Fig. 3.

Upregulation of the subunit 6 variant L₁₇₃P is subunit 9-dependent. a) Growth phenotypes. Fresh glucose cultures of the indicated strains were serially diluted and spotted on rich glucose and glycerol media with or without 5 µg/mL doxycycline (DOX) as indicated. The glucose and glycerol plates were scanned after 3 and 6 days of incubation at 28°C, respectively. b and c) In vivo labeling of mitochondrial translation products. Pulse labeling was performed for 20 min with [³⁵S]-methionine + [³⁵S]-cysteine in the presence of cycloheximide to block cytoplasmic translation, in cells freshly grown in rich galactose medium. Total protein extracts were then prepared and separated by SDS/PAGE in 2 different gels: (1) 12% polyacrylamide containing 4 M urea and 25% glycerol (to resolve Var1, Cox1, Cox2, Cytochrome b, Cox3, and Atp6); (2) 17.5% of polyacrylamide (to resolve Atp8 and Atp9). The 2 gels shown in b) were loaded with equal amounts of radioactivity from the same experiment. After drying of the gels under vacuum, the radiolabeled proteins were visualized using a PhosphorImager after one-week of exposure. The subunit 6 (Atp6) and Cox3 signals were quantified and compared to each other within each sample. The Atp6/Cox3 ratio was set to 1.0 for the WT. The identity of the band above Cox3 (designated by a hash sign) in the samples containing the 6-L₁₇₃P variant is unknown.