Figure 4. The distribution of Ltc1 to ER–mitochondria and ER–vacuolar contacts regulates Ltc1-dependent separable mitochondrial and vacuolar functions. (A) The localization of Ltc1 to ER–mitochondria, but not to ER–vacuole, contacts is essential in Δmdm34 cells. Diploid yeast heterozygous for the indicated deletions were sporulated and analyzed by tetrad dissection as described in “Fluorescence microscopy.” The green circle indicates viable Δvac8 Δmdm34 cells (the colonies immediately above and below are Δvac8 and WT, respectively). Red circles indicate nonviable Δtom70 Δtom71 Δmdm34 cells. (B) The Ltc1 GRAM domain is required for the mitochondrial localization of Ltc1. Cells expressing Ltc1(ΔGRAM)-yEGFP, Vph1-yEmCherry (vacuoles), and mtBFP (mitochondria) were grown to mid-log phase and imaged as described in “Fluorescence microscopy.” Open arrowheads indicate the localization of Ltc1 to Vph1-depleted regions on vacuoles. Dashed lines denote the enlarged region shown as two-color images to the right of the merged, three-color image. (C) The mitochondrial localization of Ltc1 is essential in Δmdm34 mutants. Diploid yeast heterozygous for Δmdm34 and Δltc1 and harboring a yeast centromeric plasmid pRS313 containing Ltc1-yEGFP or Ltc1(ΔGRAM) were sporulated and analyzed by tetrad dissection as described in “Fluorescence microscopy.” The expected frequency of viable Δmdm34 Δltc1 mutants for pRS313 plasmids containing a WT Ltc1 is 1/8 spore colonies. The observed frequency for WT Ltc1 was 12/104 (∼1/8), whereas the observed frequency for Ltc1(ΔGRAM) and an empty vector control were 1/108 and 2/92, respectively. (D) Ltc1 is required for vacuole membrane domain formation in response to cycloheximide and glucose starvation. WT and Δltc1 cells expressing Vph1-yEGFP were grown exponentially in synthetic complete media for at least 12 generations and then subjected to the indicated treatments and imaged as described in “Fluorescence microscopy.” Quantification represents triplicate biologically independent experiments. (E) Shifting the proportional localization of Ltc1 to ER–vacuole contacts is sufficient to induce vacuole membrane domain formation under normal growing conditions. WT, Δtom70 Δtom71, and Δltc1 Δtom70 Δtom71 cells were grown for at least 12 generations in nutrient-replete media, and domain formation was assessed using Vph1-yEGFP as described in “Fluorescence microscopy.” Quantification represents triplicate biologically independent experiments. The arrowhead indicates a vacuole with Vph1-GFP–labeled domains. Bars, 2 µm.
Figure 4.

The distribution of Ltc1 to ER–mitochondria and ER–vacuolar contacts regulates Ltc1-dependent separable mitochondrial and vacuolar functions. (A) The localization of Ltc1 to ER–mitochondria, but not to ER–vacuole, contacts is essential in Δmdm34 cells. Diploid yeast heterozygous for the indicated deletions were sporulated and analyzed by tetrad dissection as described in “Fluorescence microscopy.” The green circle indicates viable Δvac8 Δmdm34 cells (the colonies immediately above and below are Δvac8 and WT, respectively). Red circles indicate nonviable Δtom70 Δtom71 Δmdm34 cells. (B) The Ltc1 GRAM domain is required for the mitochondrial localization of Ltc1. Cells expressing Ltc1(ΔGRAM)-yEGFP, Vph1-yEmCherry (vacuoles), and mtBFP (mitochondria) were grown to mid-log phase and imaged as described in “Fluorescence microscopy.” Open arrowheads indicate the localization of Ltc1 to Vph1-depleted regions on vacuoles. Dashed lines denote the enlarged region shown as two-color images to the right of the merged, three-color image. (C) The mitochondrial localization of Ltc1 is essential in Δmdm34 mutants. Diploid yeast heterozygous for Δmdm34 and Δltc1 and harboring a yeast centromeric plasmid pRS313 containing Ltc1-yEGFP or Ltc1(ΔGRAM) were sporulated and analyzed by tetrad dissection as described in “Fluorescence microscopy.” The expected frequency of viable Δmdm34 Δltc1 mutants for pRS313 plasmids containing a WT Ltc1 is 1/8 spore colonies. The observed frequency for WT Ltc1 was 12/104 (∼1/8), whereas the observed frequency for Ltc1(ΔGRAM) and an empty vector control were 1/108 and 2/92, respectively. (D) Ltc1 is required for vacuole membrane domain formation in response to cycloheximide and glucose starvation. WT and Δltc1 cells expressing Vph1-yEGFP were grown exponentially in synthetic complete media for at least 12 generations and then subjected to the indicated treatments and imaged as described in “Fluorescence microscopy.” Quantification represents triplicate biologically independent experiments. (E) Shifting the proportional localization of Ltc1 to ER–vacuole contacts is sufficient to induce vacuole membrane domain formation under normal growing conditions. WT, Δtom70 Δtom71, and Δltc1 Δtom70 Δtom71 cells were grown for at least 12 generations in nutrient-replete media, and domain formation was assessed using Vph1-yEGFP as described in “Fluorescence microscopy.” Quantification represents triplicate biologically independent experiments. The arrowhead indicates a vacuole with Vph1-GFP–labeled domains. Bars, 2 µm.

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