Plant mitochondrial F₀F₁ ATP synthase

Abstract

Spinach leaf mitochondrial FoFl ATPase has been purified and is shown to consist of twelve polypeptides. Five of the polypeptides constitute the F1 part of the enzyme. The remaining polypeptides, with molecular masses of 28 kDa, 23 kDa, 18.5 kDa, 15 kDa, 10.5 kDa, 9.5 kDa and 8.5 kDa, belong to the Fo part of the enzyme. This is the first report concerning identification of the subunits of the plant mitochondrial Fo. The identification of the components is achieved on the basis of the N-terminal amino acid sequence analysis and Western blot technique using monospecific antibodies against proteins characterized in other sources. The 28-kDa protein crossreacts with antibodies against the subunit of bovine heart ATPase with N-terminal Pro-Val-Pro-which corresponds to subunit Fob of Escherichia coli FoF1. Sequence analysis of the N-terminal 32 amino acids of the 23-kDa protein reveals that this protein is similar to mammalian oligomycin-sensitivity-conferring protein and corresponds to the F16 subunit of the chloroplast and E. coli ATPases. The 18.5-kDa protein crossreacts with antibodies against subunit 6 of the beef heart F, and its N-terminal sequence of 14 amino acids shows a high degree of sequence similarity to the conserved regions at N-terminus of the ATPase subunits 6 from different sources. ATPase subunit 6 corresponds to subunit F,a of the E. coli enzyme. The 15-kDa protein and the 10.5-kDa protein crossreact with antibodies against F6 and the endogenous ATPase inhibitor protein of beef heart FoF1-ATPase, respectively. The 9.5-kDa protein is an N,N-dicyclohexylcarbodiimide-binding protein corresponding to subunit Foc of the E. coli enzyme. The 8.5-kDa protein is of unknown identity. The isolated spinach mitochondrial FoFl ATPase catalyzes oligomycin-sensitive ATPase activity of 3.5 pmol . mg-' . min-I . The enzyme catalyzes also hydrolysis of GTP (7.5 pmol . mg-l * min-') and ITP (4.4 pmol . mg-' . min-I).Hydrolysis of ATP was stimulated fivefold in the presence of amphiphilic detergents, however the hydrolysis of other nucleotides could not be stimulated by these agents. These results show that the plant mitochondrial FoF ATPase complex differs in composition from the other mitochondrial, chloroplast and bacterial ATPases. The enzyme is, however, more closely related to the yeast mitochondrial ATPase and to the animal mitochondrial ATPase than to the chloroplast enzyme. The plant mitochondrial enzyme, however, exhibits catalytic properties which are characteristic for the chloroplast enzyme.The membrane-bound H+-ATPase, F,,F,-type, of mitochondria, chloroplasts and bacteria catalyzes the terminal reaction of oxidation and photophosphorylation. For ATP synthesis, the enzyme utilizes the respiratory-chain-induced electrochemical membrane potential and catalyzes H+-backflowCorrespondence to E. Claser, Department of Biochemistry, Arrhcnius Laboratories, Stockholm Univcrsity, 106 91 Stockholm, SwedenAhbrcviutions. Fo and F,, proton-translocating and catalytic moieties of the mitochondrial ATP synthase, respec...

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