A novel 11-kDa inhibitory subunit in the F1FO ATP synthase of Paracoccus denitrificans and related α-proteobacteria

The F1FO and F-1-ATPase complexes of Paracoccus denitrificans were isolated for the first time by ion exchange, gel filtration, and density gradient centrifugation into functional native preparations. The liposome-reconstituted holoenzyme preserves its tight coupling between F-1 and F-O sectors, as evidenced by its high sensitivity to the FO inhibitors venturicidin and diciclohexylcarbodiimide. Comparison and N-terminal sequencing of the band profile in SDS-PAGE of the F-1 and F1FO preparations showed a novel 11-kDa protein in addition to the 5 canonical alpha, beta, gamma, delta, and epsilon subunits present in all known F-1-ATPase complexes. BN-PAGE followed by 2D-SDS-PAGE confirmed the presence of this 11-kDa protein bound to the native F1FO-ATP synthase of P. denitrificans, as it was observed after being isolated. The recombinant 11 kDa and epsilon subunits of P. denitrificans were cloned, overexpressed, isolated, and reconstituted in particulate F1FO and soluble F-1-ATPase complexes. The 11-kDa protein, but not the epsilon subunit, inhibited the F1FO and F-1-ATPase activities of P. denitrificans. The 11-kDa protein was also found in Rhodobacter sphaeroides associated to its native F1FO-ATPase. Taken together, the data unveil a novel inhibitory mechanism exerted by this 11-kDa protein on the F1FO-ATPase nanomotor of P. denitrificans and closely related alpha-proteobacteria.-Morales-Rios, E., de la Rosa-Morales, F., Mendoza-Hernandez, G., Rodriguez-Zavala, J. S., Celis, H., Zarco-Zavala, M., Garcia-Trejo, J. J. A novel 11-kDa inhibitory subunit in the F1FO ATP synthase of Paracoccus denitrificans and related alpha-proteobacteria. FASEB J. 24, 599-608 (2010). www.fasebj.org