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 ab 2 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.
Krebstakies
Thomas
T
Abteilung Mikrobiologie, Fachbereich Biologie/Chemie, Universität Osnabrück, D-49069 Osnabrück, Germany.
Aldag
Ingo
I
Altendorf
Karlheinz
K
Greie
Jörg-Christian
JC
Deckers-Hebestreit
Gabriele
G
D025243:Bacterial Proton-Translocating ATPases / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D021381:Protein Transport
D029968:Escherichia coli Proteins; C523987:ATP synthase subunit c, E coli; D025243:Bacterial Proton-Translocating ATPases