The yeast mitochondrial ATPase complex. Subunit composition and evidence for a latent protease contaminant

Abstract

1. 1. The subunit compositions of the F 1 (oligomycin-insensitive) and F 1–F 0 (oligomycin-sensitive) mitochondrial ATPase complexes from Saccharomyces cerevisiae have been examined by the highly resolving technique of sodium dodecyl sulphate-polyacrylamide slab gel electrophoresis using a discontinuous buffer system. When isolated in the presence of protease inhibitors, F 1 and F 1–F 0 contained five and twelve bands, respectively; this contrasts with the four- and ten-band patterns seen previously using the less resolving disc gel method. When isolated in the absence of protease inhibitors both F 1 and F 1–F 0 contain spurious polypeptides produced by proteolytic modification. 2. 2. Endogenous protein turnover in S. cerevisiae was impaired in the presence of protease inhibitors. F 1–F 0 isolated from cells grown in the presence and absence of inhibitors contained an identical polypeptide composition, suggesting that the subunits are not significantly modified by endogenous proteases prior to cell harvesting. 3. 3. Yeast F 1–F 0 prepared in the presence of protease inhibitors contains a latent, sodium dodecyl sulphate-activated protease contaminant. Sodium dodecyl sulphate-induced proteolysis is largely confined to the 52 000 dalton α subunit which degrades into polypeptides of 40 000 and 10 700 daltons. The 40 000 dalton band is apparently equivalent to the polypeptide previously designated subunit 3. 4. 4. Both F 1 and F 1–F 0 were isolated from Torulopsis glabrata, a yeast with considerably shorter mitochondrial DNA than that in S. cerevisiae. F 1–F 0 catalysed high rates of ATP- 32P i exchange when reconstituted into phospholipid vesicles, thus demonstrating the presence of a complete coupling mechanism. F 1–F 0 contained approximately twelve subunits and F 1 five, like the S. cerevisiae complexes. It therefore appears that the shorter mitochondrial DNA length does not produce a significantly simpler ATPase subunit structure.