Peroxisome biogenesis initiated by protein phase separation

Peroxisomes are organelles that perform beta-oxidation of fatty acids and amino acids. Both rare and prevalent diseases are caused by their disfunction1. Among disease-causing mutant genes are those required for protein transport into the peroxisome. The peroxisomal protein import machinery, also shared with chloroplasts, is unique in transporting folded and large, up to 10 nm in diameter, protein complexes into peroxisomes2 and current models postulate a large pore formed by transmembrane proteins3. To date, however, no pore structure has been observed. In the budding yeast Saccharomyces cerevisiae, the minimum transport machinery includes membrane proteins Pex13 and Pex14 and cargo protein-binding transport receptor, Pex5. Here we show that Pex13 undergoes liquid-liquid phase separation (LLPS) with Pex5-cargo. Intrinsically disordered regions (IDR) in Pex13 and Pex5 resemble those found in nuclear pore complex (NPC) proteins. Cargo transport into peroxisomes depends on the number but not patterns of aromatic residues in these IDRs, consistent with their roles as stickers in associative polymer models of LLPS4,5. Finally, imaging Fluorescence Cross-Correlation Spectroscopy (iFCCS) shows that the transport of cargo correlates with transient focusing of GFP-Pex13/14 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 results suggest a model in which LLPS of Pex5-cargo with Pex13/14 results in transient protein transport channels.