FIG. 6.

Native immunoprecipitation of Vam3p with Vam7p. Spheroplasts (5 OD₆₀₀ equivalents) from the following strains were generated and incubated at either 26 or 38°C as indicated for 1 h: TKSY48 (sec18-1 vam7Δ), SEY6210 (wild type), EGY118-110 (sec18-1), and CBY42 (sec18-1 vam3Δ). Spheroplasts were then lysed with 0.5% Triton X-100 and incubated with anti-Vam7p antibody for 1 h at 4°C, followed by the addition of protein A-Sepharose for an additional 1 h. Bound immunocomplexes were washed with 0.1% Triton X-100 and eluted from beads with SDS-PAGE sample buffer. Two-OD₆₀₀-unit samples were loaded onto SDS-PAGE gels, resolved by electrophoresis, and transferred to nitrocellulose paper. Vam3p and Vam7p were immunoblotted with anti-Vam3p and -Vam7p antibody, respectively, and visualized by ECL fluorography. Of the total sec18-1 38°C extract, 5% was TCA precipitated and loaded as an indication of the amount of total Vam3p bound to Vam7p.