Reversion Mutations in the β 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 β subunit of Escherichia coli F1-ATPase. These mutants had an altered molecular assembly of the α and β subunit on the membranes and lost the binding activity of the β subunit to the monoclonal antibody β31. 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 α and β 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 β subunit but that the residue contributes to a conformation around residue 40, which is important for the assembly of the α and β 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 α and β 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 α 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 α and β 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 α and β subunit into the F1F0 complex, and that this interaction is limited by the spatial arrangement of the residues.