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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 pex4Delta mutant cells contain peroxisomal membrane protein-containing peroxisomes that import residual amounts of peroxisomal matrix proteins.

Fluorescent peroxisomal probes were developed by fusing green fluorescent protein (GFP) to the matrix peroxisomal targeting signals PTS1 and PTS2, as well as to an integral peroxisomal membrane protein (IPMP). These proteins were used to identify and characterize novel peroxisome assembly (pas) mutants in the yeast Pichia pastoris. Mutant cells lacking the PAS10 gene mislocalized both PTS1-GFP and PTS2-GFP to the cytoplasm but did incorporate IPMP-GFP into peroxisome membranes. Similar distributions were observed for endogenous peroxisomal matrix and membrane proteins. While peroxisomes from translocation-competent pas mutants sediment in sucrose gradients at the density of normal peroxisomes, >98% of peroxisomes from pas10 cells migrated to a much lower density and had an extremely low ratio of matrix:membrane protein. These data indicate that Pas10p plays an important role in protein translocation across the peroxisome membrane. Consistent with this hypothesis, we find that Pas10p is an integral protein of the peroxisome membrane. In addition, Pas10p contains a cytoplasmically-oriented C3HC4 zinc binding domain that is essential for its biological activity.