Figure 6.
Authentic ER-TA proteins mistargeted to mitochondria return to the ER via the Msp1-GET pathway. (A) Schematic representation of time-lapse imaging. 3xFLAG-mNG-Frt1 was expressed under the control of the cyanamide-inducible DDI2 promoter and Msp1 from the β-estradiol–inducible Z4EV promoter. Get3-AID*-9xMyc, instead of endogenous Get3, was expressed from the chromosome under the control of its own promoter. Yeast cells were grown in SCD medium at 30°C, and Get3-AID*-9xMYC was depleted by 1 mM IAA for 30 min at 30°C. Then, 3xFLAG-mNG-ER-TA proteins were induced by 5 mM cyanamide for 3 h at 30°C. The cells were washed with fresh SCD medium to shut off the 3xFLAG-mNG-ER-TA protein expression and re-express Get3-AID*-9xMYC for 2 h at 30°C. Time-lapse imaging was recorded after addition of 1 μM β-estradiol to start expression of Msp1. (B) 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. Mitochondria were labeled with Tom70-mCherry. Arrowheads indicate the signal from mNG-Frt1 mislocalized to mitochondria. Scale bar, 5 μm. DIC, differential interference contrast microscopy. (C) Colocalization of mNG-Frt1 with mitochondria was analyzed 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. (D) Yeast cells in A were grown in SCD at 30°C and treated with 1 μM β-estradiol after expression shutoff of 3xFLAG-mNG-Frt1. Time-lapse images were then taken at 2-min intervals. Single-plane images are shown. The ER was labeled with Snd3-mCherry. Cell outlines (blue) are shown by a DIC image. Scale bar, 5 μm. (E) Colocalization of 3xFLAG-mNG-Frt1 with the ER was analyzed using Pearson’s correlation coefficient between mNG and mCherry signals. A single yeast cell was selected as a region of interest (ROI), and Pearson correlation coefficients were analyzed at each time point. Values are mean ± SD (−β-estradiol; n = 30, +β-estradiol; n = 38, +IAA, β-estradiol; n = 30) from three technical replicates; n represents the number of cells. ****, P < 0.0001, *, P = 0.0475 compared with the result of time-lapse imaging without β-estradiol treatment by one-way ANOVA with Dunnett’s multiple comparison test. (F) Yeast cells in B were grown in SCD at 30°C and treated with 1 mM IAA and 1 μM β-estradiol after expression shutoff of 3xFLAG-mNG-Frt1. Time-lapse images were then taken at 2-min intervals. Single-plane images are shown. Mitochondria was labeled with Tom70-mCherry. Scale bar, 5 μm. (G) Colocalization of 3xFLAG-mNG-Frt1 with mitochondria was analyzed using Pearson’s correlation coefficient between mNG and mCherry signals. A single yeast cell was selected as a region of interest (ROI), and Pearson correlation coefficients were analyzed at each time point. Values are mean ± SD (−β-estradiol; n = 30, +β-estradiol; n = 30, +IAA, β-estradiol; n = 30) from three independent experiments; n represents the number of cells. ****, P < 0.0001 compared with the result of time-lapse imaging without β-estradiol treatment by one-way ANOVA with Dunnett’s multiple comparison test.

Authentic ER-TA proteins mistargeted to mitochondria return to the ER via the Msp1-GET pathway. (A) Schematic representation of time-lapse imaging. 3xFLAG-mNG-Frt1 was expressed under the control of the cyanamide-inducible DDI2 promoter and Msp1 from the β-estradiol–inducible Z4EV promoter. Get3-AID*-9xMyc, instead of endogenous Get3, was expressed from the chromosome under the control of its own promoter. Yeast cells were grown in SCD medium at 30°C, and Get3-AID*-9xMYC was depleted by 1 mM IAA for 30 min at 30°C. Then, 3xFLAG-mNG-ER-TA proteins were induced by 5 mM cyanamide for 3 h at 30°C. The cells were washed with fresh SCD medium to shut off the 3xFLAG-mNG-ER-TA protein expression and re-express Get3-AID*-9xMYC for 2 h at 30°C. Time-lapse imaging was recorded after addition of 1 μM β-estradiol to start expression of Msp1. (B) 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. Mitochondria were labeled with Tom70-mCherry. Arrowheads indicate the signal from mNG-Frt1 mislocalized to mitochondria. Scale bar, 5 μm. DIC, differential interference contrast microscopy. (C) Colocalization of mNG-Frt1 with mitochondria was analyzed 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. (D) Yeast cells in A were grown in SCD at 30°C and treated with 1 μM β-estradiol after expression shutoff of 3xFLAG-mNG-Frt1. Time-lapse images were then taken at 2-min intervals. Single-plane images are shown. The ER was labeled with Snd3-mCherry. Cell outlines (blue) are shown by a DIC image. Scale bar, 5 μm. (E) Colocalization of 3xFLAG-mNG-Frt1 with the ER was analyzed using Pearson’s correlation coefficient between mNG and mCherry signals. A single yeast cell was selected as a region of interest (ROI), and Pearson correlation coefficients were analyzed at each time point. Values are mean ± SD (−β-estradiol; n = 30, +β-estradiol; n = 38, +IAA, β-estradiol; n = 30) from three technical replicates; n represents the number of cells. ****, P < 0.0001, *, P = 0.0475 compared with the result of time-lapse imaging without β-estradiol treatment by one-way ANOVA with Dunnett’s multiple comparison test. (F) Yeast cells in B were grown in SCD at 30°C and treated with 1 mM IAA and 1 μM β-estradiol after expression shutoff of 3xFLAG-mNG-Frt1. Time-lapse images were then taken at 2-min intervals. Single-plane images are shown. Mitochondria was labeled with Tom70-mCherry. Scale bar, 5 μm. (G) Colocalization of 3xFLAG-mNG-Frt1 with mitochondria was analyzed using Pearson’s correlation coefficient between mNG and mCherry signals. A single yeast cell was selected as a region of interest (ROI), and Pearson correlation coefficients were analyzed at each time point. Values are mean ± SD (−β-estradiol; n = 30, +β-estradiol; n = 30, +IAA, β-estradiol; n = 30) from three independent experiments; n represents the number of cells. ****, P < 0.0001 compared with the result of time-lapse imaging without β-estradiol treatment by one-way ANOVA with Dunnett’s multiple comparison test.

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