Figure S5.
Frt1 mislocalized to the mitochondrial OM is transferred to the ER in a manner dependent on Msp1 and Get3. (A) Localization of 3xFLAG-mNG-Frt1 was imaged by fluorescence microscopy in the presence (−IAA, upper panel) or absence (+IAA, lower panel) of Get3-AID-9xMyc. Single-plane images are shown. The ER were labeled with Snd3-mCherry. Arrowheads indicate mNG-Frt1 mislocalized to mitochondria. Scale bar, 5 μm. DIC, differential interference contrast microscopy. (B) Colocalization of mNG-Frt1 with the ER was analyzed by using Pearson’s correlation coefficient between mNG and mCherry signals. Values are mean ± SD (n = 50) from three technical replicates; n represents the number of cells. ****, P < 0.0001 compared with IAA untreated (−IAA) cells by two-tailed paired t test. (C) Yeast cells expressing Get3-AID*-9xMYC as in Fig. 6 A were grown in SCD medium. Cell extracts were prepared, and proteins were analyzed by SDS-PAGE and immunoblotting with the indicated antibodies (left). Lane 1: untreated cells, lane 2: IAA treatment for 30 min at 30°C, lane 3: cyanamide induction for 3 h at 30°C in the presence of IAA, lane 4: 1 h after washing off of IAA and cyanamide, and lane 5: 2 h after washing off of IAA and cyanamide. Protein amounts of Get3-AID*-9xMYC were normalized by that of Tom40 (right). Values are mean ± SD from four independent experiments. (D) Time-lapse microscopy images were taken at the indicated times (min) for the cells in Fig. 6 A without IAA and β-estradiol treatment. Cell outlines (blue) are shown by a DIC image. Scale bar, 5 μm. (E) Promoter shutoff chase of 3xFLAG-mNG-Frt1. Yeast cells in Fig. 6 A were grown in SCD at 30°C. Cell extracts were prepared at the indicated times in the presence or absence of 1 μM β-estradiol, and proteins were analyzed by SDS-PAGE and immunoblotting with the indicated antibodies (left). Relative amounts of 3xFLAG-mNG-Frt1 (central) and Tom40 (right) were plotted against the protein levels at t = 0, respectively. Values are mean ± SD from three independent experiments. Source data are available for this figure: SourceData FS5.

Frt1 mislocalized to the mitochondrial OM is transferred to the ER in a manner dependent on Msp1 and Get3. (A) Localization of 3xFLAG-mNG-Frt1 was imaged by fluorescence microscopy in the presence (−IAA, upper panel) or absence (+IAA, lower panel) of Get3-AID-9xMyc. Single-plane images are shown. The ER were labeled with Snd3-mCherry. Arrowheads indicate mNG-Frt1 mislocalized to mitochondria. Scale bar, 5 μm. DIC, differential interference contrast microscopy. (B) Colocalization of mNG-Frt1 with the ER was analyzed by using Pearson’s correlation coefficient between mNG and mCherry signals. Values are mean ± SD (n = 50) from three technical replicates; n represents the number of cells. ****, P < 0.0001 compared with IAA untreated (−IAA) cells by two-tailed paired t test. (C) Yeast cells expressing Get3-AID*-9xMYC as in Fig. 6 A were grown in SCD medium. Cell extracts were prepared, and proteins were analyzed by SDS-PAGE and immunoblotting with the indicated antibodies (left). Lane 1: untreated cells, lane 2: IAA treatment for 30 min at 30°C, lane 3: cyanamide induction for 3 h at 30°C in the presence of IAA, lane 4: 1 h after washing off of IAA and cyanamide, and lane 5: 2 h after washing off of IAA and cyanamide. Protein amounts of Get3-AID*-9xMYC were normalized by that of Tom40 (right). Values are mean ± SD from four independent experiments. (D) Time-lapse microscopy images were taken at the indicated times (min) for the cells in Fig. 6 A without IAA and β-estradiol treatment. Cell outlines (blue) are shown by a DIC image. Scale bar, 5 μm. (E) Promoter shutoff chase of 3xFLAG-mNG-Frt1. Yeast cells in Fig. 6 A were grown in SCD at 30°C. Cell extracts were prepared at the indicated times in the presence or absence of 1 μM β-estradiol, and proteins were analyzed by SDS-PAGE and immunoblotting with the indicated antibodies (left). Relative amounts of 3xFLAG-mNG-Frt1 (central) and Tom40 (right) were plotted against the protein levels at t = 0, respectively. Values are mean ± SD from three independent experiments. Source data are available for this figure: SourceData FS5.

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