsomes requires Sec18p function. Microsomes (sec22Δ SEY1-OE) were incubated on ice or at 27°C with GTP/ATP or GTP alone in the presence of anti-Sey1p antibodies or increasing concentrations of anti-Sec18p antibodies for 90 min. Data represent the means ± SEM (error bars; n = 3). Statistically different groups are indicated by different lowercase letters (a–d, between ATP/GTP-driven reactions, P < 0.001; and x–y, between GTP-only-driven reactions, P < 0.05).