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Figure 2.
Distinct requirements of Pex30 DysF and DUF domains at different MCS. (A) Schematic representation of the predicted Pex30 domains. RHD, reticulon-homology domain; DysF, dysferlin; DUF, domain of unknown function 4196. The ER membrane is shown to indicate Pex30 topology. (B) Effect of different Pex30 mutations on the localization of endogenous Pex32-mNG and Inp1-mCherry in exponentially growing cells. Bar, 5 µm. (C) Quantification of the number of peroxisomes per cell, in exponentially growing cells. Three independent experiments were analyzed (>30 cells/genotype/experiment were counted). Each dot corresponds to a cell, and the bars represent the mean and SD. Ordinary one-way ANOVA and Dunnett’s multiple comparisons were used to compare the number of peroxisomes between mutant and WT cells (***, P < 0.001; ns, not significant). (D) Distribution of peroxisomes in cells with the indicated genotype during exponential growth. Peroxisomes were labeled by the mCherry-PTS1 marker. Please note the increase of cytosolic fluorescence in the Pex30DysFΔ mutant cells, corresponding to non-imported mCherry-PTS1. Images correspond to maximum intensity Z-projections. Bar, 5 µm. (E) Localization of indicated Pex30 mutants tagged with mNG fluorescent protein and expressed from the endogenous Pex30 locus. The localization of Nvj1-tdTomato and LDs, stained with the neutral lipid dye MDH, was also analyzed in early stationary phase cells. NVJ-clustered and irregularly distributed LDs are indicated by yellow and magenta arrowheads, respectively. Bar, 5 µm. (F) Quantification of cells with the indicated genotype displaying defects in NVJ formation and LD clustering during the early stationary phase, as in E. Cells were classified into three categories, as depicted in the cartoons: (1) normal Nvj1 localization and clustered LDs; (2) normal Nvj1 localization and randomly distributed LDs; and (3) abnormal Nvj1 localization and randomly distributed LDs. Three independent experiments were analyzed (>100 cells/genotype/experiment were counted). The bars represent the SD. Ordinary one-way ANOVA and Dunnett’s multiple comparisons were used (****, P < 0.0001).

Distinct requirements of Pex30 DysF and DUF domains at different MCS. (A) Schematic representation of the predicted Pex30 domains. RHD, reticulon-homology domain; DysF, dysferlin; DUF, domain of unknown function 4196. The ER membrane is shown to indicate Pex30 topology. (B) Effect of different Pex30 mutations on the localization of endogenous Pex32-mNG and Inp1-mCherry in exponentially growing cells. Bar, 5 µm. (C) Quantification of the number of peroxisomes per cell, in exponentially growing cells. Three independent experiments were analyzed (>30 cells/genotype/experiment were counted). Each dot corresponds to a cell, and the bars represent the mean and SD. Ordinary one-way ANOVA and Dunnett’s multiple comparisons were used to compare the number of peroxisomes between mutant and WT cells (***, P < 0.001; ns, not significant). (D) Distribution of peroxisomes in cells with the indicated genotype during exponential growth. Peroxisomes were labeled by the mCherry-PTS1 marker. Please note the increase of cytosolic fluorescence in the Pex30DysFΔ mutant cells, corresponding to non-imported mCherry-PTS1. Images correspond to maximum intensity Z-projections. Bar, 5 µm. (E) Localization of indicated Pex30 mutants tagged with mNG fluorescent protein and expressed from the endogenous Pex30 locus. The localization of Nvj1-tdTomato and LDs, stained with the neutral lipid dye MDH, was also analyzed in early stationary phase cells. NVJ-clustered and irregularly distributed LDs are indicated by yellow and magenta arrowheads, respectively. Bar, 5 µm. (F) Quantification of cells with the indicated genotype displaying defects in NVJ formation and LD clustering during the early stationary phase, as in E. Cells were classified into three categories, as depicted in the cartoons: (1) normal Nvj1 localization and clustered LDs; (2) normal Nvj1 localization and randomly distributed LDs; and (3) abnormal Nvj1 localization and randomly distributed LDs. Three independent experiments were analyzed (>100 cells/genotype/experiment were counted). The bars represent the SD. Ordinary one-way ANOVA and Dunnett’s multiple comparisons were used (****, P < 0.0001).

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