Msp1 and Pex19-Pex3 cooperate to achieve correct localization of Pex15 to peroxisomes

Abstract

Yeast Msp1 is a dual-localized AAA-ATPase on the mitochondrial outer membrane (OM) and peroxisomal membrane. We previously showed that Msp1 transfers mistargeted tail-anchored (TA) proteins from mitochondria to the endoplasmic reticulum (ER) for degradation or delivery to their original destinations. However, the mechanism by which Msp1 in mitochondria and peroxisomes handles authentic peroxisomal TA proteins remains unclear. We show that newly synthesized Pex15 is targeted to peroxisomes primarily via the Pex19- and Pex3-dependent pathway. Mistargeted Pex15 on the mitochondrial OM is extracted by mitochondrial Msp1 and transferred to the ER via the guided-entry of TA proteins pathway for degradation or to peroxisomes via the Pex19-Pex3 pathway. Intriguingly, endogenous Pex15 localized in peroxisomes is also extracted from the membranes by peroxisomal Msp1 but returns to peroxisomes via the Pex19-Pex3 pathway. These results suggest that correct Pex15 localization to peroxisomes relies on not only the initial targeting by Pex19-Pex3 but also the constant re-routing by Msp1 and Pex19-Pex3.

Keywords: Msp1; Pex15; Pex19‐Pex3; mitochondria; peroxisome.

Fig. 1

Deletions of pex19 and pex3 cause Pex15 mistargeting to mitochondria and its destabilization. (A) WT, msp1Δ, get3Δ, and get1Δget2Δ cells expressing mNG‐Pex15 under the control of its own promoter from the chromosome were grown in SCD medium at 30 °C and imaged by fluorescence microscopy. Peroxisomes were labeled with mCherry‐PTS1. Maximum projection images of Z‐stacks are shown. Representative images from two independent experiments are shown for each yeast strain. Scale bars, 5 μm; 2 μm for zoomed‐in image. DIC, differential interference contrast microscopy. (B) WT, msp1Δ, get3Δ, and get1Δget2Δ cells expressing 3xFLAG‐Pex15 under the control of its own promoter from the chromosome were grown in YPD medium at 30 °C. At the indicated times after the addition of 100 μg·mL⁻¹ CHX, cell extracts were prepared and proteins were analyzed by SDS/PAGE and immunoblotting with the indicated antibodies (left). The normalized relative amounts of 3xFLAG‐Pex15 were plotted against the chase time. Values are the mean ± SD from three independent experiments. (C) pex19Δ and pex3Δ cells expressing mNG‐Pex15 under the control of its own promoter from the chromosome, were grown in SCD medium at 30 °C and imaged by fluorescence microscopy. Peroxisomes and mitochondria were labeled with mCherry‐PTS1 and Su9‐RFP, respectively. Single‐plane for peroxisomes and maximum projection for mitochondria images of Z‐stacks are shown. Representative images from two independent experiments are shown for each yeast strain. Scale bars, 5 μm; 2 μm for zoomed‐in image. DIC, differential interference contrast microscopy. (D) WT, pex19Δ, pex3Δ and pex19Δpex3Δ cells expressing 3xFLAG‐Pex15 under the control of its own promoter from the chromosome, were grown in YPD medium at 30 °C. At the indicated times after the addition of 100 μg/mL CHX, cell extracts were prepared and proteins were analyzed by SDS/PAGE and immunoblotting with the indicated antibodies (left). The normalized relative amounts of 3xFLAG‐Pex15 were plotted against the chase time. Values are the mean ± SD from three independent experiments.

Fig. 2

Pex19 has a chaperone function that protects degradation of mislocalized Pex15. (A) Localization of mNG‐tagged Pex19 in WT and pex3Δ cells. WT or pex3Δ cells expressing mNG‐Pex19 under the control of its own promoter from the chromosome were grown in SCD medium at 30 °C and imaged by fluorescence microscopy. Peroxisomes were labeled with mCherry‐PTS1. Maximum projection images of Z‐stacks are shown. Representative images from two independent experiments are shown for each yeast strain. Scale bar, 5 μm. DIC, differential interference contrast microscopy. (B) pex19Δ cells expressing mNG‐Pex15 under the control of its own promoter from the chromosome, were grown without (pRS303: upper panel) or with wild‐type Pex19 (PEX19: middle panel) or its non‐farnesylated mutant (pex19(C339S): lower panel), from the chromosome under the control of the own promoter in SCD medium at 30 °C and imaged by fluorescence microscopy. Peroxisomes were labeled with mCherry‐PTS1. Maximum projection images of Z‐stacks are shown. Scale bar, 5 μm. DIC, differential interference contrast microscopy. In pex19Δ cells expressing the Pex19(C339S) mutant (lower panel), white arrowheads indicate immature peroxisomes that mNG‐Pex15 foci do not co‐localize with mCherry‐PTS1, while yellow arrowheads indicate mature peroxisomes that mNG‐Pex15 foci co‐localize with mCherry‐PTS1. (C) Localization of mNG‐Pex15 in pex19Δ cells expressing the Pex19(C339S) mutant in Fig. 2B was analyzed from more than 80 cells at each experiment. Values are the mean ± SD from three independent replicates. (D) pex19Δpex3Δ cells expressing 3xFLAG‐Pex15 under the control of its own promoters from the chromosomes were grown without (pRS306) or with wild‐type Pex19 (PEX19) or its non‐farnesylated mutant (pex19(C339S)), from the chromosome under the control of the own promoter in YPD medium at 30 °C. CHX‐chase experiments were performed as in Fig. 1B. The normalized relative amounts of 3xFLAG‐Pex15 were plotted against the chase time. Values are the mean ± SD from three independent experiments.

Fig. 3

Proteasome is involved in the degradation of mislocalized Pex15. pex19Δpdr5Δ cells expressing 3xFLAG‐Pex15 under the control of its own promoters from the chromosomes were grown in YPD medium at 30 °C, and then further incubated for 30 min after the addition of 50 μm MG132. Extracts were prepared at the indicated times from cells incubated at 30 °C after the addition of 100 μg·mL⁻¹ CHX, and proteins were analyzed by SDS/PAGE and immunoblotting with the indicated antibodies (left). The normalized relative amounts of 3xFLAG‐Pex15 were plotted against the chase time. Values are the mean ± SD from three independent experiments.

Fig. 4

Mistargeted Pex15 is transferred to the ER via Msp1 and the GET pathway and then degraded by the ERAD system. (A) pex19Δ, pex19Δmsp1Δ and pex19Δget3Δ cells expressing 3xFLAG‐Pex15 under the control of its own promoter from the chromosome, were grown in YPD medium at 30 °C. CHX‐chase experiments were performed as in Fig. 1B. The normalized relative amounts of 3xFLAG‐Pex15 were plotted against the chase time. Values are the mean ± SD from three independent experiments. (B) pex19Δmsp1Δ and pex19Δget3Δ cells expressing mNG‐Pex15 under the control of its own promoter from the chromosome, were grown in SCD medium at 30 °C and imaged by fluorescence microscopy as in Fig. 1A. Representative images from two independent experiments are shown for each yeast strain. Scale bars, 5 μm; 2 μm for zoomed‐in image. (C) pex19Δget3Δ cells expressing mNG‐Pex15 and Hsp104‐mCherry under the control of their own promoters from the chromosomes were grown in SCD medium at 30 °C and imaged by fluorescence microscopy as in Fig. 1A. Representative images from two independent experiments are shown for each yeast strain. Scale bars, 5 μm; 2 μm for zoomed‐in image. (D) pex19Δ, pex19Δdoa10Δ, pex19Δhrd1Δ, and pex19Δdoa10Δhrd1Δ cells expressing 3xFLAG‐Pex15 under the control of its own promoter from the chromosome, were grown in YPD medium at 30 °C. CHX‐chase experiments were performed as in Fig. 1B. The normalized relative amounts of 3xFLAG‐Pex15 were plotted against the chase time. Values are the mean ± SD from three independent experiments. (E) pex19Δ, pex19Δubc6Δ and pex19Δubc7Δ cells expressing 3xFLAG‐Pex15 under the control of its own promoter from the chromosome, were grown in YPD medium at 30 °C. CHX‐chase experiments were performed as in Fig. 1B. The normalized relative amounts of 3xFLAG‐Pex15 were plotted against the chase time. Values are the mean ± SD from three independent experiments. (F) pex19Δ, pex19Δubc7Δ, pex19Δubc7Δmsp1Δ and pex19Δubc7Δget3Δ cells expressing mNG‐Pex15 under the control of its own chromosomal promoter from the chromosome, were grown in SCD medium at 30 °C and imaged by fluorescence microscopy. The ER was labeled with BipN‐mCherry‐HDEL (ER‐mCherry). Single‐plane images are shown. Scale bars, 5 μm; 2 μm for zoomed‐in image. DIC, differential interference contrast microscopy. (G) Co‐localization of mNG‐Pex15 with the ER was quantified by using Pearson's correlation coefficient between the mNG and ER (mCherry) signals. Values are the mean ± SD (n = 60) from three independent replicates; n represents the number of cells. ****, P < 0.0001 compared with pex19Δubc7Δ cells by one‐way ANOVA with Dunnett's multiple comparison test.

Fig. 5

Newly synthesized Pex15 is targeted to peroxisomes in a Pex19‐ and Pex3‐dependent manner. (A) A schematic representation of the acute depletion of Pex19 using the AID system. N‐terminally AID*‐tagged Pex19 was expressed under the control of its own promoter from the chromosome. 3xFLAG‐mNG‐Pex15 was expressed from the cyanamide‐inducible DDI2 promoter and OsTIR1 was expressed from the ADH1 promoter. (B) AID*‐Pex19 was depleted by a treatment with 1 mm IAA for 30 min at 30 °C, followed by the induction of 3xFLAG‐mNG‐Pex15 with 5 mm cyanamide at 30 °C for 2 h. The localization of 3xFLAG‐mNG‐Pex15 was then analyzed using fluorescence microscopy. (C) Yeast cells in (A) were grown in SCD medium at 30 °C. Cell extracts were prepared at the indicated times in the presence (−IAA) or absence (+IAA) of AID*‐Pex19. Proteins were analyzed by SDS/PAGE and immunoblotting with the indicated antibodies. The relative amounts of AID*‐Pex19 (upper panel) and Pgk1 (lower panel) were plotted against the chase time. Values are the mean ± SD from three independent experiments. (D) The localization of 3xFLAG‐mNG‐Pex15 was imaged by fluorescence microscopy in the presence (−IAA, upper panel) or absence (+IAA, lower panel) of AID*‐Pex19. Maximum projection images of Z‐stacks are shown. Peroxisomes were labeled with Pex11‐mCherry. Scale bars, 5 μm; 2 μm for zoomed‐in image. DIC, differential interference contrast microscopy. (E) Co‐localization of 3xFLAG‐mNG‐Pex15 with peroxisomes was quantified using Pearson's correlation coefficient between the mNG and mCherry signals. Values are the mean ± SD (−IAA; n = 60, +IAA; n = 60) from three independent replicates; n represents the number of cells. ****, P < 0.0001 compared with IAA untreated (−IAA) cells by two‐tailed paired t test. (F) Yeast cells expressing Pex3‐AID*‐V5 under the control of its own promoter from the chromosome were grown in SCD medium at 30 °C. Extracts were prepared at the indicated times from cells cultured in the presence (−IAA) or absence (+IAA) of Pex3‐AID*‐V5, and proteins were analyzed by SDS/PAGE and immunoblotting with the indicated antibodies. The relative amounts of Pex3‐AID*‐V5 (upper panel) and Tom40 (lower panel) were plotted against the chase time. Values are the mean ± SD from three independent experiments. (G) The localization of 3xFLAG‐mNG‐Pex15 in the presence (−IAA, upper panel) or absence (+IAA, lower panel) of Pex3‐AID*‐V5 was imaged by fluorescence microscopy as in (B). Maximum projection images of Z‐stacks are shown. Peroxisomes were labeled with Pex11‐mCherry. Scale bars, 5 μm; 2 μm for zoomed‐in image. DIC, differential interference contrast microscopy. (H) Co‐localization of 3xFLAG‐mNG‐Pex15 with peroxisomes was quantified using Pearson's correlation coefficient between the mNG and mCherry signals, as in (E). Values are the mean ± SD (−IAA; n = 60, +IAA; n = 60) from three independent replicates; n represents the number of cells. ****, P < 0.0001 compared with IAA untreated (−IAA) cells by two‐tailed paired t test.

Fig. 6

Newly synthesized Pex15 is mistargeted to mitochondria by rapid depletion of Pex19 in MSP1‐deficient cells. (A) msp1Δ cells expressing AID*‐Pex19 under the control of its own promoter from the chromosome were grown in SCD medium at 30 °C. AID*‐Pex19 was depleted by a treatment with 1 mm IAA for 30 min at 30 °C, followed by 3xFLAG‐mNG‐Pex15 induction with 5 mm cyanamide at 30 °C for 2 h. The localization of 3xFLAG‐mNG‐Pex15 was imaged by fluorescence microscopy in the presence (−IAA, upper panel) or absence (+IAA, lower panel) of AID*‐Pex19. Maximum projection images of Z‐stacks are shown. Peroxisomes were labeled with Pex11‐mCherry. Scale bars, 5 μm; 2 μm for zoomed‐in image. DIC, differential interference contrast microscopy. (B) Co‐localization of 3xFLAG‐mNG‐Pex15 with peroxisomes was analyzed using Pearson's correlation coefficient between the mNG and mCherry signals. Values are the mean ± SD (−IAA; n = 60, +IAA; n = 60) from three independent replicates; n represents the number of cells. ****, P < 0.0001 compared with IAA untreated (−IAA) cells by two‐tailed paired t test. (C) The localization of 3xFLAG‐mNG‐Pex15 was imaged by fluorescence microscopy in the presence (−IAA, upper panel) or absence (+IAA, lower panel) of AID*‐Pex19, as in (A). Maximum projection images are shown. Mitochondria were labeled with Tom70‐mCherry. Scale bars, 5 μm; 2 μm for zoomed‐in image. (D) Co‐localization of 3xFLAG‐mNG‐Pex15 with mitochondria was analyzed using Pearson's correlation coefficient between the mNG and mCherry signals, as in (B). Values are the mean ± SD (−IAA; n = 59, +IAA; n = 58) from three independent replicates; n represents the number of cells. ****, P < 0.0001 compared with IAA untreated (−IAA) cells by two‐tailed paired t test. (E) Yeast cells expressing Get3‐AID*‐9xMyc under the control of its own promoter from the chromosome were grown in SCD medium at 30 °C. Get3‐AID*‐9xMyc was depleted by treatment with 1 mm IAA for 30 min at 30 °C, followed by induction of 3xFLAG‐mNG‐Pex15 with 5 mm cyanamide at 30 °C for 2 h. Localization of 3xFLAG‐mNG‐Pex15 was imaged by fluorescence microscopy in the presence (−IAA, upper panel) or absence (+IAA, lower panel) of Get3‐AID*‐9xMyc. Maximum projection images are shown. Peroxisomes were labeled with mCherry‐PTS1. Scale bars, 5 μm; 2 μm for zoomed‐in image. (F) Co‐localization of 3xFLAG‐mNG‐Pex15 with peroxisomes was analyzed using Pearson's correlation coefficient between the mNG and mCherry signals, as in (B). Values are the mean ± SD (−IAA; n = 56, +IAA; n = 57) from three independent replicates; n represents the number of cells. **, P = 0.0061 compared with IAA untreated (−IAA) cells by two‐tailed paired t test.

Fig. 7

Msp1‐ and Pex19‐dependent transfer of mistargeted Pex15 from mitochondria to peroxisomes. (A) A schematic representation of the live‐cell imaging. As in Fig. 5A, AID*‐Pex19 and OsTIR1 were expressed under the control of its own promoter and the ADH1 promoter, respectively. 3xFLAG‐mNG‐Pex15 was expressed under the control of the cyanamide‐inducible DDI2 promoter. The endogenous MSP1 gene was deleted to block the extraction of Pex15 from peroxisomes. Instead, a mitochondria‐targeted Msp1 variant (Tom70TM‐Msp1) was expressed under the control of the β‐Estradiol‐inducible Z4EV promoter. (B) Yeast cells in (A) were grown in SCD medium at 30 °C, and AID*‐Pex19 was depleted by a treatment with 1 mm IAA for 30 min at 30 °C. 3xFLAG‐mNG‐Pex15 was then induced with 5 mm cyanamide for 2 h at 30 °C. The cells were washed with fresh SCD medium to shut off the 3xFLAG‐mNG‐Pex15 expression and allow the re‐expression of AID*‐Pex19 by growth in SCD medium for 2 h at 30 °C. Time‐lapse imaging was recorded after the addition of 1 μm β‐Estradiol to induce the expression of Tom70TM‐Msp1. (C) Yeast cells in (A) were grown in SCD at 30 °C, and treated with 1 μm β‐Estradiol after the expression of 3xFLAG‐mNG‐Pex15 was shut off. Time‐lapse images were then obtained at 20‐min intervals. Maximum projection images of Z‐stacks are shown. Peroxisomes were labeled with Pex11‐mCherry. Scale bars, 5 μm; 2 μm for zoomed‐in image. (D) Time‐lapse microscopy images were obtained at the indicated times (min) for the cells in (A) without IAA and β‐Estradiol treatment. Maximum projection images of Z‐stacks are shown. Peroxisomes were labeled with Pex11‐mCherry. Scale bars, 5 μm; 2 μm for zoomed‐in image. (E) Yeast cells in A were grown in SCD at 30 °C, and treated with 1 mm IAA and 1 μm β‐Estradiol after the expression of 3xFLAG‐mNG‐Pex15 was shut off. Maximum projection images of Z‐stacks are shown. Peroxisomes were labeled with Pex11‐mCherry. Scale bars, 5 μm; 2 μm for zoomed‐in image. (F) For the microscopic images obtained in (C–E), the fluorescence intensity of 3xFLAG‐mNG‐Pex15 on peroxisomes (labeled with Pex11‐mCherry) was quantified at each time point. A single yeast cell was selected as a region of interest (ROI), and the intensity was shown relative to Time 0 in cells extracted as ROIs. Values are the mean ± SD (+β‐Estradiol; n = 60, −β‐Estradiol; n = 60, +IAA, β‐Estradiol; n = 60) from three independent replicates; n represents the number of cells. ****, P < 0.0001; **, P = 0.0475 compared with the results of time‐lapse imaging with β‐Estradiol treatment by one‐way ANOVA with Dunnett's multiple comparison test.

Fig. 8

The GET pathway mediates Pex15 transfer from mitochondria to the ER for degradation. The promoter shutoff chase of 3xFLAG‐mNG‐Pex15 expression was performed with the yeast cells described in Fig. 7A (GET3) and yeast cells lacking GET3 (get3Δ), which were grown in SCD medium at 30 °C. AID*‐Pex19 was then depleted by a treatment with 1 mm IAA for 30 min at 30 °C, followed by the induction of 3xFLAG‐mNG‐Pex15 with 5 mm cyanamide for 2 h at 30 °C. The cells were washed with fresh SCD medium to shut off the 3xFLAG‐mNG‐Pex15 expression and induce the re‐expression of AID*‐Pex19 by an incubation in fresh SCD medium for 2 h at 30 °C. Cell extracts were prepared at the indicated times in the presence (+β‐Estradiol) of Tom70TM‐Msp1 induction, and proteins were analyzed by SDS/PAGE and immunoblotting with the indicated antibodies. The normalized relative amounts of 3xFLAG‐mNG‐Pex15 to Pgk1 were plotted against the chase time. Values are the mean ± SD from three independent experiments.

Fig. 9

Peroxisomal Msp1 mediates endogenous Pex15 degradation under rapid Pex19 depletion conditions. (A) Wild‐type (MSP1) and msp1Δ cells expressing AID*‐Pex19 and 3xFLAG‐Pex15 under the control of their own promoters from the chromosomes were grown in YPD medium at 30 °C, and then further incubated for 30 min after the addition of 1 mm IAA. Extracts were prepared at the indicated times after the addition of 100 μg·mL⁻¹ CHX, and proteins were analyzed by SDS/PAGE and immunoblotting with the indicated antibodies (left). The normalized relative amounts of 3xFLAG‐Pex15 were plotted against the chase time. Values are the mean ± SD from three independent experiments. (B) Wild‐type (MSP1) and msp1Δ cells expressing AID*‐Pex19 and mNG‐Pex15 under the control of their own promoters from the chromosomes were grown in SCD medium at 30 °C. The localization of mNG‐Pex15 was observed by fluorescence microscopy at the indicated time points after the addition of 1 mm IAA and 100 μg·mL⁻¹ CHX. Maximum projection images of Z‐stacks are shown. Peroxisomes were labeled with Pex11‐mCherry. Scale bars, 5 μm; 2 μm for zoomed‐in image. DIC, differential interference contrast microscopy. Background subtraction was first performed on the microscopic images and followed by z‐stack maximum intensity projection. The mNG‐Pex15 fluorescence signal on peroxisomes was then quantified from more than 100 cells at each time point per experiment. The normalized relative value of mNG‐Pex15 was plotted against the chase time. Values are the mean ± SD from two independent experiments. (C) Wild‐type (MSP1) and msp1Δ cells expressing AID*‐Pex19 and Pex6‐mNG under the control of their own promoters from the chromosomes were grown in SCD medium at 30 °C. The localization of Pex6‐mNG was observed by fluorescence microscopy at the indicated time points after the addition of 1 mm IAA and 100 μg·mL⁻¹ CHX. Maximum projection images of Z‐stacks are shown. Peroxisomes were labeled with Pex11‐mCherry. Scale bars, 5 μm; 2 μm for zoomed‐in image. DIC, differential interference contrast microscopy. The quantification of the Pex6‐mNG signal was performed as in (B), with values plotted relative to the signal at time 0 set to 1. The normalized relative value of Pex6‐mNG were plotted against the chase time. Values are the mean ± SD from two independent experiments.

Fig. 10

Peroxisomal Msp1 extracts endogenous Pex15 upon rapid depletion of Pex3. (A) Yeast cells expressing 3xFLAG‐Pex15 with AID*‐Pex19 or Pex3‐AID*‐V5 under the control of their own promoters from the chromosomes were grown in YPD medium at 30 °C, and then further incubated for 30 min after the addition of 1 mm IAA. Extracts were prepared at the indicated times from cells incubated at 30 °C after the addition of 100 μg·mL⁻¹ CHX, and proteins were analyzed by SDS/PAGE and immunoblotting with the indicated antibodies (left). The normalized relative amounts of 3xFLAG‐Pex15 were plotted against the chase time. Values are the mean ± SD from three independent experiments. (B) Wild‐type (MSP1) and msp1Δ cells expressing Pex3‐AID*‐V5 and mNG‐Pex15 under the control of their own promoters from the chromosomes were grown in SCD medium at 30 °C. Localization of mNG‐Pex15 was observed by fluorescence microscopy at the indicated time points after the addition of 1 mm IAA and 100 μg·mL⁻¹ CHX. Maximum projection images of Z‐stacks are shown. Peroxisomes were labeled with Pex11‐mCherry. Scale bars, 5 μm; 2 μm for zoomed‐in image. DIC, differential interference contrast microscopy. The quantification of the mNG‐Pex15 signal was performed as in Fig. 9B, with the signal at time 0 set to 1. The normalized relative value of mNG‐Pex15 were plotted against the chase time. Values are the mean ± SD from two independent experiments. (C) Yeast cells expressing AID*‐Pex19 and Pex3‐3xFLAG under the control of their own promoters from the chromosomes were grown in YPD medium at 30 °C, and were further incubated for 30 min after the addition of 1 mm IAA. Extracts were prepared at the indicated times from cells incubated at 30 °C after the addition of 100 μg·mL⁻¹ CHX, and proteins were analyzed by SDS/PAGE and immunoblotting with the indicated antibodies (left). The normalized relative amounts of Pex3‐3xFLAG were plotted against the chase time. Values are the mean ± SD from three independent experiments.

Fig. 11

Peroxisomal Msp1 transfers endogenous Pex15 from peroxisomes to mitochondria. (A) Wild‐type and msp1Δ cells expressing mNG‐Pex15 under the control of its own promoter from the chromosome were grown in SCD medium at 30 °C. In msp1Δ cells, Tom70TM‐Msp1 or a peroxisome‐targeted Msp1 variant, Pex22TM‐Msp1, was expressed under the control of its own promoter from the chromosome. Cell extracts were prepared, and proteins were analyzed by SDS/PAGE and immunoblotting with the indicated antibodies (left). Representative images from three independent experiments are shown. (B) Yeast cells in (A) were grown in SCD medium at 30 °C and imaged by fluorescence microscopy. Mitochondria were labeled with Tom70‐mCherry. Maximum projection images of Z‐stacks are shown. Scale bars, 5 μm; 2 μm for zoomed‐in image. DIC, differential interference contrast microscopy. (C) Co‐localization of mNG‐Pex15 with mitochondria was quantified by using Pearson's correlation coefficient between the mNG and mitochondria (mCherry) signals. Values are the mean ± SD (n = 60) from three independent replicates; n represents the number of cells. ****, P < 0.0001 compared with wild‐type by one‐way ANOVA with Dunnett's multiple comparison test.

Fig. 12

Wild‐type (MSP1) and msp1Δ cells without (msp1Δ) or with expression of Tom70TM‐Msp1 (msp1Δ:: TOM70TM‐MSP1) or Pex22TM‐Msp1 (msp1Δ::PEX22TM‐MSP1) used in Fig. 11 were grown on YPD and the oleic acid‐based (YNBO) agar plates at 30 °C. W303‐1A wild‐type (W303‐1A) and its pex19‐deletion mutant (pex19Δ) were shown as control strains. This experiment was performed once to verify the growth of yeast cells expressing the Msp1 mutant.

Fig. 13

Model of the Msp1‐ and Pex19‐ and Pex3‐mediated localization of Pex15 to peroxisomes. After translation, newly synthesized Pex15 binds to Pex19 in the cytosol and is directed to the peroxisomal membrane. Membrane insertion occurs through the association of Pex19 with Pex3. The absence of Pex19 or Pex3 results in the mistargeting of Pex15 to the mitochondrial OM. Mislocalized Pex15 on the mitochondrial OM is extracted by Msp1 and transferred to the ER via the GET pathway. At the ER, Pex15 is ubiquitinated by the ER‐resident E3 ligase Doa10 or Hrd1 and subsequently degraded by the proteasome. In contrast, when Pex19 and Pex3 are present, Msp1‐extracted Pex15 from the mitochondrial OM can be correctly redirected to peroxisomes. In addition, Pex15 is occasionally extracted from the peroxisomal membrane by Msp1, but is reinsertered into peroxisomes in a Pex19‐ and Pex3‐dependent manner.