DysF domain is required for recruitment of Pex30 at ER-LD contact sites. (A) Schematic of Pex30 truncations tagged with GFP. (B) WF images of sei1pex30Δ cells endogenously expressing Opi1-mCherry and truncations of Pex30 tagged with GFP on a plasmid grown to logarithmic phase. White arrowheads denote Pex30-GFP and Opi1-mCherry puncta that colocalize. Bar = 4 μm. (C and D) Quantification of cells shown in B showing percent cells with Pex30-GFP and Opi1-mCherry 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 < 0.0001). (E) 10-fold serial dilutions of sei1pex30Δ cells expressing Pex30-GFP truncation plasmids indicated in A were spotted on synthetic media without leucine. Cells were incubated for 2 days at 30°C and 37°C. (F) Western blot analysis of cell lysates from sei1pex30Δ cells expressing Pex30-GFP truncation plasmids indicated in A. 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. (G) Quantification of protein levels from F. Bars show the mean from three replicates and SEM. One-way ANOVA and Dunnett’s multiple comparison test were used to compare protein levels (*P < 0.05). Source data are available for this figure: SourceData F3. WF, widefield images.