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.

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...

Background: PEX5 binds newly synthesized peroxisomal proteins in the cytosol and releases them in the organelle matrix. Results: PEX5 binds monomeric catalase and releases it in the presence of PEX14. Conclusion: PEX14 participates in the cargo release step. Significance: Knowing how PEX5 interacts with cargo proteins and which factors disrupt this interaction are crucial for understanding this protein sorting pathway.

Background:The mammalian deubiquitinase that hydrolyzes the ubiquitin-PEX5 thioester conjugate was unknown. Results: USP9X was found to be the most active deubiquitinase acting on ubiquitin-PEX5. Conclusion:We propose that USP9X participates in the PEX5-mediated peroxisomal protein import pathway. Significance: The unbiased biochemical strategy described here will be useful to identify deubiquitinases acting on other substrates.