Fig. 2.

Ccp1 exits mitochondria as yeast begin respiring and extramitochondrial Ccp1 does not possess CCP activity. (A) Immunoblot analysis of equal volumes of denucleated (S2), mitochondrial (P10), and cytosolic (S10) fractions vs. cell age. Porin and Cyc1 are mitochondrial outer membrane and IMS markers, respectively. (B) The Ccp1 signals in A were quantified and normalized to the sum of the Coomassie bands in the same lane. (C) Dot blot analysis with anti-Ccp1 (Top row) and the ECL reagent (luminol/H₂O₂) to detect heme (Bottom row) from 2-d and 7-d P10 and S10 fractions. Myoglobin and BSA were used as positive and negative heme controls, respectively. (D) Normalized CCP activity in mitochondrial (P10) and cytosolic (S10) fractions. Specific activity was ratioed by the Ccp1 protein levels in B and normalized to the level for 2-d cells (SI Appendix, Table S2). Results in A and C are representative of three independent cultures (n = 3) and averages ± SD are plotted in B and D.