Figure 4.

Turnover of in vivo-labeled mitochondrial translation products and steady-state concentration of Cox1p in wild type and COX mutants. (A) Wild type (W303-1A) and mutants (described in Table I) were grown and labeled for 20 min at 30°C with [³⁵S]methionine. Labeling was terminated by addition of 80 μmol cold methionine and 12 μg/ml puromycin (0 time). Samples of the cultures were collected after the indicated times of incubation at 30°C and processed as in Figure 1. (B) The wild-type strain W303-1A and the cox14 and cox17 mutants were labeled and chased for the indicated times as in panel A. One-half of each culture was incubated in the presence of chloramphenicol as in Figure 1 prior to labeling. (C) The wild-type W303-1A and D273-10B and mutant strains were grown in 2% galactose, 1% yeast extract, and 2% peptone to stationary phase. Mitochondria were prepared and 10 μg of protein was separated by SDS–PAGE on a 12% polyacrylamide gel. The proteins were transferred to nitrocellulose and probed with a polyclonal antibody against yeast Cox1p. The antibody–antigen complexes were visualized by a secondary reaction with [¹²⁵I]protein A.