Figure 1.
Msp1-dependent transfer of Pex15Δ30 from mitochondria to the ER. (A) Schematic representation of time-lapse imaging. 3xFLAG-mNG-Pex15Δ30 was expressed under the control of the cyanamide-inducible DDI2 promoter, and Msp1 was from the β-estradiol-inducible Z4EV promoter. DOA10 was deleted to block the ubiquitination and degradation of Pex15Δ30. Yeast cells were grown in SCD medium at 30°C, and 3xFLAG-mNG-Pex15Δ30 was induced by 5 mM cyanamide for 4 h at 30°C. The cells were washed with fresh SCD medium to shut off 3xFLAG-mNG-Pex15Δ30 expression. Time-lapse imaging was recorded after addition of 1 μM β-estradiol to start expression of Msp1. (B) Promoter shutoff chase of 3xFLAG-mNG-Pex15Δ30. doa10Δ and its isogenic WT (DOA10) cells in A were grown in SCD medium at 30°C. Cell extracts were prepared at the indicated times after addition of 1 μM β-estradiol, and proteins were analyzed by SDS-PAGE and immunoblotting with the indicated antibodies (left). Normalized relative amounts of 3xFLAG-mNG-Pex15Δ30 were plotted against chase time (right), and the amount of 3xFLAG-mNG-Pex15Δ30 in doa10Δ cells, right after the addition of β-estradiol (chase time = 0), relative to that in DOA10 cells, is shown (center). Values are mean ± SD from three independent experiments. (C) Yeast cells in A were grown in SCD medium at 30°C and treated with 1 μM β-estradiol after expression shutoff of 3xFLAG-mNG-Pex15Δ30. Time-lapse images were then taken at 1-min intervals. Single-plane images are shown. The ER was labeled with BipN-mCherry-HDEL (ER-mCherry). Cell outlines (blue) are shown by a DIC image. Scale bar, 5 μm. (D) Colocalization of 3xFLAG-mNG-Pex15Δ30 with the ER in the presence (+β-estradiol, Fig. 1 C) and absence (−β-estradiol, Fig. S1 B) of Msp1 was analyzed using Pearson’s correlation coefficient between mNG and mCherry signals. A single cell was selected as a region of interest (ROI), and Pearson’s correlation coefficients were analyzed at each time point. Values are mean ± SD (−β-estradiol, n = 45; +β-estradiol, n = 60) from three independent experiments; n represents the number of cells. ****, P < 0.0001 compared with DOA10 cells by two-tailed paired t test. Source data are available for this figure: SourceData F1.

Msp1-dependent transfer of Pex15Δ30 from mitochondria to the ER. (A) Schematic representation of time-lapse imaging. 3xFLAG-mNG-Pex15Δ30 was expressed under the control of the cyanamide-inducible DDI2 promoter, and Msp1 was from the β-estradiol-inducible Z4EV promoter. DOA10 was deleted to block the ubiquitination and degradation of Pex15Δ30. Yeast cells were grown in SCD medium at 30°C, and 3xFLAG-mNG-Pex15Δ30 was induced by 5 mM cyanamide for 4 h at 30°C. The cells were washed with fresh SCD medium to shut off 3xFLAG-mNG-Pex15Δ30 expression. Time-lapse imaging was recorded after addition of 1 μM β-estradiol to start expression of Msp1. (B) Promoter shutoff chase of 3xFLAG-mNG-Pex15Δ30. doa10Δ and its isogenic WT (DOA10) cells in A were grown in SCD medium at 30°C. Cell extracts were prepared at the indicated times after addition of 1 μM β-estradiol, and proteins were analyzed by SDS-PAGE and immunoblotting with the indicated antibodies (left). Normalized relative amounts of 3xFLAG-mNG-Pex15Δ30 were plotted against chase time (right), and the amount of 3xFLAG-mNG-Pex15Δ30 in doa10Δ cells, right after the addition of β-estradiol (chase time = 0), relative to that in DOA10 cells, is shown (center). Values are mean ± SD from three independent experiments. (C) Yeast cells in A were grown in SCD medium at 30°C and treated with 1 μM β-estradiol after expression shutoff of 3xFLAG-mNG-Pex15Δ30. Time-lapse images were then taken at 1-min intervals. Single-plane images are shown. The ER was labeled with BipN-mCherry-HDEL (ER-mCherry). Cell outlines (blue) are shown by a DIC image. Scale bar, 5 μm. (D) Colocalization of 3xFLAG-mNG-Pex15Δ30 with the ER in the presence (+β-estradiol, Fig. 1 C) and absence (−β-estradiol, Fig. S1 B) of Msp1 was analyzed using Pearson’s correlation coefficient between mNG and mCherry signals. A single cell was selected as a region of interest (ROI), and Pearson’s correlation coefficients were analyzed at each time point. Values are mean ± SD (−β-estradiol, n = 45; +β-estradiol, n = 60) from three independent experiments; n represents the number of cells. ****, P < 0.0001 compared with DOA10 cells by two-tailed paired t test. Source data are available for this figure: SourceData F1.

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