In a recent study we established that there are at least two mechanistically distinct pathways of PMP import, a class I pathway that is dependent upon PEX19 and a class II pathway that is independent of PEX19 (Jones et al., 2004
). Here we observed that PEX3 is essential for PEX19 docking at peroxisomes and sufficient to dock PEX19 at heterologous membranes. The PEX3-binding domain of PEX19 is sufficient for docking PEX19 at the peroxisome surface, but only in the presence of PEX3 on the peroxisome surface. Disruption of the PEX19-binding site on PEX3 disrupts its ability to recruit PEX19 to membranes and eliminates PEX3 function. Finally, we show that PEX3 is essential for import of class I PMPs but is not required for either class II PMP import or peroxisomal matrix protein import. These observations support the hypothesis that PEX3 plays an essential and selective role in the import of class I PMPs and that this role includes being a docking factor for PEX19, the class I PMP import receptor. It is also consistent with the recent observation that PEX3 and PEX19 interact primarily at the surface of the peroxisome membrane (Muntau et al., 2003
The most widely accepted model of peroxisome biogenesis is that peroxisomes are formed by the growth and division of preexisting peroxisomes (Lazarow and Fujiki, 1985
). Under this model, any significant defect in PMP import would be expected to cause the loss of peroxisomes from the cell. This is precisely the phenotype of pex3
- and pex19
-null mutants in both humans and S. cerevisiae
(Matsuzono et al., 1999
; Hettema et al., 2000
; Sacksteder et al., 2000
; South et al., 2000
). Although class I PMP import might require more protein factors than PEX19 and PEX3, it is also possible that it does not. Therefore, we should not exclude additional roles for PEX19 and PEX3 in PMP import. In fact, some studies have detected interactions between class I PMPs and both PEX19 and PEX3 at the peroxisome membrane (Snyder et al., 2000
; Hazra et al., 2002
) and these observations might reflect a role for PEX3 and/or PEX19 in the insertion of class I PMPs.
Neither PEX3 nor PEX19 are required for class II PMP import (Jones et al., 2004
). Of the PMPs that have been tested, PEX3 is the only class II PMP yet identified. Given the essential role for PEX3 in PMP import and the phenotypes associated with the loss of PEX3 activity (loss of peroxisomes from the cell, rapid destruction of PMPs, and cytosolic accumulation of matrix enzymes in the cytosol), it is expected that loss of any factor involved in PEX3 import would result in phenotypes that are at least as severe as the pex3
-null mutant. No such mutants are known in yeast but the pex16
mutant of humans does meet these criteria. Future studies that test whether PEX16 acts as a PEX3 import factor, as another class I PMP import factor, or as a critical player in some other aspect of peroxisome membrane biogenesis (lipid import, peroxisome division, etc.) are a high priority. In addition to searching for factors that might mediate PEX3 import, it might be possible that PEX3 does not require any specific protein factors for import into peroxisomes, perhaps autocatalyzing its import into peroxisomes. Interestingly, Haan et al. (2002)
have observed that an engineered epitope appears exposed to the cytosol regardless of where it is inserted in PEX3, and on this basis have argued that PEX3 might not even be an integral PMP, even though it has the biochemical properties of being embedded in the membrane.
The unique targeting pathway for PEX3 and its conserved role in the import of most PMPs can also be interpreted in the context of more speculative models of peroxisome biogenesis. For example, several researchers believe that peroxisomes arise, directly or indirectly, from the ER (Mullen et al., 2001
; Titorenko and Rachubinski, 2001a
; Tabak et al., 2003
). A critical requirement of such models is the existence of a peroxin that is essential for the formation of peroxisome membranes but traffics to peroxisomes by a mechanism that is fundamentally distinct from that used by the vast majority of PMPs. Moreover, Faber et al. (2002)
have claimed that the overexpression of nonfunctional forms of PEX3 in a pex3
-null mutant can stimulate the formation of “preperoxisomal” vesicles from the ER and that these vesicles subsequently mature into fully functional peroxisomes upon the expression of WT PEX3. Our observation that PEX3 is imported differently than most other PMPs might be interpreted by some as indirect support for both an ER-to-peroxisome route for PEX3 biogenesis and a direct role for the ER in the genesis of peroxisomal vesicles.
However, we do not feel that such a conclusion is warranted at this time, and for several reasons. First, the kinetics of PEX3
-mediated peroxisome synthesis during the complementation of pex3
-null mutants is one to two orders of magnitude slower than the kinetics of PEX3 import into peroxisomes of WT cells (South et al., 2000
). This result means that virtually all PEX3 is imported into preexisting peroxisomes long before it even has the chance to mediate “de novo” peroxisome synthesis. These considerations raise serious doubts as to whether any observations of peroxisome synthesis in the absence of preexisting peroxisomes are physiologically relevant. Second, the one study that has attempted to follow the biogenesis of PEX3 in vivo found no evidence for PEX3 migration through the ER to the peroxisome in WT cells or in cells that lack peroxisomes and instead detected PEX3 first in the peroxisome (South et al., 2000
). Third, PEX3
-mediated peroxisome synthesis occurs independently of the SEC61 and SSH1 protein translocons in the ER membrane, independently of the COPII coat complex, which is essential for vesicular traffic from the ER, and independently of the COPI coat complex, which is required for other vesicle-mediated trafficking events in the early secretory pathway (South et al., 2001
). These concerns and observations, coupled with the unique function of PEX3 in peroxisome biogenesis and its enigmatic route of import, make it more important than ever to resolve the details of PEX3 biogenesis through direct analysis of endogenously expressed PEX3 in normal cells.