THE F0 COMPLEX OF THE PROTON-TRANSLOCATING F-TYPE ATPASE OF ESCHERICHIA-COLI

Abstract

The ATP synthase (F1F(o)) of Escherichia coli consists of two structurally and functionally distinct entities. The F1 part is composed of five subunits alpha, beta, gamma, delta and epsilon (3:3:1:1:1) and carries the catalytic centres of the enzyme. The membrane-bound F(o) complex functions as a proton channel and consists of the three subunits a, b and c (1:2:10+/-1). Subunit c (8288 M(r)) exhibits a hairpin-like structure within the membrane. A conserved acidic residue (Asp-61) in the C-terminal hydrophobic segment is absolutely required for proton translocation through F(o), whereas the hydrophilic loop region is necessary for F1 binding. Expression of the chloroplast proteolipid together with subunits a and b of E. coli did not produce an active F(o) hybrid complex. Therefore, the construction of hybrid c subunits consisting of parts of the proteolipid from both organisms is in progress to determine those parts of subunit c that are essential for a functional interplay with subunits a and b. Subunit a (30276 M(r)), which is also involved in proton translocation, is an extremely hydrophobic protein with 5-8 membrane-spanning helices. Studies with alkaline phosphatase fusion proteins resulted in controversial conclusions about the localization of the N and C termini of the protein. A foreign epitope (13 amino acids) has been inserted into the N- or C-terminal region of subunit a without affecting the function of F(o). Binding studies with a monoclonal antibody against this epitope are now under investigation to determine the orientation of subunit a. Subunit b (I 7 265 M(r)) is anchored in the membrane by its apolar N-terminal region, whereas the hydrophilic part protrudes into the cytoplasm. Studies with proteases and truncated b' subunits revealed that the C-terminal part of subunit b is involved in binding of F1 to F(o) and is necessary for correct assembly of F(o).