FIG. 4.

Deletion as well as overexpression of Hsp104 stabilizes Cox2 in the nam9-1 strain. (A) Stability of newly synthesized Cox2 in MB43-NAM9, MB43-nam9-1, MB43-nam9-1Δhsp104, and MB43-nam9-1[HSP104↑]. Cells were pulse-labeled with [³⁵S]methionine in the presence of cycloheximide, and a chase reaction was performed for the times indicated (see Materials and Methods). The mitochondrial translation products from cells, corresponding to an OD₆₀₀ of 0.5, were analyzed by SDS–16% PAGE. M3041 (Δcob) and V25 (Δcox2) were used as references for the migration of Cox2 and Cob, respectively. Note that Cox2 and Cob migrate in the reverse order in SDS–16% PAGE compared to SDS–12% PAGE (compare to Fig. 6). (B) Steady-state levels of Cox2 in strains MB43-NAM9, MB43-nam9-1, MB43-nam9-1 hsp104, and MB43-nam9-1[HSP104↑]. The respective strains were grown on low-glucose medium and harvested either at the point of glucose exhaustion (glucose +/−) or after an additional incubation of 12 h (glucose −) as described in Materials and Methods. Total protein was extracted from cells corresponding to an OD₆₀₀ of 0.5. Western blots were decorated with antibodies directed against the mitochondrial matrix protein aconitase (Aco) and cytochrome c oxidase subunit 2 (Cox2).