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

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.