The 24-kDa subunit of the bovine mitochondrial NADH:ubiquinone oxidoreductase is a G protein.
Based on the results obtained from GTP overlay assay, immunoprecipitation, two dimensional electrophoresis and radiolabeled GTP binding, we provide evidence that the bona fide subunit of Complex I, the long known 24 kDa protein is a G protein. Bacterially expressed 24 kDa protein with additional N-terminal methionine and alanine residues or naturally expressed truncated isoform fail to bind GTP suggesting that secondary modification/ processed N-terminal end is necessary for GTP binding. Competitive inhibition of binding of radiolabeled GTP to electroblotted 24 kDa protein with unlabelled nucleotides showed that the protein binds GTP and GDP with high affinity in presence of Mg2+, and has decreased to very low affinity for ITP, CTP, GMP and UTP. A comparative binding of [gamma-35S]-GTP to Complex I and 24 kDa protein (electroblotted) suggests that the GTP binding in the native Complex is solely due to 24 kDa protein. Further, four fold difference in the binding affinities between native Complex I and 24 kDa protein (electroblotted) as seen by Scatchard analysis of the binding data indicates that protein undergoes structural rearrangement in Complex I bound form, that presumably triggers divalent cation dependent GTPase activity in native complex. We were unable to detect the effect of GTP/ GDP on the ubiquinone/ferricyanide reductase activity. Since the subunit is found missing in tissues affected by mitochondrial respiratory chain diseases, we presume that the subunit has regulatory role in the Complex I function in the electron transport chain.
Hegde
R
R
Department of Animal Biology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia 19104, USA.
D000818:Animals; D002417:Cattle; D042967:Electron Transport Complex I; D020558:GTP Phosphohydrolases / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D008928:Mitochondria / Q000201:enzymology*; D017240:Mitochondrial Myopathies / Q000201:enzymology; D016660:NAD(P)H Dehydrogenase (Quinone) / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D009247:NADH, NADPH Oxidoreductases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000378:metabolism
D011994:Recombinant Proteins; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D009247:NADH, NADPH Oxidoreductases; D016660:NAD(P)H Dehydrogenase (Quinone); D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; D042967:Electron Transport Complex I