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Figure 4.
PA-binding residues of the DysF domain required for Pex30 function. (A) Binding of the DysF domain of Pex30 to membrane systems with increasing concentrations of DOPA in CG-MD simulations. On top, the visual inset shows the initial system setup and a representative snapshot of the DysF domain bound to the membrane after unbiased CG-MD simulations. (B) Normalized frequency of lipid interaction per residue. Representative error bars are shown for the highest binding residues. The binding regions of the protein, with their amino acid sequence shown, are highlighted in pink on the plot. The inset represents the surface of the DysF domain, and residues are colored according to their normalized frequency of interaction with the membrane. (C) Representative close-up snapshot of the DysF domain of Pex30 (blue cartoon) bound to a 60% DOPC, 40% DOPA membrane system. Residues displaying high membrane-binding frequency are highlighted in pink. Lipids: gray, PO4 and NC3 beads; yellow, DOPC lipid tails; orange, DOPA lipid tails. (D) Localization of the indicated Pex30 mutants tagged with mNG fluorescent protein and of Nvj1-tdTomato in early stationary phase cells. Bar, 5 µm. (E) Quantification of cells with Nvj1-tdTomato concentrated at the NVJ in cells grown as in D. Cells were quantified as depicted in the cartoon by exhibiting organized Nvj1 localization in the nuclear ER. 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). (F) Localization of endogenous Pex32-mNG and Inp1-mCherry in cells expressing the indicated Pex30 mutants. Cells were analyzed in the exponential phase. Bar, 5 µm. (G) Quantification of cells with the indicated genotype displaying Inp1-mCherry foci. 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).

PA-binding residues of the DysF domain required for Pex30 function. (A) Binding of the DysF domain of Pex30 to membrane systems with increasing concentrations of DOPA in CG-MD simulations. On top, the visual inset shows the initial system setup and a representative snapshot of the DysF domain bound to the membrane after unbiased CG-MD simulations. (B) Normalized frequency of lipid interaction per residue. Representative error bars are shown for the highest binding residues. The binding regions of the protein, with their amino acid sequence shown, are highlighted in pink on the plot. The inset represents the surface of the DysF domain, and residues are colored according to their normalized frequency of interaction with the membrane. (C) Representative close-up snapshot of the DysF domain of Pex30 (blue cartoon) bound to a 60% DOPC, 40% DOPA membrane system. Residues displaying high membrane-binding frequency are highlighted in pink. Lipids: gray, PO4 and NC3 beads; yellow, DOPC lipid tails; orange, DOPA lipid tails. (D) Localization of the indicated Pex30 mutants tagged with mNG fluorescent protein and of Nvj1-tdTomato in early stationary phase cells. Bar, 5 µm. (E) Quantification of cells with Nvj1-tdTomato concentrated at the NVJ in cells grown as in D. Cells were quantified as depicted in the cartoon by exhibiting organized Nvj1 localization in the nuclear ER. 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). (F) Localization of endogenous Pex32-mNG and Inp1-mCherry in cells expressing the indicated Pex30 mutants. Cells were analyzed in the exponential phase. Bar, 5 µm. (G) Quantification of cells with the indicated genotype displaying Inp1-mCherry foci. 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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