PEX14 condensates recruit receptor and cargo pairs for peroxisomal protein import.

Peroxisomal proteins are imported into peroxisomes as folded proteins bound to the receptor peroxisomal biogenesis factor 5 (PEX5) through a biomolecular condensate mainly formed by the tyrosine-glycine (YG) repeats in PEX13. PEX14, another essential component of the translocon complex, contributes to this process by interacting with PEX5 and PEX13 through its N-terminal domain. Clinical data suggest that the human PEX14 (hPEX14) C-terminal domain (CTD) is crucial for peroxisomal protein import. Here we analyze the overall structure of the hPEX14 tetramer and demonstrate that hPEX14 CTD undergoes phase separation in vitro. Replacing hPEX14 CTD with other polypeptides capable of forming condensates partially restores peroxisomal protein import. We found that electrostatic interactions and the specific sequence of the CTD are essential for peroxisomal import. hPEX14 and hPEX13 form immiscible condensates and hPEX14 condensates recruit cargoes containing peroxisome-targeting signal 1 (PTS1) or PTS2 in a PEX5-dependent manner. Overall, our study proposes that PEX14 condensates recruit the receptor-cargo complexes for subsequent partitioning into the PEX13 YG phase.