Figure S1.
Msp1-mediated transfer of Pex15Δ30 from mitochondria to the ER. (A) Yeast cells in Fig. 1 A were grown in SCD at 30°C, treated with 5 mM cyanamide for 4 h, and imaged by fluorescence microscopy. The ER and mitochondria were labeled with BipN-mRuby3-HDEL (ER-mRuby3) and Su9-mTagBFP2 (Su9-BFP2), respectively (upper panel). Peroxisomes and mitochondria were labeled with mCherry-PTS1 and Su9-BFP2, respectively (lower panel). Cell shapes are outlined with broken lines, and arrowheads indicate mNG-Pex15Δ30 in peroxisomes. Maximum-projection images reconstituted from the z-stacks are shown (13 plane stacks, 0.4-μm spacing). Scale bar, 5 μm. (B) Time-lapse microscopy images were taken at the indicated times (min) for cells in Fig. 1 A without treatment with β-estradiol. Cell outlines (blue) are shown by a DIC image. Scale bar, 5 μm. (C) Promoter shutoff chase of 3xFLAG-mNG-Pex15Δ30. Yeast cells in Fig. 1 A were grown in SCD at 30°C. Cell extracts were prepared at the indicated times in the presence (+β-estradiol) or absence (−β-estradiol) of Msp1 induction, and proteins were analyzed by SDS-PAGE and immunoblotting with the indicated antibodies. Relative amounts of 3xFLAG-mNG-Pex15Δ30 (left) and Tom40 (right) were plotted against chase time. Values are mean ± SD from three independent experiments. (D) Yeast cells in Fig. 1 A were grown in SCD at 30°C. Total RNA was isolated from yeast cells before (cyanamide) and after (wash 2 h) washing off cyanamide, and mRNA levels of 3xFLAG-mNG-Pex15Δ30 (and control ACT1) were measured by quantitative real-time (RT)-PCR. mRNA levels were normalized to ACT1 mRNA levels, and values are mean ± SD from three independent experiments. The mean value before washing was set to 1.0. **, P = 0.01 compared with cyanamide-induced cells by two-tailed paired t test. (E) Quantification of 3xFLAG-mNG-Pex15Δ30 intensities in Fig. 1 C. A single cell was selected as a region of interest (ROI), and each mNG signal was quantified at chase time = 0 and 30. Values are mean ± SD (n = 9); n represents the number of cells. The mean value at chase time = 0 was set to 1.0. Source data are available for this figure: SourceData FS1.

Msp1-mediated transfer of Pex15Δ30 from mitochondria to the ER. (A) Yeast cells in Fig. 1 A were grown in SCD at 30°C, treated with 5 mM cyanamide for 4 h, and imaged by fluorescence microscopy. The ER and mitochondria were labeled with BipN-mRuby3-HDEL (ER-mRuby3) and Su9-mTagBFP2 (Su9-BFP2), respectively (upper panel). Peroxisomes and mitochondria were labeled with mCherry-PTS1 and Su9-BFP2, respectively (lower panel). Cell shapes are outlined with broken lines, and arrowheads indicate mNG-Pex15Δ30 in peroxisomes. Maximum-projection images reconstituted from the z-stacks are shown (13 plane stacks, 0.4-μm spacing). Scale bar, 5 μm. (B) Time-lapse microscopy images were taken at the indicated times (min) for cells in Fig. 1 A without treatment with β-estradiol. Cell outlines (blue) are shown by a DIC image. Scale bar, 5 μm. (C) Promoter shutoff chase of 3xFLAG-mNG-Pex15Δ30. Yeast cells in Fig. 1 A were grown in SCD at 30°C. Cell extracts were prepared at the indicated times in the presence (+β-estradiol) or absence (−β-estradiol) of Msp1 induction, and proteins were analyzed by SDS-PAGE and immunoblotting with the indicated antibodies. Relative amounts of 3xFLAG-mNG-Pex15Δ30 (left) and Tom40 (right) were plotted against chase time. Values are mean ± SD from three independent experiments. (D) Yeast cells in Fig. 1 A were grown in SCD at 30°C. Total RNA was isolated from yeast cells before (cyanamide) and after (wash 2 h) washing off cyanamide, and mRNA levels of 3xFLAG-mNG-Pex15Δ30 (and control ACT1) were measured by quantitative real-time (RT)-PCR. mRNA levels were normalized to ACT1 mRNA levels, and values are mean ± SD from three independent experiments. The mean value before washing was set to 1.0. **, P = 0.01 compared with cyanamide-induced cells by two-tailed paired t test. (E) Quantification of 3xFLAG-mNG-Pex15Δ30 intensities in Fig. 1 C. A single cell was selected as a region of interest (ROI), and each mNG signal was quantified at chase time = 0 and 30. Values are mean ± SD (n = 9); n represents the number of cells. The mean value at chase time = 0 was set to 1.0. Source data are available for this figure: SourceData FS1.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal