Peroxisome biogenesis initiated by protein phase separation

Extended Data Fig. 1 | Predicted PLD in Pex13/Pex5. PLD Analysis of a, Pex13 b, Pex5 and c, Pex14 using PLAAC21. Predicted PLD sequence is marked in red for Pex13 and Pex5 on the left, corresponding to the outputs on the right. d, Multiple sequence alignment of the Pex13 sequence from the indicated five species, showing conserved Y residues within the predicted yeast PLD sequence.

Extended Fig. 2. PTS1 transport defective in Pex13-PLD variants a, Distribution of Y (white) to F (orange) or S (purple) substitutions introduced in the Pex13 PLD using CRISPR-Cas9. Point mutation heat map shown in shades of gray denotes the number of mCherry foci observed in the indicated cells. n = 100 cells from 3 independent experiments. Confocal images of b, GFP-Pex13-S8 (interspersed) and c, GFP-Pex13-S9 (blocky) cells taken at the indicated time points after induction of Pex5. Scale bar 5 mm. Percentage cells displaying mCherry-SKL foci are indicated below each panel. n = 100 cells per sample.

Extended Data Fig. 3 | Import kinetics in Pex13-WT and Y→S/F mutants. a, Percentage of cells displaying mCherry-SKL foci in the indicated strains following Pex5 induction. Error bars are mean ± s.e.m. for 3 independent experiments, n=100 cells. The schematic on the right shows the Y→S/F substitutions made. b, Timelapse following mCherry-SKL foci formation in Pex13 WT cells (black arrow) and c, Pex13 S8 cells (orange/blue arrows). Scale bar 5 µm. d, e, mCherry-SKL foci fluorescence intensity of the cells imaged in panels b and c, respectively, plotted as a function of time after induction of Pex5 expression.

Extended Data Fig. 4 | Representative images of cargo import defects. Spinning disc confocal images of a, GFP-Pex13-S7 and b, GFP-Pex13-S15 cells taken at the indicated time points after induction of Pex5. Scale bar 5 µm. Percentage cells displaying mCherry-SKL foci are indicated below the panels. n = 100 cells.

Extended Data Fig. 5 | LLPS of purified PTS1 peroxins. a, Coomassie stained SDS-PAGE gels of 1 µg of the indicated purified proteins. Images of condensates formed by b,c, Pex13-IDR WT labeled with AZdye 488 maleimide d,e, Pex5 labeled with AZdye 647 maleimide and f,g, Pex14 IDR labeled with AZdye 488 maleimide. Scale bar 5 µm.

Extended Data Fig. 6 | Pex5-cargo partitions into Pex13-IDR condensates. Titration of a, Pex5 and b, mCherry-SKL to determine the concentration at which they do not form condensates. 1 µM of both Pex5 and mCherry-SKL were used for in vitro reconstitution assays with Pex13-IDR. c, Reconstitution experiments to measure partitioning of Pex5 labelled with AZ 647 dye and mCherry-SKL into Pex13-IDR WT condensates immediately after mixing at room temperature. d, Cas9-AZdye 488 maleimide does not actively partition into Pex13 droplets (top) while mCherry-SKL partitions with Pex5 and Pex13 (bottom). e, Pex5 and mCherry-SKL partition into Pex13-IDR WT (10 µM) and Pex13-IDR S8 (30 µM) condensates. f, Pex13-IDR S15 does not form spherical condensates. mCherry-SKL remains diffuse in most images of Pex13-S15 aggregates (top). In some images, however, Pex5-mCherry-SKL appears to partition with Pex13-S15 (bottom). This is likely due to experimental variation, perhaps a somewhat lower concentration of Pex5-mCherry-SKL, co-aggregating with Pex13-IDR-S15. Scale bar 5 µm.

Extended Data Fig. 7 | iFCCS) workflow and controls a, Fiducial markers used to align the GFP-Pex13 and mCherry-SKL channels using MATLAB b, The centroid position of fiducial markers are selected and the coordinates are exported using a normalized cross correlation between the red and green channels. c, The centroid locations of peroxisomes of interest are localized using a particle localization analysis (dashed circle provided as guide for the eye). d, The GFP and mCherry intensities of each of the surrounding 17x17 pixels of the peroxisome centroid location are extracted. The intensities of centroid pixel boxed in c are shown in d. Using the equation in e, the intensity fluctuations, δF(t), relative to the mean intensity are calculated and used to calculate G(τ). Resulting data f, Spatial and g, Temporal, as explained in the methods section. h, Controls for laser power effect on photo bleaching. i, Averaged standard deviation of the cross correlation curves with the maximum G_XC(0) value in each peroxisome at each laser power.

Extended Data Fig. 8 | iFCCS spatial and temporal data shows transient clusters of correlated signal of mCherry-SKL and GFP-Pex13. a, Percentage of peroxisomes with GXC(0)>0.5 for GFP-Pex13 and soluble mCherry (no SKL) (gray). The-data of GFP-Pex13 with mCherry-SKL import from Fig. 4j is shown for comparison (red). b, Spatial iFCCS data from six different peroxisomes in WT 50 min after Pex5 induction where distinct transient clusters of correlated signals are observed. Images measure 1.19 µm x 1.19 µm in size. c, Transient nature of individual clusters are observed in the decay and fluctuations of the cross-correlation over time between clusters in different peroxisomes (n=27 peroxisomes). d, Gaussian distribution of NGFP-Pex13/N-mCherry-SKL obtained by autocorrelating signal from the Pex13-GFP and SKL-mCherry channels, extracting a value from the pixel exhibiting the highest GXC(0) > 0.5 in each peroxisome from the Pex13 WT strain, 50 min after Pex5 induction. e, Bimodal Gaussian fit of GFP-Pex14 same as in panel d, for data obtained 90 min after Pex5 induction. f, Distribution of NPex13-488/NPex5-647 showing the ratio of Pex13 to Pex5 in Pex13-IDR and Pex13-S8 condensates. All pixels within a single condensate of Pex13-IDR-WT or Pex13-IDR-S8 with Pex5 partitioned within the condensate were analyzed. Gaussian fit parameters µ and σ listed with 95% confidence intervals in panels d,e and f. g, An alternative transmembrane intercalation model for peroxisomal cargo import.