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Peroxisome biogenesis initiated by protein phase separation

Nature volume 617, pages 608–615 (2023)Cite this article

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Abstract

Peroxisomes are organelles that carry out β-oxidation of fatty acids and amino acids. Both rare and prevalent diseases are caused by their dysfunction1. Among disease-causing variant genes are those required for protein transport into peroxisomes. The peroxisomal protein import machinery, which also shares similarities with chloroplasts2, is unique in transporting folded and large, up to 10 nm in diameter, protein complexes into peroxisomes3. Current models postulate a large pore formed by transmembrane proteins4; however, so far, no pore structure has been observed. In the budding yeast Saccharomyces cerevisiae, the minimum transport machinery includes the membrane proteins Pex13 and Pex14 and the cargo-protein-binding transport receptor, Pex5. Here we show that Pex13 undergoes liquid–liquid phase separation (LLPS) with Pex5–cargo. Intrinsically disordered regions in Pex13 and Pex5 resemble those found in nuclear pore complex proteins. Peroxisomal protein import depends on both the number and pattern of aromatic residues in these intrinsically disordered regions, consistent with their roles as ‘stickers’ in associative polymer models of LLPS5,6. Finally, imaging fluorescence cross-correlation spectroscopy shows that cargo import correlates with transient focusing of GFP–Pex13 and GFP–Pex14 on the peroxisome membrane. Pex13 and Pex14 form foci in distinct time frames, suggesting that they may form channels at different saturating concentrations of Pex5–cargo. Our findings lead us to suggest a model in which LLPS of Pex5–cargo with Pex13 and Pex14 results in transient protein transport channels7.

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Fig. 1: Pex13 Y-to-S substitutions disrupt peroxisomal PTS1-cargo import.
Fig. 2: Pex13-PLD Y-to-S substitutions decrease rates of PTS1-cargo import.
Fig. 3: mCherry–SKL requires binding to Pex5 to partition into Pex13-IDR condensates in vitro.
Fig. 4: Clusters of transiently correlated mCherry–SKL and GFP–Pex13 or GFP–Pex14 on peroxisomes.

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Data availability

Data supporting the findings of this study are included within the Article and its extended data and supplementary figures. Videos analysed for Fig. 4b–f are included as Supplementary Videos 1–4. Uncropped immunoblots used in Fig. 2b are provided in Supplementary Fig. 1. Source data are provided with this paper.

Code availability

Custom codes developed for iFCCS analysis were central to the conclusions of the paper. The iFCCS code is publicly available on Zenodo (https://doi.org/10.5281/zenodo.7702794) and through GitHub (https://github.com/KisleyLabAtCWRU/Peroxisome-iFCCS). Further questions can be directed to lydia.kisley@case.edu. Custom code for measurement of Pex13-IDR droplet fusion rates is also available on Zenodo (https://doi.org/10.5281/zenodo.7702890) and through GitHub (https://github.com/Xavier-Castellanos-Girouard/Peroxisome-biogenesis-initiated-by-protein-phase-separation/).

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Acknowledgements

We thank M. Schuldiner for seamless SWAT strains and advice on the VPS1 knockouts, J. Vogel for providing strains from the YKO collection, E. Lemke for suggestions regarding in vitro phase separation experiments, R. Erdmann for the gift of Pex5 antibody, T. Tai for making the mCherry–SKL construct, P. Garneau for technical assistance and N. Stifani for providing expertise with microscopy. We acknowledge Canadian Institutes of Health Research grant MOP-GMX-152556 and Human Frontier Science Program grant RGP0034/2017, and the Canada Research Chairs Program (S.W.M.), a cross-disciplinary fellowship (LT000805/2018-C; I.O.L.B.), the National Science and Engineering Council (J.G.O.), the Case Western Reserve College of Arts and Sciences and the Department of Physics (L.K. and Z.Z.) and NIH NIGMS grant R35GM142466 (L.K.) for financial support of this work.

Author information

Author notes

  1. Isabel O. L. Bacellar

    Present address: Douglas Research Centre, Montreal, Quebec, Canada

  2. Zhenghao Zhang

    Present address: Mitchell Physics Building (MPHY), College Station, TX, USA

Authors and Affiliations

  1. Département de Biochimie, Université de Montréal, Montreal, Quebec, Canada

    Rini Ravindran, Isabel O. L. Bacellar, Xavier Castellanos-Girouard, Haytham M. Wahba, James G. Omichinski & Stephen W. Michnick

  2. Department of Biochemistry, Faculty of Pharmacy, Beni-Suef University, Beni-Suef, Egypt

    Haytham M. Wahba

  3. Department of Physics, Case Western Reserve University, Cleveland, OH, USA

    Zhenghao Zhang & Lydia Kisley

  4. Department of Chemistry, Case Western Reserve University, Cleveland, OH, USA

    Lydia Kisley

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Contributions

S.W.M. and I.O.L.B. conceptualized this study. I.O.L.B. generated and characterized Pex13 mutants and yeast strains for induction of Pex5 expression, standardized microscopy conditions for iFCCS and designed constructs for protein purification. R.R. generated Pex13 mutants, carried out mCherry–SKL import kinetics assays in yeast strains, purified proteins, standardized and carried out in vitro experiments, acquired data for iFCCS and interpreted data. X.C.-G. analysed Pex13 droplet fusion data. H.M.W. purified proteins and J.G.O. provided expertise in protein purification. Z.Z. and L.K. generated pipelines for iFCCS, and compiled and analysed the resulting data. R.R., I.O.L.B., X.C.-G., L.K. and S.W.M. analysed and interpreted data. R.R., I.O.L.B., L.K. and S.W.M. wrote the manuscript and all authors provided feedback and editorial support.

Corresponding author

Correspondence to Stephen W. Michnick.

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Nature thanks Ralf Erdmann, Alf Honigmann and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data figures and tables

Extended Data Fig. 1 Predicted PLD in Pex13 and 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 Data 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 μm. Percentage cells displaying mCherry-SKL foci are indicated below each panel. n = 100 cells per sample.

Source Data

Extended Data Fig. 3 Import kinetics in Pex13-WT and Y→S or F substitutions.

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 or F substitutions made. b, Timelapse following mCherry-SKL foci formation in Pex13 WT cells (black arrow) and c, Pex13 S8 cells (orange and 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.

Source Data

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.

Source Data

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 ms) > 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/NmCherry-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.

Source Data

Extended Data Table 1 Strains used in this study
Full size table
Extended Data Table 2 Plasmids used in this study
Full size table

Supplementary information

Supplementary Fig. 1

Uncropped immunoblots for Fig. 2b.

Reporting Summary

Peer Review File

Supplementary Video 1

Cross-correlation video of positive control shows high spatial correlation between dual-labelled GFP–Pex13–mCherry–SKL. A peroxisome identified by single-particle tracking algorithm is located at the centre of the image, which measures 1.19 µm × 1.19 µm in size. The time listed above the figure indicates the lag time, and the colour indicates the magnitude of the cross-correlation at the given lag time. Note the extended GXC(τ) scale from 0 to 1 compared to the scales used in Supplementary Videos 2–4.

Supplementary Video 2

Cross-correlation video of negative control shows low spatial correlation between mCherry–SKL and GFP–Ant1. A peroxisome identified by single-particle tracking algorithm is located at the centre of the image, which measures 1.19 µm × 1.19 µm in size. The time listed above the figure indicates the lag time, and the colour indicates the magnitude of the cross-correlation at the given lag time. Data obtained from a peroxisome identified 50 min after induction, the same time as the wild-type data shown in Supplementary Video 4.

Supplementary Data Video 3

Cross-correlation video of GFP–WT-Pex13 cells 10 min after induction shows low spatial correlation between mCherry–SKL and GFP–Pex13. Data are similar to those of the negative control showing low correlation. A peroxisome identified by single-particle tracking algorithm is located at the centre of the image, which measures 1.19 µm × 1.19 µm in size. The time listed above the figure indicates the lag time, and the colour indicates the magnitude of the cross-correlation at the given lag time.

Supplementary Video 4

Cross-correlation video of GFP–WT Pex13 at 50 min after induction shows higher spatial correlation between mCherry–SKL and GFP–Pex13 distributed around the edge of peroxisome. Three distinct pores can be visualized in the first frame of the video, which then quickly decay as lag time increases, indicating the transient nature of the pores. A peroxisome identified by single-particle tracking algorithm is located at the centre of the image, which measures 1.19 µm × 1.19 µm in size. The time listed above the figure indicates the lag time, and the colour indicates the magnitude of the cross-correlation at the given lag time.

Source data

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Ravindran, R., Bacellar, I.O.L., Castellanos-Girouard, X. et al. Peroxisome biogenesis initiated by protein phase separation. Nature 617, 608–615 (2023). https://doi.org/10.1038/s41586-023-06044-1

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