FIGURE 1.

Interactions among Cdc48, Vms1, and Ufd2. A, co-immunoprecipitation analysis of in vivo interactions between yeast Cdc48 and Vms1 in the presence or absence of Ufd2. Proteins were extracted from yeast cells expressing RGS/His₆-tagged Cdc48 and FLAG-tagged Vms1 and immunoprecipitated with beads coupled to various antibodies as indicated. Strains are indicated above the panels. The antibodies used for immunoprecipitation (IP) and Western blotting are indicated on the right of the panels. Equal amounts of protein extracts were used as determined by blotting with anti-Rpt5 antibody (lower panel). B, interactions between Cdc48 and Ufd2 in wild-type or vms1Δ cells. C, associations between Vms1 and Ufd2 derivatives in yeast. Ufd2 alleles defective for Cdc48 binding were described previously (4). D, purification of GST-tagged Cdc48 and His₆-tagged Vms1 from E. coli. The Coomassie Blue-stained gel shows purified Cdc48 and Vms1. E, Vms1 directly binds Cdc48 in vitro. Purified GST-Cdc48 and His₆-Vms1 were mixed in vitro in the presence of GST-Sepharose and analyzed by Western blotting using anti-His₆ antibody.