Figure 3.
SEC11Δ149 is expressed in the membrane fraction in the GPS system. Yeast membrane fractions were prepared as described before (Rosa and Correia, 1991). Twenty milliliters of yeast containing the constructs were cultured until 0.6–0.8 OD600 and harvested by centrifugation at 4°C. The pellets were quickly frozen (−70°C) after the addition of 5 mL of 100 mm Tricine, 5 mm EDTA, and 2 mm DTT. The samples were thawed, dispersed by vortexing with 1.5-mm-diameter glass beads, and diluted with 5 mL of 0.33 m Suc, 0.1 m Tris, 5 mm EDTA, and 2 mm DTT, adjusted to pH 8. After centrifugation for 3 min at 900g, the supernatants were decanted and centrifuged for 45 min at 40,000g at 4°C. The new supernatants were used as the nonmembrane fractions, and the pellets were resuspended in 20% glycerol, 10 mm Tris, 0.1 mm EDTA, and 0.1 mm DTT, adjusted to pH 7.5, and comprised the membrane fractions. Samples harvested directly from the yeast (total), the nonmembrane fraction, and the membrane fraction were loaded and separated by gel electrophoresis at 20 μg of protein per lane. Immunoblot analysis of the samples was carried out with commercial anti-Pma1 antibody and anti-VP16 antibody for the bait fusions. Ponceau S staining was used for blotting/loading control.