The Peroxisome Biogenesis Factors Pex4p, Pex22p, Pex1p, and Pex6p Act in the Terminal Steps of Peroxisomal Matrix Protein Import

To form a functional peroxisome, peroxisome membranes must be generated and the subsequent import of both membrane and matrix proteins must occur. Various studies have established that both peroxisomal membrane proteins (PMPs) and peroxisomal matrix proteins are made in the cytosol and delivered posttranslationally to the peroxisome (26). However, the similarities between PMP import and peroxisomal matrix protein import appear to end there. Peroxisomal matrix proteins are targeted to the peroxisome lumen via the presence of either a PTS1 or a PTS2 targeting signal (40). The PTS1 signal, which consists of a -SKL sequence (or a conserved variant thereof) at the extreme carboxy terminus of the protein (16), is present on the vast majority of peroxisomal matrix proteins. The PTS2 signal consists of the sequence R/K-L/V/I-X5-H/Q-L/A near the amino terminus (41) and has so far been identified in only a handful of proteins. Both types of signals are recognized by specific receptors: Pex5p for the PTS1 signal (6, 29) and Pex7p for the PTS2 signal (27, 31). After binding to the receptor, matrix proteins are taken to the peroxisome surface and inserted into the organelle lumen through an as yet unknown mechanism. For Pex5p, it has been proposed that the receptor is then recycled back to the cytosol and can undergo further rounds of import (7). In contrast, PMPs lack PTS1 and PTS2 motifs (40), are imported independently of Pex5p and Pex7p (2), and require a distinct PMP-binding protein, Pex19p, for their import (19, 28, 34). In addition to Pex5p, Pex7p, and Pex19p, a variety of other proteins required for peroxisome biogenesis have been identified (19). These include peroxins necessary for the biogenesis of peroxisome membranes (Pex3p), docking factors for the PTS receptors (Pex13p and Pex14p), proteins that act downstream of receptor docking but are required for translocation of proteins across the peroxisome membrane (Pex8p, Pex10p, and Pex12p), and other proteins whose functions are less clear (Pex1p, Pex2p, Pex4p, Pex6p, Pex17p, and Pex22p). Despite the identification of so many components of peroxisome biogenesis, there is still little direct evidence as to the order in which these peroxins act. In the course of investigating the effects that different peroxins have on Pex5p, the PTS1 receptor, we observed that the loss of Pex1p or Pex6p in either yeast or human cells leads to an accelerated turnover of Pex5p and a significant drop in steady-state levels of Pex5p (7, 49). Koller et al. (25) have recently reported a similar phenotype for Pichia pastoris pex4 and pex22 mutants. Here we show that the phenotype of reduced Pex5p abundance in P. pastoris is unique to selected pex mutants and can be used to determine the epistatic relationships among the different peroxins. The results of our epistasis analysis suggest that Pex4p, Pex22p, Pex1p, and Pex6p act in the terminal steps of peroxisomal matrix protein import. This hypothesis is consistent with the reported phenotypes of P. pastoris pex1, pex6, and pex22 mutants, all of which contain peroxisomes and import detectable levels of peroxisomal matrix proteins. Similarly, we show here that the pex4 mutant also contains peroxisomes that are capable of peroxisomal matrix protein import.