Overexpression of Msp1 leads to accumulation of Pex15Δ30 in the cytosol and ER in the GET mutants. (A–C) doa10Δ (A), doa10Δget3Δ (B), and doa10Δget1Δget2Δ (C) cells expressing 3xFLAG-mNG-Pex15Δ30 from the GAL1 promoter with overexpression of Msp1 (Msp1O/E) from the ADH1 promoter were grown in SCD at 30°C and in SCGal for 4 h at 30°C and were imaged by fluorescence microscopy. Single-plane images are shown. The ER and mitochondria were labeled with ER-mCherry (lower panels in A, B, and C) and Su9-RFP (upper panels in A, B, and C), respectively. Arrowheads indicate mNG-Pex15Δ30 localized in mitochondria (upper panels in B and C) and the ER (lower panels in B and C). Scale bars, 5 μm. DIC, differential interference contrast microscopy. (D and E) Colocalization of 3xFLAG-mNG-Pex15Δ30 with mitochondria (D) or the ER (E) was measured by 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 doa10ΔMsp1O/E cells and doa10Δget3ΔMsp1O/E or doa10Δget1Δget2ΔMsp1O/E cells by one-way ANOVA with Dunnett’s multiple comparison test. (F)doa10Δget1Δget2Δ cells expressing Get3-mCherry (upper panel) or Hsp104-mCherry (lower panel) from their own promoters and 3xFLAG-mNG-Pex15Δ30 from the GAL1 promoter with overexpressed Msp1 (Msp1 O/E) from the ADH1 promoter were grown in SCD at 30°C and in SCGal for 4 h at 30°C and were imaged by fluorescence microscopy. Single-plane images are shown. Arrowheads indicate 3xFLAG-mNG-Pex15Δ30 foci that were merged with Get3-mCherry or Hsp104-mCherry. Scale bar, 5 μm. The percentages and numbers of cells showing 3xFLAG-mNG-Pex15Δ30 foci colocalized with Get3 (n = 75) and Hsp104 (n = 119) are indicated on the right. DIC, differential interference contrast microscopy.