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 F1-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 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 alpha 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.(ABSTRACT TRUNCATED AT 400 WORDS)
Miki
J
J
Department of Biotechnology, Faculty of Engineering Sciences, Okayama University, Japan.
Tsugumi
S
S
Kanazawa
H
H
D001483:Base Sequence; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005971:Glutamates / Q000235:genetics; D018698:Glutamic Acid; D007930:Leucine / Q000235:genetics; D008566:Membranes / Q000201:enzymology; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D006180:Proton-Translocating ATPases / Q000235:genetics* / Q000378:metabolism*; D015183:Restriction Mapping; D013329:Structure-Activity Relationship; D013489:Suppression, Genetic
D005971:Glutamates; D018698:Glutamic Acid; D006180:Proton-Translocating ATPases; D007930:Leucine