We have identified human PEX16, and established that an inactivating mutation in this gene abolishes the synthesis of peroxisomes in a Zellweger syndrome patient, PBD061. These results expand the range of phenotypes and genotypes that are associated with Zellweger syndrome, and thus, expand our understanding of this lethal disease. However, this study also enhances our understanding of the molecular events involved in formation of peroxisome membranes. Most importantly, the rescue of peroxisome synthesis in PBD061 cells by PEX16 expression demonstrates that preexisting peroxisomes, defined here as PMP-containing vesicles, are not required for the formation of new peroxisomes. While the elucidation of PEX16 function clearly requires additional studies our results do provide some insight into the biogenesis of peroxisome membranes, at least in humans.
Perhaps the simplest model for peroxisome synthesis in rescued PBD061 cells would be that PEX16 expression allows the synthesis of peroxisomes from phospholipids and PMPs rather than preexisting vesicles. However, this appears unlikely since there is no precedent for the synthesis of biological membranes other than from a preexisting lipid bilayer. An alternative hypothesis is that PEX16, either alone or in conjunction with other proteins, converts a preperoxisomal vesicle to recognizable peroxisomes by mediating the import of other PMPs. This in turn would lead to assembly of the matrix protein import apparatus, and the subsequent import of peroxisomal matrix proteins, completing the formation of mature peroxisomes.
Although this may explain the formation of peroxisomes during the rescue of PBD061 cells it is questionable whether such a simple model can explain peroxisome synthesis under all conditions. For instance, if peroxisomes were typically formed by such a process we would expect that the biogenesis of peroxisomes in PBD061 cells would occur on roughly the same time course as the import of PEX16 into peroxisomes of normal cells. However, this is clearly not the case as PEX16 is imported into virtually all peroxisomes of normal cells in <2 h whereas the reconstitution of normal peroxisomes in PBD061 cells requires 18–24 h. Furthermore, this model fails to incorporate roles for the proliferation factors PEX11α and PEX11β (Schrader et al., 1998
) in the formation of peroxisomes.
These and other lines of evidence suggested instead that peroxisome formation may occur by two distinct, yet overlapping pathways (Fig. ). In this model, peroxisomes would typically form by division of preexisting peroxisomes in a process mediated by PEX11α and PEX11β, not PEX16. This would explain the hyperabundance of peroxisomes in cells overexpressing PEX11α or PEX11β, and the fact that overexpression of PEX16 does not induce any detectable proliferation of peroxisomes. In this model, PEX16 would function normally in importing PMPs into preexisting peroxisomes, and would itself be imported into preexisting peroxisomes. In the absence of PEX16, PMP import would cease, peroxisomes could not divide, and the organelle would eventually be lost from the cell population. In addition to this common route of peroxisome formation we propose that PEX16 may, at low efficiency, be targeted to an unknown preperoxisomal structure. The incorporation of PEX16 into such vesicles may convert them to nascent peroxisomes that are able to import other PMPs, and eventually to mature peroxisomes. This model would not only explain the ability of PEX16 to rescue PBD061 cells but would also explain: the presence of a few, large peroxisomes in mutants lacking the PEX11
gene (Erdmann and Blobel, 1995
; Marshall et al., 1995
); the peroxisome proliferation promoting activity of PEX11 proteins; and the lack of peroxisome proliferation promoting activity for PEX16.
Figure 11 A model of peroxisome biogenesis in the absence (top) and presence (bottom) of preexisting peroxisomes. During rescue of PBD061 cells, PEX16 creates nascent peroxisomes, possibly from a preperoxisomal vesicle. These nascent PEX16-containing peroxisomes (more ...)
This model is consistent with virtually all available data on the formation of peroxisomes. However, it proposes the existence of a structure that has yet to be identified, the preperoxisome, and proposes an activity for PEX16 for which there is no direct evidence, PMP import. Nevertheless, this model serves as a useful starting point for considering how peroxisomes are formed. The preperoxisomal structure need not be a dedicated structure awaiting PEX16 for conversion into a nascent peroxisome, only a subcellular vesicle that happens to incorporate a PEX16 molecule. As for the function of PEX16, our only evidence that this protein is involved in PMP import is the inability of PBD061 cells to import PMPs into peroxisomes, a phenotype that is also consistent with a defect in the synthesis of the preperoxisomal structure of our model. Nevertheless, PEX16 has the appropriate structural requirements for a role in PMP import. PEX16 is itself an integral PMP and extends its NH2
and COOH termini into the cytoplasm where they may facilitate the recognition of PMPs and/or their insertion into the peroxisome membrane. In addition, PEX16 appears to be transported efficiently to peroxisomes, and is detected only in peroxisomes, not some other compartment, at steady state. It should be noted that similar models have been proposed that implicate the ER as the source of the preperoxisomal structure (Kunau and Erdmann, 1998
; Titorenko and Rachubinski, 1998a
). However, our data do not provide evidence for a role of the ER in peroxisome biogenesis.
In addition to improving our understanding of peroxisome synthesis and the molecular basis of Zellweger syndrome, this report also makes clear that homologues of the same peroxisome biogenesis factor may have disparate roles in different organisms. Although we identified human PEX16 by its sequence similarity to Y. lipolytica
PEX16, there were significant differences between these proteins. Prior reports have established that YlPEX16 is peripherally associated with the inner face of the peroxisome membrane, and transported to peroxisomes via the ER in a PEX1
- and PEX6
-dependent manner. Despite it playing a role in the import of a subset of peroxisomal matrix proteins, YlPEX16 is not required for synthesis of peroxisome membranes nor PMP import (Eitzen et al., 1997
; Titorenko et al., 1997
; Titorenko and Rachubinski, 1998b
). In contrast, human PEX16, an integral PMP with its NH2
and COOH termini exposed to the cytoplasm, is required for PMP import and peroxisome synthesis, and appears to traffic from the cytoplasm to the peroxisome in a PEX1
-independent manner. These differences emphasize the need for caution in extrapolating the functions of a particular peroxisome biogenesis factor to its homologue in divergent species. Discretion in this matter is emphasized further by the fact that the Saccharomyces cerevisiae
genome lacks a PEX16 homologue, even though the YlPEX16 and human PEX16 proteins share 24% amino acid identity.