A cryptic protease couples deubiquitination and degradation by the proteasome

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Abstract

The 26S proteasome is responsible for most intracellular proteolysis in eukaryotes1,2. Efficient substrate recognition relies on conjugation of substrates with multiple ubiquitin molecules and recognition of the polyubiquitin moiety by the 19S regulatory complex—a multisubunit assembly that is bound to either end of the cylindrical 20S proteasome core. Only unfolded proteins can pass through narrow axial channels into the central proteolytic chamber of the 20S core, so the attached polyubiquitin chain must be released to allow full translocation of the substrate polypeptide. Whereas unfolding is rate-limiting for the degradation of some substrates and appears to involve chaperone-like activities associated with the proteasome3,4,5, the importance and mechanism of degradation-associated deubiquitination has remained unclear. Here we report that the POH1 (also known as Rpn11 in yeast) subunit of the 19S complex is responsible for substrate deubiquitination during proteasomal degradation. The inability to remove ubiquitin can be rate-limiting for degradation in vitro and is lethal to yeast. Unlike all other known deubiquitinating enzymes (DUBs) that are cysteine proteases6,7, POH1 appears to be a Zn2+-dependent protease.

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Figure 1: Proteasomal degradation of UbOM is targeted by means of the unfolded ovomucoid moiety.
Figure 2: A ubiquitin-aldehyde (Ubal)-insensitive DUB couples release of ubiquitin with substrate degradation (western blots).
Figure 3: Alignment of the MPN domain of MPN family proteins.
Figure 4: Evidence that Rpn11/POH1 is a Zn2+-dependent DUB and characterization of rpn11 mutants.

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Acknowledgements

We thank D. Finley and D. Leggett for yeast strains, advice on proteasome purification, and a sample of yeast proteasomes; G. DeMartino for bovine PA700; A. Robertson for ovomucoid protein; C. Pickart for the Ub(V76)DHFR plasmid; K. Wilkinson, H. Ploegh and A. Borodovsky for UbVS, recombinant Ubp6 protein and anti-human Ubp6; C. Pickart, A. Lam and K. Wilkinson for discussions; and L. Weisman and C. Pickart for comments on the manuscript. This work was supported by a grant from the NIH and a UI Biocatalysis and Bioprocessing Fellowship to T.Y.

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Correspondence to Robert E. Cohen.

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Yao, T., Cohen, R. A cryptic protease couples deubiquitination and degradation by the proteasome. Nature 419, 403–407 (2002) doi:10.1038/nature01071

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