ERMES function is differentially required for formation of MICOS subcomplexes. (A) Images of Δmic19 Δmmm1 cells expressing Atp2-mCherry (magenta) and Mic27-EGFP (green). (B) As in A for cells expressing Mic60-EGFP (green). White arrows mark Mic60 assemblies. (C) Images of Δmic19 cells expressing Mic27-mCherry (magenta) and Mic60-EGFP (green) with wild-type MMM1 or the temperature-sensitive allele mmm1-1 and and grown at the indicated temperatures and times. White arrows mark colocalized Mic60 and Mic27 assemblies, and green arrows indicate Mic60 assemblies with no detectable Mic27 assembly colocalized. (D) Graph of the percentage of Mic60 assemblies that colocalize with Mic27 from cells grown as in C. Data are the summation of three independent experiments with >130 Mic60 assemblies per condition. (E) Graph of the total spectral matches identified by immunopurification (IP) of Mic27-EGFP and MS analysis for each MICOS subunit in the indicated strain backgrounds relative to wild-type cells. Spectral matches of Mic27 were used to normalize each strain background to wild type and data represent the average of two experimental replicates. See Table S1. (F) Images are shown of Δmic60 MMM1 cells (top) or Δmic60 Δmmm1 cells expressing pVps13(L1627S) (bottom) and coexpressing Tim23-mCherry (magenta) and Mic27-EGFP (green). See Fig. S3 G for quantification. Cells are outlined with dashed lines. Scale bars = 2 µm.