A yeast mitochondrial ATPase inhibitor interacts with three proteins that are easy to dissociate from the mitochondrial inner membrane.
A mitochondrial ATPase inhibitor is a 7.4 kDa protein that regulates the catalytic activity of ATP synthase (F(1)F(o)-ATPase). In the present study, we examined the binding sites of the inhibitor on the mitochondrial membrane using chemical cross-linkers, disuccinimidyl suberate (DSS) and N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ). Most of the inhibitors were recovered from the inner membrane fraction of mitochondria, indicating that the inhibitor binds to the membrane. Seven different cross-linked products that reacted with the antibody against the inhibitor were detected. The apparent molecular masses of the products were 61, 58, 47, 41, 28, 27, and 26 kDa. The 61 and 58 kDa products were attributed to the inhibitor+alpha and inhibitor+beta adducts on immunoblotting. The proteins cross-linked to the inhibitor in the 28, 27, and 26 kDa products were distinguished from subunit 4 (23 kDa), oligomycin sensitivity conferring protein (21 kDa), and subunit d (20 kDa) of F(1)F(o)-ATPase by analysis of the cross-linked products of mutant mitochondria in which the three proteins were replaced by hemagglutinin-tagged versions. The 28, 27, and 26 kDa products could be gradually dissociated from the mitochondrial membrane by increasing the salt concentration. These results shows that the endogenous inhibitor binds not only to the catalytic part of the enzyme, but also to the 19-21 kDa proteins that loosely associate with the mitochondrial inner membrane.
D000595:Amino Acid Sequence; D001665:Binding Sites; D003432:Cross-Linking Reagents / Q000737:chemistry; D004791:Enzyme Inhibitors / Q000378:metabolism* / Q000494:pharmacology; D000939:Epitopes; D006388:Hemagglutinins / Q000276:immunology; D015151:Immunoblotting; D007425:Intracellular Membranes / Q000187:drug effects / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism*; D008928:Mitochondria / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism; D025261:Mitochondrial Proton-Translocating ATPases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009840:Oligomycins / Q000494:pharmacology; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D011804:Quinolines / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D012995:Solubility; D013388:Succinimides / Q000737:chemistry
D003432:Cross-Linking Reagents; D004791:Enzyme Inhibitors; D000939:Epitopes; D006388:Hemagglutinins; D008565:Membrane Proteins; D009840:Oligomycins; D021122:Protein Subunits; D011804:Quinolines; D029701:Saccharomyces cerevisiae Proteins; D013388:Succinimides; C000470:EEDQ; D025261:Mitochondrial Proton-Translocating ATPases; C019358:disuccinimidyl suberate