Orientation of the epsilon subunit in Escherichia coli ATP synthase.
Deletion mutations in the NH2- and COOH-terminal regions of the epsilon subunit of Escherichia coli ATP synthase were constructed making use of the AatII and HincII restriction enzyme sites. The resultant mutated epsilon species were analyzed for in vivo functionality and for recognition by anti-epsilon monoclonal antibodies. Deletion of residues Asp-7 through Gln-14 (epsilon delta D7-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 overexpression, the NH2-terminal deletion mutants were expressed at levels comparable to the wild-type epsilon 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 epsilon-1, which cannot bind to epsilon when it is part of F1-ATPase, recognized the COOH-terminal deletions well, but the NH2-terminal deletions poorly. Additional epitope mapping using epsilon fusion proteins revealed that residues required for the epsilon-1 epitope extend to between Thr-77 and Arg-85. Monoclonal antibody epsilon-4, which can bind to epsilon 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 epsilon-4 begin in the region between Val-78 and Met-95. These results imply a two-domain structure of epsilon and orient the subunit within the enzyme.
Skakoon
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Department of Biochemistry, University of Western Ontario, London, Canada.
Dunn
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D000911:Antibodies, Monoclonal; D001483:Base Sequence; D015153:Blotting, Western; D015252:Deoxyribonucleases, Type II Site-Specific; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes / Q000737:chemistry / Q000276:immunology; D004926:Escherichia coli / Q000201:enzymology*; D017353:Gene Deletion; D016296:Mutagenesis; D006180:Proton-Translocating ATPases / Q000737:chemistry* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000276:immunology; D015183:Restriction Mapping; D017422:Sequence Analysis, DNA
D000911:Antibodies, Monoclonal; D000939:Epitopes; D011993:Recombinant Fusion Proteins; D015252:Deoxyribonucleases, Type II Site-Specific; C069035:GACGTC-specific type II deoxyribonucleases; C024400:GTYRAC-specific type II deoxyribonucleases; D006180:Proton-Translocating ATPases