Specificity of the ubiquitin isopeptidase in the PA700 regulatory complex of 26 S proteasomes

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 Lys(48)-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 Leu(8) or Ile(44) 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 H-1 NMR spectroscopy. This and the finding that di-Ub with a single proximal Ub(L8A,I44A) is cleaved efficiently suggest that Leu(8) and Ile(44) in the distal-end Ub contact the isopeptidase directly. In addition to Lys(48)-linked chains, PA700 also could disassemble Lys(6)- 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 Leu(8), Ile(44), and Lys(48) on the distal Ub and, for poly-Ub, some features of the Ub-Ub linkage itself.