Interaction of the δ and b Subunits Contributes to F1 and F0 Interaction in the Escherichia coli F1F0-ATPase

Interactions of the F 1 F 0 -ATPase subunits between the cytoplasmic domain of the b subunit (residues 26–156, b cyt ) and other membrane peripheral subunits including α, β, γ, δ, ε, and putative cytoplasmic domains of the a subunit were analyzed with the yeast two-hybrid system and in vitro reconstitution of ATPase from the purified subunits as well. Only the combination of b cyt fused to the activation domain of the yeast GAL-4, and δ subunit fused to the DNA binding domain resulted in the strong expression of the β-galactosidase reporter gene, suggesting a specific interaction of these subunits. Expression of b cyt fused to glutathione S -transferase (GST) together with the δ subunit in Escherichia coli resulted in the overproduction of these subunits in soluble form, whereas expression of the GST-b cyt fusion alone had no such effect, indicating that GST-b cyt was protected by the co-expressed δ subunit from proteolytic attack in the cell. These results indicated that the membrane peripheral domain of b subunit stably interacted with the δ subunit in the cell. The affinity purified GST-b cyt did not contain significant amounts of δ, suggesting that the interaction of these subunits was relatively weak. Binding of these subunits observed in a direct binding assay significantly supported the capability of binding of the subunits. The ATPase activity was reconstituted from the purified b cyt together with α, β, γ, δ, and ε, or with the same combination except ε. Specific elution of the ATPase activity from glutathione affinity column with the addition of glutathione after reconstitution demonstrated that the reconstituted ATPase formed a complex. The result indicated that interaction of b and δ was stabilized by F 1 subunits other than ε and also suggested that b-δ interaction was important for F 1 -F 0 interaction.