Peroxisomal Protein Import: The Paradigm Shifts

Dammai and Subramani (2001)xDammai, V. and Subramani, S. Cell. 2001; 105: 187–196Abstract | Full Text | Full Text PDF | PubMed | Scopus (163)See all ReferencesDammai and Subramani (2001) have tested the two models of Pex5p dynamics during peroxisomal protein import. They present convincing evidence that Pex5p uses the mechanism predicted by the extended shuttle model during targeting of matrix proteins. Cleverly designed fusion proteins expressed in HeLa and peroxisome import-deficient human cell lines allowed the authors to distinguish between unimported Pex5p fusion protein that remained in the cytoplasm, Pex5p that was imported into and remained in the peroxisome matrix, and Pex5p that had been imported into the matrix and subsequently exported back into the cytosol. The fusion protein consisted of Pex5p linked to an N-terminal PTS2 signal followed in tandem by a FLAG epitope. A prethiolase processing site (PPS) that is cleaved by a specific peroxisomal matrix protease separated the PTS2 signal and FLAG epitope. Cleavage at PPS upon entry into the peroxisomal matrix resulted in a detectable molecular weight shift and the exposure of the N-terminal FLAG epitope. M1 and M2 monoclonal antibodies capable of distinguishing N-terminal from internal FLAG epitopes, respectively, provided an independent means of assaying PPS processing. The location and import status of the Pex5p fusion proteins was determined using a combination of subcellular fractionation, reactivity to the FLAG epitope-specific antibodies, and the molecular weight of the fusion proteins.In transfected cell lines expressing the PTS2-FLAG-Pex5p fusion, the majority of the processed, M1-reactive fusion protein (FLAG-Pex5p) was cytoplasmic. This finding indicated that Pex5p was imported into the matrix and efficiently exported back to the cytoplasm where it presumably is made available for further rounds of import. A PTS2-FLAG fusion in which Pex5p was replaced with GFP was processed and localized exclusively in peroxisomes, providing evidence that cycling was specific for Pex5p. Pex5p shuttling was unlikely to be an artifact of the PTS2-FLAG-Pex5p construct because the fusion was able to complement a Pex5p-defective mutant cell line, indicating that the receptor fusion was fully functional in vivo. Protease protection assays and immunofluorescence microscopy using differential detergent extractions demonstrated that the majority of imported FLAG-Pex5p that remained associated with the peroxisome was indeed localized to the matrix although some remained associated with the peroxisome surface. The presence of surface bound FLAG-Pex5p suggested that the receptor was docked at the Pex13p, 14p, 17p complex, poised to undergo further rounds of targeting.Finally, the authors used pulse-chase experiments to explore the kinetics of shuttling. They were able to demonstrate by quantitative analysis that both newly synthesized PTS2-FLAG-Pex5p as well as cytosolic FLAG-Pex5p contributed to the accumulation of FLAG-Pex5p in the matrix. Therefore, the processed FLAG-Pex5p was capable of reentering the peroxisome following a cycle of import and export.It remains to be demonstrated that the shuttling of the Pex5p receptor is a function of a productive cycle of cargo delivery from the cytoplasm to the matrix. Nevertheless, this study provides convincing evidence to support the extended shuttling model for protein import in peroxisomes, and raises a number of important questions concerning the molecular mechanism of protein trafficking to this organelle. First, what regulates binding and dissociation of cargo from the receptor to provide directionality of import? Signal cleavage would provide a convenient mechanism for dissociation of cargo from the receptor, but the PTS1 signal is not cleaved upon import. Therefore, there is likely to be another active mechanism for receptor dissociation in the matrix. Receptor recycling at the surface of the ER and chloroplasts is regulated by cycles of GTP binding and hydrolysis at the SRP (Song et al., 2000xSong, W., Raden, D., Mandon, E.C., and Gilmore, R. Cell. 2000; 100: 333–343Abstract | Full Text | Full Text PDF | PubMedSee all References(Song et al., 2000) and the Toc159-Toc34 receptors (Chen et al., 2000xBiochem. Chen, K., Chen, X., and Schnell, D.J. Soc. Trans. 2000; 28: 485–491PubMedSee all References(Chen et al., 2000), respectively. A GTP cycle also drives the directionality of protein trafficking in and out of the nucleus via association of the importin/karyopherin receptors with the Ran GTPase (Gorlich and Kutay, 1999xGorlich, D. and Kutay, U. Annu. Rev. Cell Dev. Biol. 1999; 15: 607–660Crossref | PubMed | Scopus (1398)See all References(Gorlich and Kutay, 1999). ATP hydrolysis is required for the import of peroxisomal matrix proteins, providing the potential for a nucleotide-hydrolysis-driven import cycle. Two peroxins, Pex1p and Pex6p, are ATPases, but their direct or indirect association with Pex5p has not been established, and their precise roles in import are unknown. The ability of the Pex5p receptor to shuttle also implies that an active protein export system functions at the peroxisomal membrane. Does the translocation apparatus act as a passive pore allowing translocation in both directions as do the Sec61p complex of the ER and the nuclear pore complex (NPC) of the nuclear envelope? And if so, what is the signal on Pex5p for export?Perhaps the most intriguing mystery of the peroxisomal import system is the nature of the protein translocon itself. Although the extended shuttling model resembles the model proposed for nucleocytoplasmic transport, there is one significant difference between the two organelles. The nucleus is not a sealed compartment; NPCs contain 9 nm diameter pores that allow the free diffusion of molecules with sizes less than 40 kDa across the nuclear envelope (Figure 1C(Figure 1C). Therefore, as the NPC expands to accommodate cargo as large as ribosomal subunits, there is no threat of compromising the integrity of the organelle. In contrast, peroxisomes are more similar to mitochondria, chloroplasts, and organelles of the endomembrane system in that they are sealed compartments that regulate the transport of metabolites and ions across a semipermeable boundary membrane (Van der Klei and Veenhuis, 1997xVan der Klei, I.J. and Veenhuis, M. Trends Microbiol. 1997; 5: 502–509Abstract | Full Text PDF | PubMed | Scopus (42)See all References(Van der Klei and Veenhuis, 1997). How can the diameter of the peroxisome translocon adapt to accommodate receptor-cargo complexes containing Pex5p associated with folded and oligomeric proteins (Figure 1B(Figure 1B)? There is no clear precedent for a similar translocon, but one possibility is that the translocon assembles at the site of translocation in response to the docking of the receptor-cargo complex. This would allow the formation of protein-conducting channels of a variety of sizes in response to the size of the transport substrate. Disassembly of the translocon immediately following membrane translocation would minimize the free diffusion of molecules across the channel, thereby maintaining the critical permeability barrier of the organelle. Other models for protein translocation involving vesicle transport via a system resembling modified endocytosis of the peroxisomal membrane have been proposed (McNew and Goodman, 1994xMcNew, J.A. and Goodman, J.M. J. Cell Biol. 1994; 127: 1245–1257Crossref | PubMed | Scopus (232)See all References(McNew and Goodman, 1994), but there is little evidence to support such a transport mechanism. The adaptability of the peroxisome translocon might account for the numerous peroxins involved in the import of matrix proteins, a number far exceeding the number of proteins required by other translocation systems with the exception of nucleocytoplasmic transport. Many of the peroxins have been shown to interact directly or indirectly by two-hybrid and coimmunoprecipitation assays, consistent with a complex network of protein-protein interactions within the translocon.It should be noted that the peroxisomal membrane is not alone in possessing a protein translocation system that accommodates folded proteins. The Tat pathway operating at bacterial plasma membranes (Berks et al., 2000xBerks, B.C., Sargent, F., and Palmer, T. Mol. Microbiol. 2000; 35: 260–274Crossref | PubMed | Scopus (407)See all References(Berks et al., 2000) and the homologous ΔpH pathway operating at the chloroplast thylakoid membrane (Keegstra and Cline, 1999xKeegstra, K. and Cline, K. Plant Cell. 1999; 11: 557–570PubMedSee all References(Keegstra and Cline, 1999) are both involved in the translocation of proteins that fold and associate with redox cofactors prior to membrane translocation (Figure 1B(Figure 1B). The nature of these translocons is not understood, but their existence in conjunction with the evidence from peroxisomes indicates that protein unfolding no longer stands as a prerequisite for membrane translocation. To date, the analysis of peroxisomal protein import has relied heavily on genetic analysis. The recent development of in vitro systems that reproduce peroxisomal protein import (Terlecky et al., 2001xTerlecky, S.R., Legakis, J.E., Hueni, S.E., and Subramani, S. Exp. Cell Res. 2001; 263: 98–106Crossref | PubMed | Scopus (21)See all References(Terlecky et al., 2001) provides the necessary tools for a detailed biochemical analysis of the nature and dynamics of this novel protein translocation system.