Specificity of the Ubiquitin Isopeptidase in the PA700 Regulatory Complex of 26 S Proteasomes

Y Amy Lam,George N Demartino,Cecile M Pickart,Robert E Cohen

doi:10.1074/jbc.272.45.28438

Abstract

The specificity of the ubiquitin (Ub) isopeptidase in the PA700 regulatory complex of the bovine 26 S proteasome was investigated. Disassembly of poly-Ub by this enzyme is restricted to the distal-end Ub of the substrate, i.e. the Ub farthest from the site of protein attachment in poly-Ub-protein conjugates. The determinants recognized by the isopeptidase were probed by the use of mutant ubiquitins incorporated into Lys48-linked poly-Ub substrates. PA700 could not disassemble poly-Ub chains that contained a distal Ub(L8A,I44A). This suggested either that the enzyme interacts directly with Leu8 or Ile44 or that it recognizes a higher order structure that caps the distal end of a poly-Ub substrate and is destabilized by Ub(L8A,I44A). The previously determined di-Ub crystal structure (Cook, W. J., Jeffrey, L. C., Carson, M., Chen, Z., and Pickart, C. M. (1992) J. Biol. Chem. 267, 16467-16471) offered a candidate for such a "cap." In solution, however, this structure was not observed by 1H NMR spectroscopy. This and the finding that di-Ub with a single proximal Ub(L8A,I44A) is cleaved efficiently suggest that Leu8 and Ile44 in the distal-end Ub contact the isopeptidase directly. In addition to Lys48-linked chains, PA700 also could disassemble Lys6- and Lys-11-linked poly-Ub, but, surprisingly, not alpha-linked di-Ub. Results with these and other substrates suggest that specificity determinants for the PA700 isopeptidase include Leu8, Ile44, and Lys48 on the distal Ub and, for poly-Ub, some features of the Ub-Ub linkage itself.

Highlights

In ubiquitin (Ub)1-mediated proteolysis, the attachment of Ub marks a protein for ATP-dependent degradation by the 26 S proteasome complex
Ub Residues Leu8 and Ile44 Are Involved in Substrate Recognition by the PA700 Isopeptidase—We have examined whether Leu8 and Ile44, residues on Ub that affect the targeting of conjugates to the 26 S proteasome [21], are required for recognition by the Ub isopeptidase in the PA700 regulatory complex of the 26 S particle
By the incorporation of Ub(L8A,I44A) into the distal position of fluorescent Lucifer Yellow (LY)-labeled poly-Ub chains, we developed substrates to test whether Leu8 or Ile44 is involved in processing by the PA700 isopeptidase

Summary

The abbreviations used are

Ubiquitin; Ubal, ubiquitin aldehyde; Ubdiol, ubiquitin C-terminal diol; E1, Ub-activating enzyme; E2, ubiquitin carrier protein; Aec, S-aminoethyl-L-cysteine; HPLC, high pressure liquid chromatography; LY, Lucifer Yellow; HyTEMPO, 4-hydroxy-2,2,6,6-tetramethylpiperidinyl-1-oxy; DQF-COSY, doublequantum filtered correlated spectroscopy. Many molecules of ubiquitin can be conjugated to a single protein target [3], and a distinction can be made between substrates with multiple lysines that are ubiquitinated and those in which multiple ubiquitins are elaborated from a single lysine via secondary Ub–Ub isopeptide linkages [4] This latter “poly-Ub” structure has been implicated as an essential determinant for efficient recognition by the 26 S proteasome of many [4, 5], but perhaps not all [3, 6, 7], Ub-dependent degradation substrates. This paper is available on line at http://www.jbc.org editing isopeptidase has been found within the PA700 (19 S) regulatory complex of bovine 26 S proteasomes [20] This enzyme, which is a tightly bound, stoichiometric component of the PA700 complex, was found to promote the selective rescue of poorly ubiquitinated proteins from degradation by reconstituted 26 S proteasomes in vitro. We examined the ability of the PA700 isopeptidase to cleave ␣-linked di-Ub and poly-Ub conjugates that contain isopeptide linkages through Lys or Lys

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION