Figure 3.
Doa10 is active in the GET mutants. (A–C) WT (A), get3Δ (B), and get1Δget2Δ (C) cells, expressing mNG-Ubc6 from the own promoter of Ubc6, were grown in SCD and imaged by fluorescence microscopy. Mitochondria and the ER were labeled with Su9-RFP and ER-mCherry, respectively. Single-plane images are shown. Scale bar, 5 μm. DIC, differential interference contrast microscopy. (D and E) Colocalization of mNG-Ubc6 with the ER (D) and mitochondria (E) was analyzed using Pearson’s correlation coefficient between mNG and RFP/mCherry signals. Values are mean ± SD (n = 50) from three technical replicates; n represents the number of cells. ****, P < 0.0001 compared with WT cells and get3Δ or get1Δget2Δ cells by one-way ANOVA with Dunnett’s multiple comparison test. (F) WT, get3Δ, and get1Δget2Δ cells expressing Ubc6 or mNG-Ubc6 from their own promoters were grown in SCD. Cell extracts were prepared, and proteins were analyzed by SDS-PAGE and immunoblotting with the indicated antibodies (upper panel). Protein amounts of endogenous Ubc6 and mNG-Ubc6 were normalized to that of Tom40 (lower panel). Values are mean ± SD from three independent experiments. One-way ANOVA with Dunnett’s multiple comparison test was used for comparison with WT-Ubc6 cells. (G) WT, get3Δ, and doa10Δ cells expressing Ste6*-3xHA from the GAL1 promoter were grown in SCD at 30°C and then in SCGal for 5 h at 30°C. Cell extracts were prepared at the indicated times at 30°C after addition of 100 µg/ml CHX, and proteins were analyzed by SDS-PAGE and immunoblotting with the indicated antibodies (left). Normalized relative amounts of Ste6*-3xHA were plotted against chase time as in Fig. 1 B. Values are mean ± SD from three independent experiments. Source data are available for this figure: SourceData F3.

Doa10 is active in the GET mutants. (A–C) WT (A), get3Δ (B), and get1Δget2Δ (C) cells, expressing mNG-Ubc6 from the own promoter of Ubc6, were grown in SCD and imaged by fluorescence microscopy. Mitochondria and the ER were labeled with Su9-RFP and ER-mCherry, respectively. Single-plane images are shown. Scale bar, 5 μm. DIC, differential interference contrast microscopy. (D and E) Colocalization of mNG-Ubc6 with the ER (D) and mitochondria (E) was analyzed using Pearson’s correlation coefficient between mNG and RFP/mCherry signals. Values are mean ± SD (n = 50) from three technical replicates; n represents the number of cells. ****, P < 0.0001 compared with WT cells and get3Δ or get1Δget2Δ cells by one-way ANOVA with Dunnett’s multiple comparison test. (F) WT, get3Δ, and get1Δget2Δ cells expressing Ubc6 or mNG-Ubc6 from their own promoters were grown in SCD. Cell extracts were prepared, and proteins were analyzed by SDS-PAGE and immunoblotting with the indicated antibodies (upper panel). Protein amounts of endogenous Ubc6 and mNG-Ubc6 were normalized to that of Tom40 (lower panel). Values are mean ± SD from three independent experiments. One-way ANOVA with Dunnett’s multiple comparison test was used for comparison with WT-Ubc6 cells. (G) WT, get3Δ, and doa10Δ cells expressing Ste6*-3xHA from the GAL1 promoter were grown in SCD at 30°C and then in SCGal for 5 h at 30°C. Cell extracts were prepared at the indicated times at 30°C after addition of 100 µg/ml CHX, and proteins were analyzed by SDS-PAGE and immunoblotting with the indicated antibodies (left). Normalized relative amounts of Ste6*-3xHA were plotted against chase time as in Fig. 1 B. Values are mean ± SD from three independent experiments. Source data are available for this figure: SourceData F3.

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