FIGURE 1:

Cox4 interacts with mtHsp70 and Mge1 in an ATP-sensitive manner. (A) Yeast cells were labeled with heavy (H) isotopes [¹⁵N₂¹³C₆]lysine/[¹⁵N₄¹³C₆]arginine (wild type) or their corresponding light (L) [¹⁴N¹²C]-containing variants (Cox4_His strain). Mitochondria were isolated, lysed, and subjected to affinity purification. Elution fractions were mixed and analyzed by quantitative mass spectrometry. The mean log₁₀(L/H) ratio and −log₁₀ p values of three independent experiments were determined and plotted against each other. Red dots, Cox4, mtHsp70, and Mge1; yellow, subunits of complex IV; green, subunits of complex III; black, other proteins; gray, unspecific proteins (see Supplemental Table S1). (B) Isolated mitochondria from wild-type (WT), Cox6_His, and Cox4_His yeast strains were lysed in digitonin and incubated with Ni²⁺-NTA agarose. Bound complexes were eluted with imidazole, and samples were analyzed by SDS–PAGE and Western blotting. Load, 3%; elution, 100%. (C, D) Wild-type (WT) and mtHsp70_His mitochondria were analyzed as described for B. Where indicated, lysis and purification of the samples were performed in the presence of 5 mM MgCl₂ and 5 mM ADP or ATP. Load, 3%; elution, 100%. AAC, ADP/ATP carrier; DIC, dicarboxylate carrier.