Subunit organization and structure in the F0 sector of Escherichia coli F1F0 ATP synthase.

In this review, we summarize recent work from our laboratory which establishes the topology and nearest neighbor organization of subunits in the F0 sector of the H+ transporting ATP synthase of Escherichia coli. The E. coli F0 sector is composed of three subunits in an a1b2c12 stoichiometric ratio. Crosslinking experiments with genetically introduced Cys establish a ring-like organization of the 12 c subunits with subunits a and b lying to the outside of the ring. The results are interpreted using an atomic resolution structural model of monomeric subunit c in a chloroform-methanol-water (4:4:1, v/v/v) solution, derived by heteronuclear NMR (M.E. Girvin, F. Abildgaard, V. Rastogi, J. Markley, R.H. Fillingame, in press). The crosslinking results validate many predictions of the structural model and confirm a front-to-back-type packing of two subunit c into a functional dimer, as was first predicted from genetic studies. Aspartyl-61, the proton translocating residue, lies at the center of the four transmembrane helices of the functional dimer, rather than at the periphery of the subunit c ring. Subunit a is shown to fold with five transmembrane helices, and a functionally important interaction of transmembrane helix-4 with transmembrane helix-2 of subunit c is established. The single transmembrane helices of the two subunit b dimerize in the membrane. The structure of the transmembrane segment of subunit b is predicted from the NMR structure of the monomeric peptide.

Transmembrane topography of subunit a in the Escherichia coli F1F0 ATP synthase.

Subunit a is the least understood of the three subunits that compose the F0 sector in the Escherichia coli F0F1 ATP synthase. In this study, we have substituted Cys into predicted extramembranous loops of the protein and used chemical modification to obtain topographical information on the folding of subunit a. The extent of labeling of the substituted Cys residues by fluorescein-5'-maleimide was determined. The localization of reactive Cys residues was inferred from differences in the extent of labeling in inside out and right side out membrane vesicles. The NH2-terminal segment of subunit a was localized to the outside (periplasmic) surface and the COOH terminus to the cytoplasmic surface by these procedures. Loop residues in two periplasmic extramembranous loops and in two cytoplasmic extramembranous loops were also localized. The localization of two cytoplasmic Cys residues was confirmed by using 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid to block fluorescein-5'-maleimide labeling. From the localization of the Cys residues, a model for the topography is proposed that consists of five transmembrane segments with the NH2 terminus periplasmic and the COOH terminus cytoplasmic. The positions of second site suppressors, including several isolated here to the nonfunctional E219C and H245C substitutions, provide support for the topographical model proposed.

On the role of Arg-210 and Glu-219 of subunit a in proton translocation by the Escherichia coli F0F1-ATP synthase.

A strain of Escherichia coli was constructed which had a complete deletion of the chromosomal uncB gene encoding subunit a of the F0F1-ATP synthase. Gene replacement was facilitated by a selection protocol that utilized the sacB gene of Bacillus subtilis cloned in a kanamycin resistance cartridge (Ried, J. L., and Collmer, A. (1987) Gene (Amst.) 57, 239-246). F0 subunits b and c inserted normally into the membrane in the DeltauncB strain. This observation confirms a previous report (Hermolin, J., and Fillingame, R. H. (1995) J. Biol. Chem. 270, 2815-2817) that subunit a is not required for the insertion of subunits b and c. The DeltauncB strain has been used to characterize mutations in Arg-210 and Glu-219 of subunit a, residues previously postulated to be essential in proton translocation. The aE219G and aE219K mutants grew on a succinate carbon source via oxidative phosphorylation and membranes from these mutants exhibited ATPase-coupled proton translocation (i.e. ATP driven 9-amino-6-chloromethoxyacridine quenching responses that were 60-80% of wild type membranes). We conclude that the aGlu-219 residue cannot play a critical role in proton translocation. The aR210A mutant did not grow on succinate and membranes exhibited no ATPase-coupled proton translocation. However, on removal of F1 from membrane, the aR210A mutant F0 was active in passive proton translocation, i.e. in dissipating the DeltapH normally established by NADH oxidation with these membrane vesicles. aR210A membranes with F1 bound were also proton permeable. Arg-210 of subunit a may play a critical role in active H+ transport that is coupled to ATP synthesis or hydrolysis, but is not essential for the translocation of protons across the membranes.