Identification of Ubiquitin-specific Protease 9X (USP9X) as a Deubiquitinase Acting on Ubiquitin-Peroxin 5 (PEX5) Thioester Conjugate

Abstract

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.

A Cargo-centered Perspective on the PEX5 Receptor-mediated Peroxisomal Protein Import Pathway

“…Thus, a 10-fold molar excess of AMP-PNP over ATP does not result in an inhibition of SCPx import, although export of monoubiquitinated PEX5 is blocked under these conditions (Fig. 2B), as expected (39).…”

“…AMP-PNP is a potent inhibitor of ATPases cleaving the bond between the ␤-and ␥-phosphate groups of ATP. Note that ubiquitination of PEX5 at the DTM still occurs in the presence of AMP-PNP because the ubiquitin-activating enzyme uses this ATP analog quite efficiently (39,55). However, export of monoubiquitinated PEX5 from the DTM to the cytosol, a process catalyzed by the ATPases PEX1/PEX6, is completely blocked by AMP-PNP (39).…”

“…4A, lanes 7 and 8). A considerable fraction of these proteins is specifically adsorbed to the peroxisome because their amounts in assays containing recombinant ⌬C1PEX5, a PEX5 protein that lacks the PTS1-binding domain but that is still competent in entering the DTM (30,39), are smaller than those observed in the presence of PEX19, a protein involved in another aspect of peroxisome biogenesis (59) and used here as a negative control (Fig. 4B, compare lanes 2 and 4 with 6 and 8, respectively).…”

“…After 7 min at 37°C, the import reactions were centrifuged to obtain an organelle pellet (P) and a supernatant (S), and both fractions were analyzed by SDS-PAGE/Western blotting. Note that AMP-PNP allows PEX5(C11K) ubiquitination but not its export into the soluble phase of the import reaction (39). C, 35 S-labeled PEX5(C11K) was incubated in the absence (Ϫ) or presence (ϩ) of apyrase (lanes 1 and 2, respectively).…”

“…This step is catalyzed by the receptor export module, a protein complex comprising the two ATPases, PEX1 and PEX6, and their membrane anchor, PEX26 (35,36). Finally, monoubiquitinated PEX5 is deubiquitinated probably by a combination of enzymatic and nonenzymatic mechanisms (37)(38)(39). As is evident from the description above, our knowledge of the pathway followed by PEX5 during the protein import process is reasonably detailed.…”