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.
This is a preview of subscription content, access via your institution
Access options
Access Nature and 54 other Nature Portfolio journals
Get Nature+, our best-value online-access subscription
$29.99 / 30 days
cancel any time
Subscribe to this journal
Receive 51 print issues and online access
$199.00 per year
only $3.90 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
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/).
References
Zalckvar, E. & Schuldiner, M. Beyond rare disorders: a new era for peroxisomal pathophysiology. Mol. Cell 82, 2228–2235 (2022).
Ganesan, I., Shi, L. X., Labs, M. & Theg, S. M. Evaluating the functional pore size of chloroplast TOC and TIC protein translocons: import of folded proteins. Plant Cell 30, 2161–2173 (2018).
Walton, P. A., Hill, P. E. & Subramani, S. Import of stably folded proteins into peroxisomes. Mol. Biol. Cell 6, 675–683 (1995).
Meinecke, M. et al. The peroxisomal importomer constitutes a large and highly dynamic pore. Nat. Cell Biol. 12, 273–277 (2010).
Martin, E. W. et al. Valence and patterning of aromatic residues determine the phase behavior of prion-like domains. Science 367, 694–699 (2020).
Wang, J. et al. A molecular grammar governing the driving forces for phase separation of prion-like RNA binding proteins. Cell 174, 688–699 (2018).
Erdmann, R. & Schliebs, W. Peroxisomal matrix protein import: the transient pore model. Nat. Rev. Mol. Cell Biol. 6, 738–742 (2005).
Gatto, G. J. Jr, Geisbrecht, B. V., Gould, S. J. & Berg, J. M. Peroxisomal targeting signal-1 recognition by the TPR domains of human PEX5. Nat. Struct. Biol. 7, 1091–1095 (2000).
Elgersma, Y. et al. The SH3 domain of the Saccharomyces cerevisiae peroxisomal membrane protein Pex13p functions as a docking site for Pex5p, a mobile receptor for the import PTS1-containing proteins. J. Cell Biol. 135, 97–109 (1996).
Erdmann, R. & Blobel, G. Identification of Pex13p a peroxisomal membrane receptor for the PTS1 recognition factor. J. Cell Biol. 135, 111–121 (1996).
Albertini, M. et al. Pex14p, a peroxisomal membrane protein binding both receptors of the two PTS-dependent import pathways. Cell 89, 83–92 (1997).
Brocard, C., Lametschwandtner, G., Koudelka, R. & Hartig, A. Pex14p is a member of the protein linkage map of Pex5p. EMBO J. 16, 5491–5500 (1997).
Francisco, T. et al. Protein transport into peroxisomes: knowns and unknowns. Bioessays https://doi.org/10.1002/bies.201700047 (2017).
Frey, S., Richter, R. P. & Görlich, D. FG-rich repeats of nuclear pore proteins form a three-dimensional meshwork with hydrogel-like properties. Science 314, 815–817 (2006).
Celetti, G. et al. The liquid state of FG-nucleoporins mimics permeability barrier properties of nuclear pore complexes. J. Cell Biol. https://doi.org/10.1083/jcb.201907157 (2020).
Paci, G., Caria, J. & Lemke, E. A. Cargo transport through the nuclear pore complex at a glance. J. Cell Sci. 134, jcs247874 (2021).
Barros-Barbosa, A. et al. The intrinsically disordered nature of the peroxisomal protein translocation machinery. FEBS J. 286, 24–38 (2019).
Emmanouilidis, L., Gopalswamy, M., Passon, D. M., Wilmanns, M. & Sattler, M. Structural biology of the import pathways of peroxisomal matrix proteins. Biochim. Biophys. Acta 1863, 804–813 (2016).
Gould, S. J. & Collins, C. S. Opinion: peroxisomal-protein import: is it really that complex. Nat. Rev. Mol. Cell Biol. 3, 382–389 (2002).
Carvalho, A. F. et al. The N-terminal half of the peroxisomal cycling receptor Pex5p is a natively unfolded domain. J. Mol. Biol. 356, 864–875 (2006).
Lancaster, A. K., Nutter-Upham, A., Lindquist, S. & King, O. D. PLAAC: a web and command-line application to identify proteins with prion-like amino acid composition. Bioinformatics 30, 2501–2502 (2014).
Hoepfner, D., van den Berg, M., Philippsen, P., Tabak, H. F. & Hettema, E. H. A role for Vps1p, actin, and the Myo2p motor in peroxisome abundance and inheritance in Saccharomyces cerevisiae. J. Cell Biol. 155, 979–990 (2001).
Yofe, I. et al. Pex35 is a regulator of peroxisome abundance. J. Cell Sci. 130, 791–804 (2017).
DeLoache, W. C., Russ, Z. N. & Dueber, J. E. Towards repurposing the yeast peroxisome for compartmentalizing heterologous metabolic pathways. Nat. Commun. 7, 11152 (2016).
Bremer, A. et al. Deciphering how naturally occurring sequence features impact the phase behaviours of disordered prion-like domains. Nat. Chem. 14, 196–207 (2022).
Huh, W. K. et al. Global analysis of protein localization in budding yeast. Nature 425, 686–691 (2003).
Nishimasu, H. et al. Crystal structure of Cas9 in complex with guide RNA and target DNA. Cell 156, 935–949 (2014).
Bergeron-Sandoval, L. P. et al. Endocytic proteins with prion-like domains form viscoelastic condensates that enable membrane remodeling. Proc. Natl Acad. Sci. USA https://doi.org/10.1073/pnas.2113789118 (2021).
Cooper, J. T. & Harris, J. M. Imaging fluorescence-correlation spectroscopy for measuring fast surface diffusion at liquid/solid interfaces. Anal. Chem. 86, 7618–7626 (2014).
Singh, A. P. & Wohland, T. Applications of imaging fluorescence correlation spectroscopy. Curr. Opin. Chem. Biol. 20, 29–35 (2014).
Shuang, B., Chen, J., Kisley, L. & Landes, C. F. Troika of single particle tracking programing: SNR enhancement, particle identification, and mapping. Phys. Chem. Chem. Phys. 16, 624–634 (2014).
Slaughter, B. D., Schwartz, J. W. & Li, R. Mapping dynamic protein interactions in MAP kinase signaling using live-cell fluorescence fluctuation spectroscopy and imaging. Proc. Natl Acad. Sci. USA 104, 20320–20325 (2007).
Cherry, J. M. et al. Saccharomyces Genome Database: the genomics resource of budding yeast. Nucleic Acids Res. 40, D700–705 (2012).
Bacia, K., Kim, S. A. & Schwille, P. Fluorescence cross-correlation spectroscopy in living cells. Nat. Methods 3, 83–89 (2006).
Kisley, L. et al. Characterization of porous materials by fluorescence correlation spectroscopy super-resolution optical fluctuation imaging. ACS Nano 9, 9158–9166 (2015).
Ouyang, M. et al. Liquid-liquid phase transition drives intra-chloroplast cargo sorting. Cell 180, 1144–1159 (2020).
Gould, S. J. et al. Pex13p is an SH3 protein of the peroxisome membrane and a docking factor for the predominantly cytoplasmic PTs1 receptor. J. Cell Biol. 135, 85–95 (1996).
Girzalsky, W. et al. Involvement of Pex13p in Pex14p localization and peroxisomal targeting signal 2-dependent protein import into peroxisomes. J. Cell Biol. 144, 1151–1162 (1999).
Kerssen, D. et al. Membrane association of the cycling peroxisome import receptor Pex5p. J. Biol. Chem. 281, 27003–27015 (2006).
Banjade, S. & Rosen, M. K. Phase transitions of multivalent proteins can promote clustering of membrane receptors. Elife 3, e04123 (2014).
Gaussmann, S. et al. Membrane interactions of the peroxisomal proteins PEX5 and PEX14. Front. Cell Dev. Biol. 9, 651449 (2021).
Feng, P. et al. A peroxisomal ubiquitin ligase complex forms a retrotranslocation channel. Nature 607, 374–380 (2022).
Pedrosa, A. G. et al. Peroxisomal monoubiquitinated PEX5 interacts with the AAA ATPases PEX1 and PEX6 and is unfolded during its dislocation into the cytosol. J. Biol. Chem. 293, 11553–11563 (2018).
Gao, Y., Skowyra, M. L., Feng, P. & Rapoport, T. A. Protein import into peroxisomes occurs through a nuclear pore-like phase. Science 378, eadf3971 (2022).
Lazard, M., Blanquet, S., Fisicaro, P., Labarraque, G. & Plateau, P. Uptake of selenite by Saccharomyces cerevisiae involves the high and low affinity orthophosphate transporters. J. Biol. Chem. 285, 32029–32037 (2010).
Felice, M. R. et al. Post-transcriptional regulation of the yeast high affinity iron transport system. J. Biol. Chem. 280, 22181–22190 (2005).
Salomons, F. A., Kiel, J. A., Faber, K. N., Veenhuis, M. & van der Klei, I. J. Overproduction of Pex5p stimulates import of alcohol oxidase and dihydroxyacetone synthase in a Hansenula polymorpha Pex14 null mutant. J. Biol. Chem. 275, 12603–12611 (2000).
Steinberg, S. et al. The PEX Gene Screen: molecular diagnosis of peroxisome biogenesis disorders in the Zellweger syndrome spectrum. Mol. Genet. Metab. 83, 252–263 (2004).
Ebberink, M. S. et al. Genetic classification and mutational spectrum of more than 600 patients with a Zellweger syndrome spectrum disorder. Hum. Mutat. 32, 59–69 (2011).
Jansen, R. L. M., Santana-Molina, C., van den Noort, M., Devos, D. P. & van der Klei, I. J. Comparative genomics of peroxisome biogenesis proteins: making sense of the PEX proteins. Front. Cell Dev. Biol. 9, 654163 (2021).
Yofe, I. et al. One library to make them all: streamlining the creation of yeast libraries via a SWAp-Tag strategy. Nat. Methods 13, 371–378 (2016).
Sherman, F. Getting started with yeast. Methods Enzymol. 194, 3–21 (1991).
Shaw, W. M. et al. Engineering a model cell for rational tuning of GPCR signaling. Cell 177, 782–796 (2019).
Davis, N. G., Horecka, J. L. & Sprague, G. F. Jr. Cis- and trans-acting functions required for endocytosis of the yeast pheromone receptors. J. Cell Biol. 122, 53–65 (1993).
Mascle, X. H. et al. Acetylation of SUMO1 alters interactions with the SIMs of PML and Daxx in a protein-specific manner. Structure 28 157–168 (2020).
Ceballos, A. V., McDonald, C. J. & Elbaum-Garfinkle, S. Methods and strategies to quantify phase separation of disordered proteins. Methods Enzymol. 611, 31–50 (2018).
Schindelin, J. et al. Fiji: an open-source platform for biological-image analysis. Nat. Methods 9, 676–682 (2012).
Kisley L. PeroxisomeCorrelationMain_LK20221006.m Zenodo https://doi.org/10.5281/zenodo.7702794 (2023).
Xavier-Castellanos-Girouard. Xavier-Castellanos-Girouard/Peroxisome-biogenesis-initiated-by-protein-phase-separation: Droplet_Fusion v0.1. Zenodo https://doi.org/10.5281/zenodo.7702890 (2023).
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
Authors and Affiliations
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
Ethics declarations
Competing interests
The authors declare no competing interests.
Peer review
Peer review information
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.
Additional information
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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.
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.
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 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.
Supplementary information
Supplementary Fig. 1
Uncropped immunoblots for Fig. 2b.
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.
Rights and permissions
Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.
About this article
Cite this article
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
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/s41586-023-06044-1
This article is cited by
-
Modulation of peroxisomal import by the PEX13 SH3 domain and a proximal FxxxF binding motif
Nature Communications (2024)
-
The peroxisome: an update on mysteries 3.0
Histochemistry and Cell Biology (2024)
Comments
By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.