Abstract
The yeast F1F0-ATP synthase forms dimeric complexes in the mitochondrial inner membrane and in a manner that is supported by the F0-sector subunits, Su e and Su g. Furthermore, it has recently been demonstrated that the binding of the F1F0-ATPase natural inhibitor protein to purified bovine F1-sectors can promote their dimerization in solution (Cabezon, E., Arechaga, I., Jonathan P., Butler, G., and Walker J. E. (2000) J. Biol. Chem. 275, 28353-28355). It was unclear until now whether the binding of the inhibitor protein to the F1 domains contributes to the process of F1F0-ATP synthase dimerization in intact mitochondria. Here we have directly addressed the involvement of the yeast inhibitor protein, Inh1, and its known accessory proteins, Stf1 and Stf2, in the formation of the yeast F1F0-ATP synthase dimer. Using mitochondria isolated from null mutants deficient in Inh1, Stf1, and Stf2, we demonstrate that formation of the F(1)F(0)-ATP synthase dimers is not adversely affected by the absence of these proteins. Furthermore, we demonstrate that the F1F0-ATPase monomers present in su e null mutant mitochondria can be as effectively inhibited by Inh1, as its dimeric counterpart in wild-type mitochondria. We conclude that dimerization of the F1F0-ATP synthase complexes involves a physical interaction of the membrane-embedded F0 sectors from two monomeric complexes and in a manner that is independent of inhibitory activity of the Inh1 and accessory proteins.
Highlights
Mitochondria, eukaryotic organelles, produce energy in the form of adenosine 5Ј-triphosphate (ATP) in a process termed oxidative phosphorylation
We demonstrate that the F1F0ATPase monomers present in su e null mutant mitochondria can be as effectively inhibited by Inh1, as its dimeric counterpart in wild-type mitochondria
Mitochondrial membrane proteins were solubilized with the detergent digitonin and the dimeric state of the F1F0-ATP synthase was directly analyzed by blue-native polyacrylamide gel electrophoresis (BN-PAGE) (Fig. 1)
Summary
Yeast strains used in this study were wild-type W303-1A (Mata, leu, trp, ura, his, ade2) [20] and the su e null mutant, ⌬su e (W303-1A, leu, trp, ura, ade, TIM11::HIS3) [10]. The introduction of the kanamycin resistance gene (KANr) into the INH1, STF1, STF2, and SFL2 (YLR327c) gene loci of wild-type cells, resulting in the complete or partial deletion of the respective open reading frames was performed. S1: 5Ј-caacagtaacaaaccgctcaagtgtacaaccaatcagaaaaaatgcgtacgctgcaggtcgac-3Ј (corresponds to nucleotides Ϫ42 to ϩ3 of the STF2 gene locus and nucleotides in the MCS of the pFA6a-KANMX6 plasmid, as described above), and S2: 5Ј-atcaatctcatcgcctggcttaccccaattacccttgccggaaccatcgatgaattcgagctcg-3Ј (corresponds to nucleotides ϩ106 to ϩ150 of the STF2 gene (ϩ1 to ϩ255 bp), which are located in the open reading frame of the STF2 locus and nucleotides of the MCS of the pFA6aKANMX6 plasmid, as described above). For the ⌬inh1/⌬stf Strain, ⌬inh1::KANr/⌬stf1::HIS3—The STF1 gene locus was deleted in the ⌬inh1::KAN yeast strain, as follows. The mitochondrial membranes were collected by centrifugation at 18,000 ϫ g for 20 min at 4 °C and were stored at Ϫ80 °C in a sucrosecontaining buffer
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