FIGURE 5.
Synthesis and degradation of mitochondrial translation products. A, RFY5-1 strain grown in the absence (-dox) or in the presence (+dox) of doxycycline (dox) (10 μg·ml-1) was labeled in vivo with 35S-labeled (methionine + cysteine) for 5 min in the presence of cycloheximide to inhibit cytosolic protein synthesis. Excesses of unlabeled methionine and cysteine were added, and samples were taken after the indicated times of chase at 28 °C as described under “Experimental Procedures.” Proteins were separated on a 12.5% gel containing 4 m urea and 25% glycerol. The gel was dried and analyzed with a PhosphorImager. B, for a better resolution of Atp6p, the contrast of the gel area containing Cox3p and Atp6p was enhanced. C, at time 0 of the chase, the level of each subunit was quantified relatively to the total radioactivity incorporated. The ratios between signals obtained for growth in the presence and absence of doxycycline were calculated for each subunit. D, at each time of the chase, Atp6p level was quantified relatively to the total radioactivity incorporated and expressed as a percentage of signal obtained at time 0 of the chase. (Gray circles, absence of doxycycline in the growth medium. Black circles, presence of doxycycline in the growth medium). a.u., arbitrary units.