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Figure 1.
Pex30 is required for the integrity of the NVJ and ER–peroxisome MCS. (A) Schematic representation of the NVJ and the MCS between the ER and peroxisomes. Left: the Pex30–Pex29 complex accumulates at the NVJ. The binding of Nvj1 (in the ER) to Vac8 (in the vacuole) defines the prototypical NVJ tether. Right: the Pex30–Pex28-Pex32 complex accumulates at the ER–peroxisome MCS. The cytosolic protein Inp1 bridges Pex3 molecules in the ER and peroxisomes and is part of the tether between the two organelles. (B) Single Z-slices of WT, pex30Δ, pex29Δ, and nvj1Δ spheroplasts during the diauxic shift from volumes acquired using SBF-SEM 3View. The site of the minimal distance between the nucleus and the vacuole is indicated by an arrowhead. N, nucleus; V, vacuole; LD, lipid droplet; SBF-SEM, serial block-face scanning electron microscopy. Bars, 1 µm. (C) Quantification of the minimal distance between the nucleus and the closest vacuole in cells grown as in B. 20 cells per genotype and per condition were analyzed. Box and whiskers represent the distribution of the values (minimum, 25th percentile, median, 75th percentile, and maximum). Ordinary one-way ANOVA and Dunnett’s multiple comparisons were used to compare the minimal distance between the organelles with the WT (DS) condition (****, P < 0.0001; ***, P < 0.001; **, P < 0.01; *, P < 0.05). (D) Quantification of the number of vacuoles per cell, in cells treated as in B. Percentage of cells with 1, 2–4, or >4 vacuoles is indicated for each condition. 50 cells per genotype and condition were analyzed. Ordinary one-way ANOVA and Dunnett’s multiple comparisons were used to compare the percentage of cells with one vacuole with the WT (DS) condition (****, P < 0.0001; ***, P < 0.001; **, P < 0.01). (E) Localization of endogenous Pex32-mNG and Inp1-mCherry in exponentially growing cells with the indicated genotype. Bar, 5 µm. (F) Steady-state levels of endogenously tagged Pex3-mNG and Inp1-mCherry in cells with the indicated genotype. Whole-cell extracts were prepared from exponentially growing cells, separated by SDS-PAGE, and analyzed by western blotting. Pex3-mNG, Inp1-mCherry and Dpm1, used as a loading control, were detected with anti-mNG, anti-mCherry, and anti-Dpm1 antibodies, respectively. IB, immunoblot. The position of molecular weight markers (75 and 25 kDa) is shown. Source data are available for this figure: SourceData F1.

Pex30 is required for the integrity of the NVJ and ER–peroxisome MCS. (A) Schematic representation of the NVJ and the MCS between the ER and peroxisomes. Left: the Pex30–Pex29 complex accumulates at the NVJ. The binding of Nvj1 (in the ER) to Vac8 (in the vacuole) defines the prototypical NVJ tether. Right: the Pex30–Pex28-Pex32 complex accumulates at the ER–peroxisome MCS. The cytosolic protein Inp1 bridges Pex3 molecules in the ER and peroxisomes and is part of the tether between the two organelles. (B) Single Z-slices of WT, pex30Δ, pex29Δ, and nvj1Δ spheroplasts during the diauxic shift from volumes acquired using SBF-SEM 3View. The site of the minimal distance between the nucleus and the vacuole is indicated by an arrowhead. N, nucleus; V, vacuole; LD, lipid droplet; SBF-SEM, serial block-face scanning electron microscopy. Bars, 1 µm. (C) Quantification of the minimal distance between the nucleus and the closest vacuole in cells grown as in B. 20 cells per genotype and per condition were analyzed. Box and whiskers represent the distribution of the values (minimum, 25th percentile, median, 75th percentile, and maximum). Ordinary one-way ANOVA and Dunnett’s multiple comparisons were used to compare the minimal distance between the organelles with the WT (DS) condition (****, P < 0.0001; ***, P < 0.001; **, P < 0.01; *, P < 0.05). (D) Quantification of the number of vacuoles per cell, in cells treated as in B. Percentage of cells with 1, 2–4, or >4 vacuoles is indicated for each condition. 50 cells per genotype and condition were analyzed. Ordinary one-way ANOVA and Dunnett’s multiple comparisons were used to compare the percentage of cells with one vacuole with the WT (DS) condition (****, P < 0.0001; ***, P < 0.001; **, P < 0.01). (E) Localization of endogenous Pex32-mNG and Inp1-mCherry in exponentially growing cells with the indicated genotype. Bar, 5 µm. (F) Steady-state levels of endogenously tagged Pex3-mNG and Inp1-mCherry in cells with the indicated genotype. Whole-cell extracts were prepared from exponentially growing cells, separated by SDS-PAGE, and analyzed by western blotting. Pex3-mNG, Inp1-mCherry and Dpm1, used as a loading control, were detected with anti-mNG, anti-mCherry, and anti-Dpm1 antibodies, respectively. IB, immunoblot. The position of molecular weight markers (75 and 25 kDa) is shown. Source data are available for this figure: SourceData F1.

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