AMINO-ACID REPLACEMENTS AT BINDING-SITES OF MONOCLONAL-ANTIBODY IN THE F1-ATPASE BETA-SUBUNIT FROM ESCHERICHIA-COLI CAUSED ALTERED SUBUNIT INTERACTIONS

The monoclonal antibodies (mAb), beta 12 and beta 31, against the beta subunit of Escherichia coli F-1-ATPase (Miki, J., Matsuda, T., Kariya , H., Ohmori, H., Tsuchiya, T, Futai, M., and Kanazawa, H. (1992) Arch , Biochem. Biophys. 294, 373-381) recognize the amino-terminal portion of the maximal 104 residues, that are not exposed to the surface of t he F-1-ATPase. To identify the epitope residues within these 104 resid ues, we introduced random mutations in the region, and clones without binding activity to the mAb, beta 12 and beta 31, were screened. beta subunits defective in binding with mAb beta 12 or beta 31 had amino ac id replacements at residues 26, 40, 52, 71, and 74 or at residues 40, 41, 43, 44, 46, and 52, respectively. These residues could be part of the epitope and are possibly located close together. We tested the eff ects of the mutations on oxidative phosphorylation-dependent growth by introducing expression plasmids of the beta subunit gene with the poi nt mutations into the beta-less mutant, JP17. The alpha and beta subun its were missing from the JP17 membranes, and both subunits were assem bled functionally after expression of the beta subunit was induced by introducing the expression plasmid. The replacement of Leu-40 by Pro c aused the amounts of the a and p subunits on the membranes to be reduc ed less than 20% of the amounts in wild type. The replacement of Glu-4 1 by Lys caused a loss of the alpha subunit on the membranes, without any decrease in the beta subunit. These results indicated that the mut ation of Leu-40 to Pro affects the essential role of the beta subunit in the assembly of the alpha and beta subunits on the membranes and th at the mutation of Glu-41 to Lys partly affects it. The amino-terminal region of the beta subunit, in particular its ternary structure, incl uding residues 40 and 41, plays an important role in the molecular ass embly of the alpha and beta subunits on the membranes.