The membrane-bound ATP synthase (F(1)F(o)) from mitochondria, chloroplasts and bacteria plays a crucial role in energy-transducing reactions. In the case of Escherichia coli, the reversible, proton-translocating ATPase complex consists of two different entities, F(1) and F(o). The water-soluble F(1) part carries the catalytic sites for ATP synthesis and hydrolysis. It is associated with the membrane-embedded F(o) complex, which functions as a proton channel and consists of subunits a, b and c present in a stoichiometry of 1:2:12.Subunit b was isolated by preparative gel electrophoresis, acetone-precipitated and renatured in a cholate-containing buffer. Reconstituted subunit b together with purified ac subcomplex is active in proton translocation and F(1) binding, thereby demonstrating that subunit b had recovered its native conformation. Circular dichroism spectroscopy of subunit b reconstituted into liposomes revealed a rather high degree of alpha -helical conformation of 80%. After addition of a His(6)-tag to the N terminus of subunit a, a stable ab(2) subcomplex was purified instead of a single subunit a, arguing in favour of a direct interaction between these subunits. After addition of subunit c and reconstitution into phospholipid vesicles, an F(o) complex was obtained exhibiting rates of proton translocation and F(1) binding comparable with those of wild-type F(o).The epitopes of monoclonal antibodies against subunit c are located in the hydrophilic loop region (cL31-Q42) as mapped by enzyme-linked immunosorbent assay using overlapping synthetic heptapeptides. Binding studies revealed that all monoclonal antibodies (mAbs) bind to everted membrane vesicles irrespective of the presence or absence of F(1). Although the hydrophilic region of subunit c, and especially the highly conserved residues cA40, cR41, cQ42 and cP43, are known to interact with subunits gamma and epsilon of the F(1) part, the mAb molecules have no effect on the function of F(o), either in proton translocation or in F(1) binding. However, the F(1) part and the mAb molecule(s) are bound simultaneously to the F(o) complex, suggesting that not all c subunits are involved in the interaction with F(1).
Influence of subunit-specific antibodies on the activity of the F0 complex of the ATP synthase of Escherichia coli. II. Effects of subunit c-specific polyclonal antibodies.
