(A) Z-projections of representative deconvolved fluorescence microscopy images are shown from wild type yeast cells expressing the indicated integrated GFP-tagged MICOS protein and mito-DsRed. The yellow box indicates the region of the cell shown in the inset below. The inset displays a single plane. (B) Representative images are shown as in (A) for ∆MICOS cells expressing the indicated GFP-tagged MICOS proteins re-integrated at their endogenous loci. The arrows mark sites of Mic60 localization to foci. (C) The distribution of Mic60-EGFP in ∆MICOS cells was determined as in (B) with the indicated untagged MICOS proteins re-integrated at their endogenous loci. (D) Representative Western blot analysis with the indicated antibodies are shown of whole cell lysates (left panel) and immunoprecipitation eluates (IPs; right panel) from ∆MICOS cells expressing either Mic60-FLAG or Mic60-EGFP at the MIC60 locus, and where indicated, expressing Mic60-EGFP from the ura3 locus using the MIC60 promoter (pMic60-EGFP). G6PDH antibody was used as a loading control. IPs were performed with the indicated antibodies. The asterisk marks a band consistent with the size of IgG heavy chain. (E) Western blot analysis with the indicated antibodies of total (T), supernatant (S), and insoluble (P) fractions of detergent-solubilized mitochondria isolated from wild type (left) or ∆MICOS (right) cells expressing Mic60-EGFP and centrifuged at 50,000×g for 1 hr, a condition that pellets particles of 60S and greater. (F) A graph showing the percentage of cells with detectable Mic60 foci from ∆MICOS cells without and with Mic19 expression as shown in (B) and (C). Approximately 75 cells from three independent experiments were quantified and data are represented as mean ± SEM. (G) Table describing the number of total spectra and protein coverage for the indicated proteins (top) from purifications and mass spectrometry analysis using FLAG antibody from ∆MICOS cell lysate expressing the indicated combinations of Mic60 and Mic19 (left) expressed at their endogenous loci. Data shown are the mean of two independent experiments. Scale bars: (A–B) 3 μm; (C) 2 μm. See also Figure 2—figure supplement 1.