Localization of Ltc1 to ER–mitochondria and ER–vacuole contact sites requires Tom70/71 and Vac8, respectively. (A) Ltc1, marked by Ltc1-yEmCherry, colocalizes with Tom71, marked by Tom71-yEGFP, in foci on the mitochondrial outer membrane. Cells expressing Ltc1-yEmCherry and Tom71-yEGFP were grown to mid-log phase and imaged as described in “Fluorescence microscopy.” Dashed lines and numbers denote enlarged regions shown as separate grayscale images of each channel to the right of the merged image. (B) Vac8 and Tom70/71 are required for Ltc1 foci localization to vacuoles and mitochondria, respectively. Cells of the indicated genotypes expressing Ltc1-yEGFP and mtDsRed were grown to mid-log phase and imaged as described in “Fluorescence microscopy.” Images represent a maximum intensity z-projection. Dashed lines denote enlarged regions shown below. (C) Ltc1 is an ER membrane–associated protein. Δtom70 Δtom71 Δvac8 cells expressing Ltc1-yEGFP were grown to mid-log phase and treated with 1 µg/ml DAPI for 20 min to label the nucleus, washed twice in SD media, and imaged as described in “Fluorescence microscopy.” Extranuclear DAPI-stained structures are likely mtDNA. Bars, 2 µm.