Protein translocation across the peroxisomal membrane requires the concerted action of numerous peroxins. One central component of this machinery is Pex5p, the cycling receptor for matrix proteins. Pex5p recognizes newly synthesized proteins in the cytosol and promotes their translocation across the peroxisomal membrane. After this translocation step, Pex5p is recycled back into the cytosol to start a new protein transport cycle. Here, we show that mammalian Pex5p is ubiquitinated at the peroxisomal membrane. Two different types of ubiquitination were detected, one of which is thiol-sensitive, involves Cys 11 of Pex5p, and is necessary for the export of the receptor back into the cytosol. Together with mechanistic data recently described for yeast Pex5p, these findings provide strong evidence for the existence of Pex4p-and Pex22p-like proteins in mammals.

The peroxisome represents a ubiquitous single membrane-bound key organelle that executes various metabolic pathways such as fatty acid degradation by ␣-and ␤-oxidation, ether-phospholipid biosynthesis, metabolism of reactive oxygen species, and detoxification of glyoxylate in mammals. To fulfil this vast array of metabolic functions, peroxisomes accommodate ϳ50 different enzymes at least as identified until now. Interest in peroxisomes has been fueled by the discovery of a group of genetic diseases in humans, which are caused by either a defect in peroxisome biogenesis or the deficient activity of a distinct peroxisomal enzyme or transporter. Although this research has greatly improved our understanding of peroxisomes and their role in mammalian metabolism, deeper insight into biochemistry and functions of peroxisomes is required to expand our knowledge of this low abundance but vital organelle. In this work, we used classical subcellular fractionation in combination with MS-based proteomics methodologies to characterize the proteome of mouse kidney peroxisomes. We could identify virtually all known components involved in peroxisomal metabolism and biogenesis. Moreover through protein localization studies by using a quantitative MS screen combined with statistical analyses, we identified 15 new peroxisomal candidates. Of these, we further investigated five candidates by immunocytochemistry, which confirmed their localization in peroxisomes. As a result of this joint effort, we believe to have compiled the so far most comprehensive protein catalogue of mammalian peroxisomes.

Newly synthesized peroxisomal matrix proteins are targeted to the organelle by PEX5, the peroxisomal cycling receptor. Over the last few years, valuable data on the mechanism of this process have been obtained using a PEX5-centered in vitro system. The data gathered until now suggest that cytosolic PEX5⅐ cargo protein complexes dock at the peroxisomal docking/ translocation machinery, where PEX5 becomes subsequently inserted in an ATP-independent manner. This PEX5 species is then monoubiquitinated at a conserved cysteine residue, a mandatory modification for the next step of the pathway, the ATPdependent dislocation of the ubiquitin-PEX5 conjugate back into the cytosol. Finally, the ubiquitin moiety is removed, yielding free PEX5. Despite its usefulness, there are many unsolved mechanistic aspects that cannot be addressed with this in vitro system and that call for a cargo protein-centered perspective instead. Here we describe a robust peroxisomal in vitro import system that provides this perspective. The data obtained with it suggest that translocation of a cargo protein across the peroxisomal membrane, including its release into the organelle matrix, occurs prior to PEX5 ubiquitination.Peroxisomal matrix proteins are synthesized in cytosolic ribosomes and post-translationally targeted to the organelle (1, 2). The vast majority of proteins destined to this compartment possess the so-called peroxisomal targeting sequence type 1 (PTS1), 3 a short domain present at their extreme C termini and frequently ending with the sequence SKL (3, 4). A small number of matrix proteins lack this domain and contain instead a PTS2, a degenerated nonapeptide with the sequence (R/K)(L/V/I)X 5 (H/Q)(L/A) present at their N termini (5, 6). In contrast to the PTS1, which is not cleaved upon peroxisomal import, the PTS2 signal is proteolytically removed in the peroxisomal matrix of many organisms by a peroxisomal processing peptidase (1,7,8).In mammals and many other organisms, both PTS1-containing and PTS2-containing proteins are targeted to the organelle by PEX5, the peroxisomal cycling receptor (9 -12). PTS1 proteins interact directly with the C-terminal half of PEX5, a region comprising seven tetratricopeptide repeats arranged into a ring-like structure, whereas the PEX5-PTS2 interaction is bridged by the adaptor protein PEX7 (13-19). This adaptor protein interacts with a small region within the largely unfolded N-terminal half of PEX5 (17,18,20). Interestingly, not all proteins derived from the mammalian PEX5 gene have the capacity to bind PEX7. This is due to alternative splicing of the PEX5 transcript yielding two major mRNAs, one encoding the so-called large isoform of PEX5 (PEX5L) and the other coding for the small PEX5 isoform (PEX5S). PEX5S lacks a 37-amino-acid region that is involved in the PEX7 interaction, and so it is incompetent in the peroxisomal targeting of PTS2 proteins (16 -18).In recent years, valuable data on the mechanistic details of the PEX5-mediated protein import pathway in mammals have been obtained using...

According to current models of peroxisomal biogenesis, newly synthesized peroxisomal matrix proteins are transported into the organelle by Pex5p. Pex5p recognizes these proteins in the cytosol, mediates their membrane translocation, and is exported back into the cytosol in an ATP-dependent manner. We have previously shown that export of Pex5p is preceded by (and requires) monoubiquitination of a conserved cysteine residue present at its N terminus. In yeasts, and probably also in plants, ubiquitination of Pex5p is mediated by a specialized ubiquitin-conjugating enzyme, Pex4p. In mammals, the identity of this enzyme has remained unknown for many years. Here, we provide evidence suggesting that E2D1/2/3 (UbcH5a/b/c) are the mammalian functional counterparts of yeast/plant Pex4p. The mechanistic implications of these findings are discussed.

Pex5p, the peroxisomal protein cycling receptor, binds newly synthesized peroxisomal matrix proteins in the cytosol and promotes their translocation across the organelle membrane. During its transient passage through the membrane, Pex5p is monoubiquitinated at a conserved cysteine residue, a requisite for its subsequent ATP-dependent export back into the cytosol. Here we describe the properties of the soluble and membrane-bound monoubiquitinated Pex5p species (Ub-Pex5p). Our data suggest that 1) Ub-Pex5p is deubiquitinated by a combination of context-dependent enzymatic and nonenzymatic mechanisms; 2) soluble Ub-Pex5p retains the capacity to interact with the peroxisomal import machinery in a cargo-dependent manner; and 3) substitution of the conserved cysteine residue of Pex5p by a lysine results in a quite functional protein both in vitro and in vivo. Additionally, we show that MG132, a proteasome inhibitor, blocks the import of a peroxisomal reporter protein in vivo.Since the discovery of the ubiquitin-conjugating cascade nearly 30 years ago, thousands of proteins have been shown to be modified by ubiquitin (1, 2). In many cases ubiquitination of a protein is linked to its proteasomal degradation (3), whereas in a growing number of examples, ubiquitination of a protein is used as a transient modification to modulate its biological properties (for a review see Ref. 4). Regardless of the final outcome, it is generally assumed and in many cases demonstrated that ubiquitin is covalently attached through an amide bond involving the carboxyl group of the last glycine of ubiquitin on one hand, and an amino group of the targeted protein on the other (5). Recent findings from several laboratories, however, suggest that this rule is not always valid, and proteins ubiquitinated at serines and threonines (yielding oxyesters) or even cysteines (forming thiol esters) have been identified (6 -10).Protein ubiquitination at cysteine residues is a particularly puzzling phenomenon for two reasons. First, on a thermodynamic basis it is the least favorable event (the approximate free energy changes for acyl shifts from a thiol ester to a thiol, alcohol, and amine are 0, Ϫ2.4, and Ϫ11 kcal/mol, respectively (11, 12)). Second, although data on the half-lives of ubiquitin-protein thiol ester conjugates under physiologically relevant conditions are scarce, it is known that ubiquitin thiol esters are easily disrupted by nucleophiles such as GSH (13), raising the possibility that, to some degree, proteins subjected to this kind of conjugation may undergo futile ubiquitination/deubiquitination cycles. Thus, a thiol ester bond appears not to be the most efficient way to link ubiquitin to a protein, unless, of course, the aim is to create an activated (easily transferable) form of ubiquitin, as is in fact the case with ubiquitin-activating enzymes (E1s), 4 ubiquitin-conjugating enzymes (E2s), and some ubiquitin ligases (E3s) (2).In the last years we have been characterizing Pex5p, one of the three presently known proteins claimed to be ub...