Mic60 assembles in proximity to the ER independently of known ER–mitochondria tethers and targets via an N-terminal region. (A) Images of Δmic19 cells expressing mito-TagBFP (blue), Mic60-mCherry (magenta), and Lam6-EGFP (green). Arrows mark examples of colocalization between Mic60 assemblies and Lam6. See Fig. S3 B for quantification. (B) Images of the indicated mmm1-1 strains expressing Atp2-mCherry (magenta) and Mic60-EGFP (green) after growth for 2 h at 37°C. Arrows mark examples of Mic60 assemblies. (C) Images of Δmic19 Δlam6 mmm1-1 cells expressing Atp2-mCherry (blue), Mic60-EGFP (green), and HaloTag-HDEL (magenta) after growth at 37°C for 2 h and 30 min. Arrows mark examples of Mic60 assemblies that appear in proximity to the ER. (D) Time-lapse images of cells as in C. White arrows indicate the original Mic60 assembly position, and yellow arrows track its movement. See also Video 5 and Video 6. (E) Single-plane and maximum-intensity projection images of Δmic60 cells expressing the indicated Mic60-EGFP variants (green) and mito-DsRed (magenta). Arrows indicate sites of EGFP assemblies. The schematic depicts the domain organization of Mic60 and each variant. (F) Graph of the frequency of cells as in E with the indicated number of foci per cell. Data are the average of three independent experiments with >45 cells per strain per experiment. Error bars indicate SEM. (G) Images of Δmic19 cells expressing Mic60-EGFP tagged at its endogenous locus or of Δmic60 cells expressing chromosomally integrated Mic60(1–365)-EGFP and coexpressing mito-dsRed (blue) and Mdm34-HaloTag (magenta). Arrows indicate sites of EGFP assembly colocalized with Mdm34. See Fig. S3 G for quantification. Cells are outlined with dashed lines. Scale bars = 2 µm.