Overexpression of the Inhibitor Protein IF₁ in AS-30D Hepatoma Produces a Higher Association with Mitochondrial F₁F₀ ATP Synthase Compared to Normal Rat Liver: Functional and Cross-Linking Studies

Abstract

According to functional studies, the higher IF₁ content reported in mitochondria of cancerous cells is supposed to induce a higher association with the F₁F₀ complex than in normal cells and therefore a better inhibition of its ATPase activity. The first structural evidence supporting this prediction is here presented. Densitometric analyses of Western blotting experiments indicated a 2-fold increase in IF₁ content of AS-30D submitochondrial particles compared to normal rat liver controls. The ratio of IF₁/F₁ α subunit increased similarly as judged by Westernblot analyses. This IF₁ overexpression correlated with a slower rate of IF₁ release (F₁F₀-ATPase activation) from the F₁F₀ complex in AS-30D than in normal rat liver submitochondrial particles. The IF₁-IF₁, γ-IF₁, and α-IF₁ cross-linkages previously formed with dithiobis(succinimidylpropionate) in bovine F₁F₀I and IF₁ complexes (Minauro-Sanmiguel, F., Bravo, C., and García, J. J. (2002). J. Bioenerg. Biomembr. 34, 433–443) were reproduced in the F₁F₀I-ATP synthase of hepatoma AS-30D cells. However, a much lower yield of IF₁ cross-linkages was found in normal rat liver particles which made them almost undetectable in SMP as well as in the immunoprecipitated F₁F₀I complex. Modeling in vivo IF₁ overexpression of cancerous cells by in vitro reconstitution of excess recombinant IF₁ with rat liver submitochondrial particles devoid of IF₁ reproduced the same IF₁ cross-linkages observed in AS-30D particles.