Subunit Interactions ofEscherichia coliF1–ATPase: Mutants of the γ Subunits Defective in Interaction with the ϵ Subunit Isolated by the Yeast Two-Hybrid System

Previously, we established a method to detect subunit interactions of F1-ATPase by the yeast two-hybrid system (Moritani, C.,et al. Biochim. Biophys. Acta1274, 67–72, 1996). Here, we isolated mutants of the γ subunits defective in interaction with the ϵ subunit by this new procedure to study the molecular basis of coupling mechanisms of the F1F0-ATPase. Based on the intensities of the reporter gene expression in this system, five mutants of the γ subunit with different levels of γ–ϵ interactions were isolated and their single base substitutions were determined. Mutants with a substitution of Pro-55 for Leu, Thr-102 for Met, Val-141 for Asp, or Gln-235 for Leu exhibited decreased reporter gene expression, suggesting decreased levels of interaction, while Asp-85 for Gly mutation caused a higher level of expression, suggesting increased interaction. Among these point mutations, G85D, M102T, or D141V mutations were introduced into the γ subunit gene in the plasmid carrying wholeuncoperon. Transformants carrying a deletion mutant of the wholeuncoperon with these expression plasmids were analyzed. Mutations M102T and D141V with decreased γ–ϵ interaction caused increases of membrane-bound F1-ATPase activity and proton pumping activity, while G85D with increased γ–ϵ interaction exhibited lower levels of F1-ATPase activity in the membranes. Molecular assembly of the F1subunits on the mutant membranes detected by Western blotting exhibited no defect for all three mutants. These results suggested that the correlation between the ATPase activity and γ–ϵ interaction is reciprocal and this interaction may regulate the ATPase activity. The topological and functional importance of Gly-85, Met-102, and Asp-141 together with Leu-55 and Leu-235 in γ–ϵ interaction is discussed.