Fig. 3.

Impact of removal of either subunit F₆ or d on the composition of vestigial ATP synthase complexes. Relative abundances of subunits and the inhibitor protein IF₁ in HAP1-ΔF₆ or HAP1-Δd cells, respectively, A and B in immunopurified vestigial complexes of ATP synthase or C and D in digitonin solubilized mitoplasts. IF₁-M1 and IF₁-M3 are specific mature forms of IF₁ (5), and f-1 and f-2 are isoforms of subunit f (Swiss-Prot P56134). The digitonin extracts of mitoplasts were prepared from 1:1 mixtures of differentially SILAC-labeled HAP1-WT cells plus either HAP1-ΔF₆ or HAP1-Δd cells, and complexes were purified from these samples. The purified samples were digested separately in solution with trypsin or chymotrypsin, and mitoplast samples were separated by SDS-PAGE and digested in gel with trypsin. Peptide mixtures were fractionated by reverse phase HPLC and analyzed by mass spectrometry. The histograms are the median values of both relative abundance ratios determined for proteins found in the complementary stable isotope labeling with amino acids in cell culture (SILAC) experiments. The protein ratio is derived from a minimum of two peptide ratios from each experiment except for ATP6 (B and D, control heavy isotope experiments), f-2 (D, both experiments), and IF₁-M1 (D, control light isotope experiment) where all values are from a single peptide ratio. In mitoplast samples, no ratios were obtained for subunit c or IF₁-M3 (C and D). The data for the identified proteins are given in Datasets S1–S8. Error bars show the range of the two values.