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Figure 5.
All-atom MD simulations to characterize DysF domain and its membrane interactions. (A) Amino acid sequence of the DysF domain, with residues involved in membrane anchoring underlined, and β1 and β6 strands depicted by bolded text. (B) Snapshot from a simulation of the DysF domain with a 70:30 DOPC:DOPA bilayer. The tails of DOPC and DOPA are shown in grey and blue, respectively. (C) Surface and cartoon representation of the DysF domain. Each residue is colored based on its average number of lipid contacts, with a contact defined as a lipid within 4.5 Å of the residue. (D) WF images of sei1pex30Δ cells endogenously expressing Opi1-mCherry and GFP-tagged Pex30 (296–315Δ) or Pex30 (378–398Δ) plasmids grown to logarithmic phase. Bar = 4 μm. (E) Quantification of cells shown in D showing percent cells with Pex30-GFP accumulation in the indicated strains. Bars show mean from three independent experiments and SEM. 100 cells per genotype from each replicate were analyzed and compared using one-way ANOVA and Dunnett’s multiple comparison test (***P < 0.001). (F) 10-fold serial dilutions of sei1pex30Δ cells expressing Pex30-GFP truncation plasmids were spotted on synthetic media without leucine. Cells were incubated for 2 days at 30°C and 37°C. (G) Western blot analysis of cell lysates from sei1pex30Δ cells expressing Pex30-GFP and DysF truncation plasmids in D. Anti-GFP monoclonal antibody was used to detect Pex30 protein levels, and anti-Porin1 monoclonal antibody was used to detect porin levels as a control. (H) Quantification of protein levels from G. Bars show the mean from two replicates and SEM. One-way ANOVA and Dunnett’s multiple comparison test were used to compare protein levels. Source data are available for this figure: SourceData F5. WF, widefield images.

All-atom MD simulations to characterize DysF domain and its membrane interactions. (A) Amino acid sequence of the DysF domain, with residues involved in membrane anchoring underlined, and β1 and β6 strands depicted by bolded text. (B) Snapshot from a simulation of the DysF domain with a 70:30 DOPC:DOPA bilayer. The tails of DOPC and DOPA are shown in grey and blue, respectively. (C) Surface and cartoon representation of the DysF domain. Each residue is colored based on its average number of lipid contacts, with a contact defined as a lipid within 4.5 Å of the residue. (D) WF images of sei1pex30Δ cells endogenously expressing Opi1-mCherry and GFP-tagged Pex30 (296–315Δ) or Pex30 (378–398Δ) plasmids grown to logarithmic phase. Bar = 4 μm. (E) Quantification of cells shown in D showing percent cells with Pex30-GFP accumulation in the indicated strains. Bars show mean from three independent experiments and SEM. 100 cells per genotype from each replicate were analyzed and compared using one-way ANOVA and Dunnett’s multiple comparison test (***P < 0.001). (F) 10-fold serial dilutions of sei1pex30Δ cells expressing Pex30-GFP truncation plasmids were spotted on synthetic media without leucine. Cells were incubated for 2 days at 30°C and 37°C. (G) Western blot analysis of cell lysates from sei1pex30Δ cells expressing Pex30-GFP and DysF truncation plasmids in D. Anti-GFP monoclonal antibody was used to detect Pex30 protein levels, and anti-Porin1 monoclonal antibody was used to detect porin levels as a control. (H) Quantification of protein levels from G. Bars show the mean from two replicates and SEM. One-way ANOVA and Dunnett’s multiple comparison test were used to compare protein levels. Source data are available for this figure: SourceData F5. WF, widefield images.

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