Abstract. Import of newly synthesized PTS1 proteins into the peroxisome requires the PTS1 receptor (Pex5p), a predominantly cytoplasmic protein that cycles between the cytoplasm and peroxisome. We have identified Pex13p, a novel integral peroxisomal membrane from both yeast and humans that binds the PTS1 receptor via a cytoplasmically oriented SH3 domain. Although only a small amount of Pex5p is bound to peroxisomes at steady state (<5%), loss of Pex13p further reduces the amount of peroxisome-associated Pex5p by ~40-fold. Furthermore, loss of Pex13p eliminates import of peroxisomal matrix proteins that contain either the type-1 or type-2 peroxisomal targeting signal but does not affect targeting and insertion of integral peroxisomal membrane proteins. We conclude that Pexl3p functions as a docking factor for the predominantly cytoplasmic PTS1 receptor.

Peroxisomes are independent organelles found in virtually all eukaryotic cells. Genetic studies have identified more than 20 PEX genes that are required for peroxisome biogenesis. The role of most PEX gene products, peroxins, remains to be determined, but a variety of studies have established that Pex5p binds the type 1 peroxisomal targeting signal and is the import receptor for most newly synthesized peroxisomal matrix proteins. The steady-state abundance of Pex5p is unaffected in most pex mutants of the yeast Pichia pastoris but is severely reduced in pex4 and pex22 mutants and moderately reduced in pex1 and pex6 mutants. We used these subphenotypes to determine the epistatic relationships among several groups of pex mutants. Our results demonstrate that Pex4p acts after the peroxisome membrane synthesis factor Pex3p, the Pex5p docking factors Pex13p and Pex14p, the matrix protein import factors Pex8p, Pex10p, and Pex12p, and two other peroxins, Pex2p and Pex17p. Pex22p and the interacting AAA ATPases Pex1p and Pex6p were also found to act after Pex10p. Furthermore, Pex1p and Pex6p were found to act upstream of Pex4p and Pex22p. These results suggest that Pex1p, Pex4p, Pex6p, and Pex22p act late in peroxisomal matrix protein import, after matrix protein translocation. This hypothesis is supported by the phenotypes of the corresponding mutant strains. As has been shown previously for P. pastoris pex1, pex6, and pex22 mutant cells, we show here that pex4⌬ mutant cells contain peroxisomal membrane protein-containing peroxisomes that import residual amounts of peroxisomal matrix proteins.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-X 5 -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 ...

In humans, defects in peroxisome assembly result in the peroxisome biogenesis disorders (PBDs), a group of genetically heterogeneous, lethal recessive diseases. We have identified the human gene PXAAA1 based upon its similarity to PpPAS5, a gene required for peroxisome assembly in the yeast Pichia pastoris. Expression of PXAAA1 restored peroxisomal protein import in fibroblasts from 16 unrelated members of complementation group 4 (CG4) of the PBD. Consistent with this observation, CG4 patients carry mutations in PXAAA1. The product of this gene, Pxaaa1p, belongs to the AAA family of ATPases and appears to be a predominantly cytoplasmic protein. Substitution of an arginine for the conserved lysine residue in the ATPase domain of Pxaaa1p abolished its biological activity, suggesting that Pxaaa1p is an ATPase. Furthermore, Pxaaa1p is required for stability of the predominantly cytoplasmic PTS1 receptor, Pxr1p. We conclude that Pxaaa1p plays a direct role in peroxisomal protein import and is required for PTS1 receptor activity.