A novel 11-kDa inhibitory subunit in the F1FO ATP synthase of Paracoccus denitrificans and related alpha-proteobacteria.
The F(1)F(O) 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 F(O) inhibitors venturicidin and diciclohexylcarbodiimide. Comparison and N-terminal sequencing of the band profile in SDS-PAGE of the F(1) and F(1)F(O) 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 F(1)F(O)-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 F(1)F(O) and soluble F(1)-ATPase complexes. The 11-kDa protein, but not the epsilon subunit, inhibited the F(1)F(O) and F(1)-ATPase activities of P. denitrificans. The 11-kDa protein was also found in Rhodobacter sphaeroides associated to its native F(1)F(O)-ATPase. Taken together, the data unveil a novel inhibitory mechanism exerted by this 11-kDa protein on the F(1)F(O)-ATPase nanomotor of P. denitrificans and closely related alpha-proteobacteria.
Biology Department, Chemistry Faculty, National Autonomous University of Mexico (UNAM), Mexico City, Mexico.
Edgar
E
Morales-Ríos
Fernanda
F
de la Rosa-Morales
Guillermo
G
Mendoza-Hernández
José S
JS
Rodríguez-Zavala
Heliodoro
H
Celis
Mariel
M
Zarco-Zavala
José J
JJ
García-Trejo
D004791:Enzyme Inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D008970:Molecular Weight; D010231:Paracoccus denitrificans / Q000201:enzymology; D021122:Protein Subunits / Q000378:metabolism*; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000302:isolation & purification; D012242:Rhodobacter sphaeroides / Q000201:enzymology
D004791:Enzyme Inhibitors; D021122:Protein Subunits; D006180:Proton-Translocating ATPases