Figure 5.

Complementation of the atp23 mutant with the E168Q mutant ATP23 gene. (A) Growth phenotype of the atp23 null mutant transformed with mutant ATP23. The parental wild-type W303-1B, the atp23 null mutant W303ΔATP23 (ΔATP23), and the null mutant expressing the E168Q mutation either from the chromosomally integrated gene [ΔATP23 + E→Q (i)] or from the gene on a multicopy plasmid [ΔATP23 + E→Q (e)] were grown in liquid YPD. Serial dilutions of the cultures were spotted on rich glucose (YPD) and rich ethanol/glycerol (YEPG) plates and incubated at 30°C for 2 d. (B) The strains used in panel A were labeled with [³⁵S]methionine in the presence of cycloheximide. Total cellular proteins were separated on a 12.5% polyacrylamide gel containing 4 M urea and 25% glycerol and transferred to nitrocellulose, and the blot was exposed to an x-ray film overnight as described in the legend to Figure 3A. The positions of mature (Atp6) and novel form of subunit 6 (pAtp6) are indicated by the arrows. (C) Growth of atp23 revertants on glycerol/ethanol. The parental wild-type W303-1B, the atp23 null mutant W303ΔATP23 (ΔATP23), and two independent revertants of the null mutant (ΔATP23/R1 and R2) were grown in liquid YPD. Serial dilutions of the cultures were spotted on rich glucose (YPD) and rich ethanol/glycerol (YEPG) plates and incubated at 30°C for 2 d. (D) The wild type and mutant strains shown in panel C were labeled, and the radioactive translation products were visualized as in panel B. (E) The top part of the panel shows the turnover of subunit 6 in wild type and in atp23 mutants. The parental wild-type W303-1B, the atp23 null mutant W303ΔATP23 (ΔATP23), and a revertant of the null mutant (W303ΔATP23/R2) were labeled in vivo for 20 min with [³⁵S]methionine in the presence of cycloheximide. Puromycin and excess unlabeled methionine were added, and samples were taken after the indicated times of chase at 30°C. Proteins were separated on a 12.5% gel containing 4 M urea and 25% glycerol. The radioactivity associated with Atp6p was quantified with a PhosphorImager. The results normalized to the values obtained at time zero of the chase are shown in the bottom part of panel E.