The F0 complex of the proton-translocating F-type ATPase of Escherichia coli

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Abstract
The ATP synthase (F1F0 ) of Escherichia coli consists of two structurally and functionally distinct entities. The F1 part is composed of five subunits α, β, γ, δ and ϵ (3:3:1:1:1) and carries the catalytic centres of the enzyme. The membrane-bound F0complex functions as a proton channel and consists of the three subunits a, b and c (l:2:10±l). Subunit c (8288 Mr) 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 F0, whereas the hydrophilic loop region is necessary for F, binding. Expression of the chloroplast proteolipid together with subunits a and b of E. coli did not produce an active F0 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 Mr), 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 Fo. Binding studies with a monoclonal antibody against this epitope are now under investigation to determine the orientation of subunit a. Subunit b (17265 Mr) 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 F0 and is necessary for correct assembly of F0.