Figure 1.

MYC-Sec1p binds to all three exocytic SNAREs. (A) Ssop, Sec9p, and Sncp coprecipitate with MYC-Sec1p. 15 A₆₀₀ units of either untagged (NY1491) or MYC-SEC1 (NY1689) cells were harvested and diluted 10-fold into ice-cold wash buffer (20 mM Tris-Cl, pH 7.5, plus 20 mM NaN₃, and 20 mM NaF) to inhibit membrane fusion and to deplete intracellular ATP. Cells were lysed in ice-cold IP buffer (50 mM Hepes, pH 7.4, 150 mM KCl, 1 mM EDTA, 0.5% NP-40 plus protease inhibitors) to solubilize proteins and inhibit ATP hydrolysis. Anti-MYC IPs were probed for coprecipitated SNARE proteins with antibodies to Sec9p, Ssop, and Sncp using standard Western blot analysis. (B) MYC-Sec1p coprecipitates with HA-Sncp. Lysates of untagged (NY1491) and MYC-SEC1 HA-SNC (NY1702) were prepared as in A. To detect coprecipitation of HA-Sncp with MYC-Sec1p, MYC-Sec1p IPs were blotted for HA-Sncp using the monoclonal HA antibody. To detect coprecipitation of MYC-Sec1p with HA-Sncp, HA-Sncp IPs were blotted for MYC-Sec1p using the Sec1p antibody. (C) Nonexocytic SNAREs do not coprecipitate with MYC-Sec1p. IPs from lysates of untagged (NY1491) and MYC-SEC1 (NY1689) strains were divided into four samples and analyzed for coprecipitated SNARE proteins with antibodies to Ssop, Pep12p, Sec22p, and Bos1p. The lysate shown is 2% of the protein used for the IP. (D) Stoichiometry of SNARE proteins bound to MYC-Sec1p. IPs from lysates of untagged (NY1491) and MYC-SEC1 (NY1689) were probed with antibodies to Ssop and Sncp. The ratio of Ssop to Sncp coprecipitated with MYC-Sec1p was compared with twofold serial dilutions of purified, soluble SNARE complexes, in which the ratio of soluble SNAREs, SsopΔTM to SncpΔTM to Sec9p (SNAP-25–like domain), is 1:1:1.