Role of F0 and F1 subunits in the gating and coupling function of mitochondrial H(+)-ATP synthase. The effect of dithiol reagents.
A study is presented on the role of F0 and F1 subunits in oligomycin-sensitive H+ conduction and energy transfer reactions of bovine heart mitochondrial F0F1 H(+)-ATP synthase. Mild treatment with azodicarboxylic acid bis(dimethylamide) (diamide) enhanced oligomycin-sensitive H+ conduction in submitochondrial particles containing F1 attached to F0. This effect was associated with stimulation of the ATPase activity, with no effect on its inhibition by oligomycin, and depression of the 32Pi-ATP exchange. The stimulatory effect of diamide on H+ conduction decreased in particles from which F1 subunits were partially removed by urea. The stimulatory effect exerted by diamide in the submitochondrial particles with F1 attached to F0 was directly correlated with a decrease of the original electrophoretic bands of a subunit of F0 (F0I-PVP protein) and the gamma subunit of F1, with corresponding formation of their cross-linking product. In F0 liposomes, devoid of gamma subunit, diamide failed to stimulate H+ conduction and to cause disappearance of F0I-PVP protein, unless purified gamma subunit was added back. The addition to F0 liposomes of gamma subunit, but not that of alpha and beta subunits, caused per se inhibition of H+ conduction. It is concluded that F0I-PVP and gamma subunits are directly involved in the gate of the F0F1 H(+)-ATP synthase. Data are also presented indicating contribution to the gate of oligomycin-sensitivity conferral protein and of another protein subunit of F0, F6.
Zanotti
F
F
Institute of Medical Biochemistry and Chemistry, Consiglio Nazionale delle Ricerche, University of Bari, Italy.
Guerrieri
F
F
Capozza
G
G
Fiermonte
M
M
Berden
J
J
Papa
S
S
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002417:Cattle; D003958:Diamide / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology; D007425:Intracellular Membranes / Q000201:enzymology; D007700:Kinetics; D008081:Liposomes; D046911:Macromolecular Substances; D008929:Mitochondria, Heart / Q000201:enzymology*; D010710:Phosphates / Q000378:metabolism; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D013367:Submitochondrial Particles / Q000201:enzymology*; D013439:Sulfhydryl Reagents / Q000494:pharmacology*
D008081:Liposomes; D046911:Macromolecular Substances; D010710:Phosphates; D013439:Sulfhydryl Reagents; D003958:Diamide; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases; D005033:Ethylmaleimide