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Cross-Linking of the Endogenous Inhibitor Protein (IF1) with Rotor (γ,ε) and Stator (α) Subunits of the Mitochondrial ATP Synthase

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Abstract

The location of the endogenous inhibitor protein ( IF1) in the rotor/stator architecture of the bovine mitochondrial ATP synthase was studied by reversible cross-linking with dithiobis(succinimidylpropionate) in soluble F1I and intact F1F0I complexes of submitochondrial particles. Reducing two-dimensional electrophoresis, Western blotting, and fluorescent cysteine labeling showed formation of α–IF1, IF1–IF1, γ–IF1, and ε–IF1 cross-linkages in soluble F1I and in native F1F0I complexes. Cross-linking blocked the release of IF1 from its inhibitory site and therefore the activation of F1I and F1F0I complexes in a dithiothreitol-sensitive process. These results show that the endogenous IF1 is at a distance ≤12 Å,to γ and ε subunits of the central rotor of the native mitochondrial ATP synthase. This finding strongly suggests that, without excluding the classical assumption that IF1 inhibits conformational changes of the catalytic β subunits, the inhibitory mechanism of IF1 may involve the interference with rotation of the central stalk.

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Authors and Affiliations

  1. Departamento de Bioquímica, Instituto Nacional de Cardiología, “Ignacio Chávez,”, México, D.F, 14080, México

    Fernando Minauro-Sanmiguel, Concepción Bravo & José J. García

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  1. Fernando Minauro-Sanmiguel
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  2. Concepción Bravo
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  3. José J. García
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Correspondence to José J. García.

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Minauro-Sanmiguel, F., Bravo, C. & García, J.J. Cross-Linking of the Endogenous Inhibitor Protein (IF1) with Rotor (γ,ε) and Stator (α) Subunits of the Mitochondrial ATP Synthase. J Bioenerg Biomembr 34, 433–443 (2002). https://doi.org/10.1023/A:1022514008462

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