REVERSION MUTATIONS IN THE BETA-SUBUNIT MUTANTS OF ESCHERICHIA-COLI F1-ATPASE WITH DEFECTIVE SUBUNIT ASSEMBLY - IMPLICATIONS FOR STRUCTURE AND FUNCTION OF THE AMINO-TERMINAL REGION

We analyzed reversion mutations of the mutants with a substitution of Leu-40 by Pro or Glu-41 by Lys in the beta subunit of Escherichia coli F-1-ATPase. These mutants had an altered molecular assembly of the alpha and beta subunit on the membranes and lost the binding activity of the beta subunit to the monoclonal antibody beta 31. For the mutation of Leu-40 to Pro, we found that all reversion mutations occured at the original mutation site as Pro-40 to Ala, Gln, or Ser besides the wild-type Leu. These pseudo reversion mutations restored the cell growth on minimal agar supplemented with succinate as the sole carbon source, the assembly of the alpha and beta subunits, and also the ATPase activities in the membranes. These results suggested that Leu-40 itself is not an essential residue for the function of the beta subunit but that the residue contributes to a conformation around residue 40, which is important for the assembly of the alpha and beta subunits onto the membranes. For the mutation of Glu-41 to Lys, pseudo reversion mutations were found at the original residue 41 as Lys to Gln or Asn and also at Arg-218 to Cys or His in addition to the original Glu-41 to Lys mutation. These suppression mutations recovered the cell growth, indicating the recovery of ATP synthesis. The ATPase activity was high in cells with the Lys-41 to Glu mutation but those were relatively low in those with other reversion mutations. The assembly of the alpha and beta subunits recovered in the revertants to the wild-type level, except for the Lys-41 to Asn mutation, which resulted in a decreased amount of the a subunit on the membranes. These results suggested that though Glu-41 is not an essential residue, it plays an important role in the assembly of the alpha and beta subunit on the membranes. The results also suggested that residues Arg-218 and Glu-41 are located close together and interact with each other, either directly or indirectly. By using site-directed mutagenesis to introduce more mutations into residues 41 and 218, mutants with a substitution of Glu-41 to Arg or Leu and of Arg-218 to Glu, Val, or Gln were obtained. Combinations of the residues with bigger side chains, Arg-41 and Arg-218, Lys-41 and Arg-218, and Leu-41 and Arg-218, caused the loss of cell growth on succinate agar. Therefore, it was suggested that the interaction between residues 41 and 218 is important for the assembly of the alpha and beta subunit into the F1F0 complex, and that this interaction is limited by the spatial arrangement of the residues. (C) 1994 Academic Press, Inc.