The Stoichiometry of Subunit c of Escherichia coli ATP Synthase Is Independent of Its Rate of Synthesis^†

Immunoblot quantitation of Escherichia coli ATP synthase isolated from atp wildtype and mutant cells, the latter comprising a reduced expression of the atpE gene coding for subunit c due to a point mutation within its Shine−Dalgarno sequence, suggested a variable stoichiometry of subunit c [Schemidt et al. (1995) Arch. Biochem. Biophys. 323, 423−428]. To study the c ring of the mutant strain and its stoichiometry in more detail, F_O isolated from wildtype and mutant were investigated by quantitation, reconstitution, and cross-linking. Direct quantitation by staining with SYPRO Ruby revealed a reduction of subunit c in the mutant by a factor of 2 compared to F_O subunits a and b. Rates of passive H⁺ translocation correlated with the amount of subunit c present. Lower rates for mutant F_O could be increased by addition of subunit c, whereas translocation rates remained constant by coreconstitution with nonfunctional subunit cD61G arguing against the presence of smaller c rings that are filled up with coreconstituted subunit c. Intermolecular cross-linking by oxidation of bicysteine-substituted subunit c (cA21C/cM65C) revealed an equal pattern of oligomer formation in wildtype and mutant also favoring a comparable subunit c stoichiometry. Cross-linking of membrane vesicles containing cysteine-substituted subunits a (aN214C) and c (cM65C) characterized the mutant F_O preparation as a heterogeneous population, which consists of assembled F_O and free ab₂ subcomplexes each present to approximately 50%. Thus, these data clearly demonstrate that the stoichiometry of the subunit c rings remains constant even after reduction of the synthesis of subunit c.