The ATP synthase of Escherichia coli: structure and function of F(0) subunits.

In this review we discuss recent work from our laboratory concerning the structure and/or function of the F(0) subunits of the proton-translocating ATP synthase of Escherichia coli. For the topology of subunit a a brief discussion gives (i) a detailed picture of the C-terminal two-thirds of the protein with four transmembrane helices and the C terminus exposed to the cytoplasm and (ii) an evaluation of the controversial results obtained for the localization of the N-terminal region of subunit a including its consequences on the number of transmembrane helices. The structure of membrane-bound subunit b has been determined by circular dichroism spectroscopy to be at least 75% alpha-helical. For this purpose a method was developed, which allows the determination of the structure composition of membrane proteins in proteoliposomes. Subunit b was purified to homogeneity by preparative SDS gel electrophoresis, precipitated with acetone, and redissolved in cholate-containing buffer, thereby retaining its native conformation as shown by functional coreconstitution with an ac subcomplex. Monoclonal antibodies, which have their epitopes located within the hydrophilic loop region of subunit c, and the F(1) part are bound simultaneously to the F(0) complex without an effect on the function of F(0), indicating that not all c subunits are involved in F(1) interaction. Consequences on the coupling mechanism between ATP synthesis/hydrolysis and proton translocation are discussed.

Structure and function of the F(o) complex of the ATP synthase from Escherichia coli.

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).

Topology of subunit a of the Escherichia coli ATP synthase.

The antigenic determinants of mAbs against subunit a of the Escherichia coli ATP synthase were mapped by ELISA using overlapping synthetic decapeptides. For two of the mAbs the epitopes are E4NMTPQD10 (GDH 14-5C6) and V29DPQ32 (GDH 8-8B3). Binding of these mAbs to membrane vesicles of different orientation revealed that both epitopes are accessible in vesicles with inside-out orientation. These results demonstrate that at least the N-terminal amino acids 1-32 of subunit a are located at the cytoplasmic side of the membrane. A further determination of the topology of subunit a was performed by inserting the reporter epitope DYKDDDDK (FLAG epitope) at different positions of the polypeptide chain. 10 of 13 insertions led to a functional F0F1 ATP synthase and allowed specific detection of the modified subunit a by immunoblotting using an mAb against the FLAG epitope. In addition, polyclonal anti-FLAG IgG was applied for the recognition of the mutant FLAG epitope DYKDDVDK. Cells carrying this mutant FLAG epitope at the C terminus of subunit a were able to grow on succinate as sole carbon and energy source, revealing a functional ATP synthase, in contrast to those carrying the original FLAG epitope at the same position. Binding studies with membrane vesicles of different orientation and anti-FLAG Ig demonstrated that both termini of the protein are located at the cytoplasmic side of the membrane, indicating that an even number of membrane-spanning segments is present in subunit a. In addition, insertion of two FLAG epitopes in tandem after K66, or one epitope after H95, and Q181 revealed that the polypeptide regions including these residues are accessible from the cytoplasmic surface of the membrane. These results support the view that the polypeptide chain of subunit a traverses the membrane six times.