Figure 1. The UBA domain of Dsk2 functions as a stabilization signal.

(a) Steady-state levels of FLAG-tagged Dsk2 determined by western blotting with a FLAG-specific antibody. ^FLAGDsk2, ^FLAGDsk2^L368,369A and ^FLAGDsk2^{ΔUbL/L368,369A} were ectopically expressed from a GAL1 promoter. Yeast was grown until early log phase. Wherever indicated, proteasome inhibitor was added to a final concentration of 50 μM, 2 h before harvesting. β-Actin is shown as loading control. Molecular weight markers are indicated. Asterisk marks a non-specific band. (b) Turnover of ^FLAGDsk2 (closed circles), ^FLAGDsk2^L368,369A (open circles) and ^FLAGDsk2^{ΔUbL/L368,369A} (closed squares). Samples were taken at the indicated time points and probed with a FLAG-specific antibody. Densitometric quantification of the western blot is shown. (c) Turnover of ^FLAGDsk2 (closed circles), ^FLAGDsk2^L368,369A (open circles) and ^FLAGDsk2^{ΔUbL/L368,369A} (closed squares) in the presence of 50 μM proteasome inhibitor MG132, as shown in b.