GENETIC FUSIONS OF SUBUNIT-C IN THE F-0 SECTOR OF H-TRANSPORTING ATP SYNTHASE - FUNCTIONAL DIMERS AND TRIMERS AND DETERMINATION OF STOICHIOMETRY BY CROSS-LINKING ANALYSIS()

The multicopy c subunit of the H+-transporting ATP synthase of Escheri chia coli folds through the transmembrane F-0 sector as a hairpin of t wo hydrophobic alpha-helices with the proton-translocating aspartyl-61 side chain centered in the second transmembrane helix. The number of subunits c in the F-0 complex, which is thought to determine the H+-pu mping/ATP stoichiometry, was previously not determined with exactness but thought to range from 9-12. The studies described here indicate th at the exact number is 12. Based upon the precedent of the subunit c i n vacuolar-type ATPases, which are composed of four transmembrane heli ces and seem to have evolved by gene duplication of an F-0-type progen itor gene, we constructed genetically fused dimers and trimers off. co li subunit c. Both the dimeric and trimeric forms proved to be functio nal. These results indicate that the total number of subunit c in F-0 should be a multiple of 2 and 3. Based upon a previous study in which the oligomeric organization of c subunits in F-0 was determined by cro ss-linking of Cys-substituted subunits (Jones, P. C., Jiang, W., and F illingame, R. H. (1998) J. Biol. Chem. 273, 17178-17185), we introduce d Cys into the first and last transmembrane helices of subunit c monom ers, dimers, and trimers and attempted to generate cross-linked produc ts by oxidation with Cu(II)-(1,10-phenanthroline)(2). Double Cys subst itutions at two sets of positions gave rise to extensive cross linked multimers. Multimers of the monomer that extended up to the position o f c(12) were correlated and calibrated with distinct cross-linked spec ies of the appropriate doubly Cys-substituted dimers (i.e. c(2), c(4). ..c(12)) and doubly Cys-substituted trimers (i.e. c(3), c(6), c(9), c( 12)). The results show that there are 12 copies of subunit c per F-0 i n E. coli, the exact number having both mechanistic and structural sig nificance.