Sbh2 degradation requires Doa10 E3 ligase activity and proceeds via the 26S proteasome. (A) Doa10 E3 ligase activity is required for Sbh2 degradation. chx chase assay of ectopically expressed (low-copy plasmid; MET25 promoter) HA-Sbh2 in WT, doa10Δ, and doa10-C39S cells (DF5 strain background). (B) Sbh2 is ubiquitylated in cells. In vivo ubiquitylation of HA-Sbh2: HA- or FLAG-tagged Sbh2 was ectopically expressed (low-copy plasmid; GPD promoter) in ssh1Δ and doa10Δ ssh1Δ cells together with MYC-ubiquitin. HA-Sbh2 was precipitated from the cell lysates with anti-HA agarose beads. Precipitates were analyzed by immunoblotting with anti-HA and anti-MYC. Asterisks indicate cross-reactive bands (IgG heavy and light chain, respectively) recognized by the secondary antibody (anti–rabbit peroxidase). IB, immunoblotting. (C) Proteasomal degradation of Sbh2. ssh1Δ pdr5Δ cells ectopically expressing HA-Sbh2 (low-copy plasmid; MET25 promoter) were grown to log phase (0 h time point), divided into two cultures, and treated for 3 h with either the proteasome inhibitor MG132 (50 µM) or the solvent DMSO. Samples were normalized for equal amounts of the stable protein Pgk1 before gel loading. (D) Ubc6 and Ubc7 are required for efficient Sbh2 degradation. Degradation of ectopically overexpressed HA-Sbh2 (low-copy plasmid; GPD promoter) in WT, ubc6Δ, ubc7Δ, and ubc6Δ ubc7Δ cells (DF5 strain background). chx chase analysis was performed as in Fig. 1 B. Relative protein levels listed below the blots were determined by quantification of pixel densities of HA-Sbh2 bands relative to those of Pgk1. 0 h time point was in each case set to 100%. (E) The AAA-ATPase Cdc48 is required for Sbh2 degradation. Degradation of ectopically overexpressed HA-Sbh2 (low-copy plasmid; GPD promoter) in WT and cdc48-3 cells (W303-1A strain background). Cells were grown to log phase at 25°C and shifted to the nonpermissive temperature of 37°C 30 min before addition of chx.