FIGURE 3.
Membrane association of Sss1p domain mutants. A, FKY198 cells bearing the Sss1ΔCp plasmid were grown exponentially in galactose-containing medium allowing expression of both Sss1p and Sss1ΔCp. These cells were fractionated into crude cytosolic (C) and membrane (M) fractions. 2.0 A600 nm cell equivalents of each fraction and a total sample (T) were analyzed by 8–16% SDS-PAGE and immunoblotting with Sss1p (upper panel) and Sec63p (lower panel) antisera. The Sss1 protein and the ER integral membrane protein Sec63p (17) were found largely in the membrane fraction (third lane) as expected. However, some Sss1ΔCp was found in the cytosolic fraction (second lane). B, membranes prepared from FKY198 cells transformed with Sss1p (WT) NSUp- and USCp-expressing plasmids grown for 6 h in glucose medium were subjected to carbonate extraction. Total (T), carbonate-soluble (S), and carbonate-resistant pellet (P) samples were analyzed by 12.5% SDS-PAGE and immunoblotted with antisera against Kar2p, Sec63p, and Sss1p and Ubc6p as appropriate. Kar2p and Sec63p provide carbonate-extractable and -resistant ER protein species, respectively (17). These data are representative of two independent experiments.