ORIENTATION OF THE EPSILON-SUBUNIT IN ESCHERICHIA-COLI ATP SYNTHASE

Deletion mutations in the NH2- and COOH-terminal regions of the ε{lunate} subunit of Escherichia coli ATP synthase were constructed making use of the AatII and HincII restriction enzyme sites. The resultant mutated species were analyzed for in vivo functionality and for recognition by anti-ε{lunate} monoclonal antibodies. Deletion of residues Asp-7 through Gln-14 (ε{lunate}ΔD7-Q14) resulted in reduced ability to complement uncC mutants as determined by growth yields on limiting glucose medium and by formation of small colonies on plates with succinate as the source of carbon and energy. None of the other mutants was notably impaired. Upon induction to obtain over-expression, the NH2-terminal deletion mutants were expressed at levels comparable to the wild-type ε{lunate} subunit, but the COOH-terminal deletion mutants were expressed less strongly, suggesting that residues in the latter region are important for protein stability. Monoclonal antibody ε{lunate}-1, which cannot bind to ε{lunate} when it is part of F1-ATPase, recognized the COOH-terminal deletions well, but the NH2-terminal deletions poorly. Additional epitope mapping using ε{lunate} fusion proteins revealed that residues required for the ε{lunate}-1 epitope extend to between Thr-77 and Arg-85. Monoclonal antibody ε{lunate}-4, which can bind to ε{lunate} when it is part of F1-ATPase, recognized the NH2-terminal deletions well, but hardly recognized the COOH-terminal deletions, indicating a role of residues located COOH-terminal to Ile-131 in recognition by this antibody. Epitope mapping using the fusion proteins revealed that the residues required by (ε{lunate}-4 begin in the region between Val-78 and Met-95. These results imply a two-domain structure of ε{lunate} and orient the subunit within the enzyme. © 1993 Academic Press. All rights reserved.