Mic60 assembles in proximity to the ER independently of known ER–mitochondria tethers and targets via an N-terminal region. (A) Deconvolved fluorescence microscopy images are shown of Δmic19 Δmmm1 yeast cells expressing Atp2-TagBFP (blue), Mic60-EGFP (green), and mCherry-HDEL (magenta). Arrows indicate regions of Mic60 assembly in proximity to the ER. (B) Graphical depiction of the extent of colocalization between assemblies of Mic60 and Lam6 from cells as in Fig. 3 A. Data displayed are the summation of the indicated number of foci from at least three independent experiments. (C) Maximum-intensity projections of images from the indicated strains expressing mito-dsRed (magenta) and Mic60-EGFP (green). Arrows mark examples of Mic60 assemblies. (D) Western blot analysis of whole-cell lysates prepared from the indicated strains coexpressing Mic19-HA tagged at its endogenous locus and probed with the indicated antibodies. (E) Images of Δmic60 cells expressing chromosomally integrated Mic60-EGFP (top) or Mic60(LBS*)-EGFP (bottom) and coexpressing mito-dsRed (magenta). Arrows mark focal Mic60(LBS*)-EGFP. (F) As in D for Δmic60 cells expressing chromosomally integrated wild-type or LBS* Mic60-EGFP. (G) Graphical depiction of the extent of colocalization between assemblies of Mic60 and Mdm34 from cells as in Fig. 3 G. Data displayed are the summation of the categorization of the indicated number of foci from at least three independent experiments. Cell boundaries are indicated with dotted lines. Scale bars = 2 µm.