Ylr072w/Ltc1 is localized to ER–mitochondria and ER–vacuole contact sites. (A) Deletion of YLR072W causes no significant growth defect on fermentable or nonfermentable carbon sources. Cells were grown to mid-log phase and plated on synthetic complete media containing 2% dextrose (SD) or 3% glycerol + 2% ethanol (SEG). (B) Deletion of YLR072W causes no significant change in mitochondrial morphology. Cells expressing mitochondria-targeted DsRed were grown to mid-log phase and imaged as described in “Fluorescence microscopy.” (C) Deletion of YLR072W causes a synthetic sick/lethal phenotype in Δmdm34 cells. Expression of Ylr072w-yEGFP restores Δmdm34 growth phenotype. Yeast diploids with the indicated genotypes were sporulated, subjected to tetrad dissection, and the resulting spore colonies were genotyped based on segregation pattern of markers. Red circles indicate inviable Δmdm34 Δylr072w cells. Green circles indicate viable Δmdm34 YLR072W-yEGFP cells. (D–F) Ylr072w localization was assessed in WT cells using Ylr072w-yEGFP integrated at its endogenous locus relative to ERMES marked by Mdm34-mCherry (D), ER and mitochondria marked by DsRed-HDEL and mitochondrial-targeted mtBFP, respectively (E), and ER marked by DsRed-HDEL and vacuoles marked by Pho8-3XBFP (F). Cells were grown to mid-log phase and imaged as described in “Fluorescence microscopy.” Yellow arrowheads in E mark ER–mitochondria contact sites, open white arrowheads in E and F mark NVJs, and closed white arrowheads in F mark ER–vacuole contacts. (G) Quantification of Ltc1 foci localization from D–F. Dashed lines in B and D demarcate cell boundaries. Dashed lines in E and F indicate enlarged regions shown as separate grayscale images to the right. Bars, 2 µm.