Figure 10.

Sro7p interacts specifically with Sec9p in the cytoplasm and at the plasma membrane. (A) Cells containing high copy SEC9 (pB35) were grown to logarithmic phase, spheroplasted, and lysed. The lysate was centrifuged at 30,000 g to separate cytosol and membrane fractions, the samples were adjusted to 0.5% NP-40, centrifuged at 13,000 g for 10 min to eliminate insoluble material, and subjected to coimmunoprecipitation analysis as in Fig. 8. Samples were analyzed by SDS-PAGE and immunoblotted with α-Sec9p, α-Sro7p, α-Sso1/2p, and α-Snc1/2p antibodies. (B) Serial coprecipitation analysis demonstrates that Sro7p interacts with Sso1/2p and Snc1/2p only through the interaction with Sec9p. A 30,000 g membrane fraction was solubilized with detergent as above and was precleared of Sec9 protein by immunoprecipitation with saturating amounts of affinity-purified α-Sec9p antibody or equivalent amounts of preimmune IgG as a control. Fractions were subjected to a second round of immunoprecipitations with α-Sec9p, α-Sro7p, α-Sso1/2p, and α-Snc1/2p polyclonal antibodies and analyzed as above.