FIGURE 6.

Increased levels of ubiquitinated proteins are associated with the Cdc48p complex in yeast lacking Vms1p. A, the indicated yeast strains expressing CFTR were grown to log phase, collected, and processed for an anti-ubiquitin (Ub; top) and anti-HA (CFTR; bottom) immunoprecipitation as described under “Experimental Procedures.” Note that polyubiquitinated CFTR resolves by SDS-PAGE as a smear ≥140 kDa, which is the approximate molecular mass of CFTR. B, WT and vms1Δ cells in which the PDR5 locus was also disrupted were transformed with either an untagged or HA-tagged version of Cdc48p. After treatment of each indicated cell type with MG132 or an equal volume of DMSO for 1 h, ER fractions were isolated, and the Cdc48p complex was immunoprecipitated (IP) with anti-HA-agarose from Triton X-100-solubilized fractions. The isolated proteins were resolved by SDS-PAGE and processed for immunoblot analysis (WB) as described under “Experimental Procedures.” The top panel depicts the results of the immunoblot with anti-ubiquitin antibody, and the bottom panel depicts the results of the immunoblot with anti-HA antibodies.