TOPOLOGY OF SUBUNIT-ALPHA OF THE ESCHERICHIA-COLI ATP SYNTHASE

The antigenic determinants of mAbs against subunit a of the Escherichi a coli ATP synthase were mapped by ELISA using overlapping synthetic d ecapeptides. For two of the mAbs the epitopes are E4NMTPQD10 (GDH 14-5 C6) and V29DPQ32 (GDH 8-8B3). Binding of these mAbs to membrane vesicl es of different orientation revealed that both epitopes are accessible in vesicles with inside-out orientation. These results demonstrate th at at least the N-terminal amino acids 1-32 of subunit a are located a t 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 cha in. 10 of 13 insertions led to a functional F0F1 ATP synthase and allo wed specific detection of the modified subunit a by immunoblotting usi ng an mAb against the FLAG epitope. In addition, polyclonal anti-FLAG IgG was applied for the recognition of the mutant FLAG epitope DYKDDVD K. Cells carrying this mutant FLAG epitope at the C terminus of subuni t a were able to grow on succinate as sole carbon and energy source, r evealing a functional ATP synthase, in contrast to those carrying the original FLAG epitope at the same position. Binding studies with membr ane vesicles of different orientation and anti-FLAG Ig demonstrated th at both termini of the protein are located at the cytoplasmic side of the membrane, indicating that an even number of membrane-spanning segm ents is present in subunit a. In addition, insertion of two FLAG epito pes in tandem after K66, or one epitope after H95, and Q181 revealed t hat the polypeptide regions including these residues are accessible fr om the cytoplasmic surface of the membrane. These results support the view that the polypeptide chain of subunit a traverses the membrane si x times.