Figure 4. VMS1 maintains 26S proteasome stability after prolonged incubation at the stationary phase.

(A) The indicated yeast strains were cultured with constant aeration for 25 days at 25°C. Samples were taken over time, lysates were prepared and equal amounts of protein were resolved by native PAGE. Proteasomes were detected with the fluorogenic substrate Suc-LLVY-AMC in the presence of 0.02% SDS (left-hand panels) and by Western blot analysis (right-hand panels) using antibodies directed against proteins in the 20S core particle (CP). Note the more rapid disappearance of the singly (RP-CP) and doubly (RP₂-CP) capped core particles in the vms1Δ strain. Data are representative of three independent experiments. (B) Quantification of singly and doubly capped proteasomes (upper panel) and free core particle (lower panel) from (A). Results are means ± S.D. from three independent experiments. (C–E) A Western blot analysis was performed using antibodies directed against the indicated components in the 19S particle. As in (A), note the more rapid disappearance of components in the RP-CP and RP₂-CP particles (denoted by arrowheads), especially at days 18 and 25, in the vms1Δ strain. The asterisk denotes the faster-migrating species observed in Figure 3. WT, wild-type.