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

Research output: Contribution to journalArticle

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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(LSA,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 α- 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.

Original languageEnglish (US)
Pages (from-to)28438-28446
Number of pages9
JournalJournal of Biological Chemistry
Volume272
Issue number45
DOIs
StatePublished - Nov 7 1997

Fingerprint

Proteasome Endopeptidase Complex
Ubiquitin
Polyubiquitin
Substrates
PA700 proteasome activator
ubiquitin isopeptidase
ATP dependent 26S protease
Ubiquitins
Enzymes
Substrate Specificity
Nuclear magnetic resonance spectroscopy
Proteins
Magnetic Resonance Spectroscopy
Crystal structure

ASJC Scopus subject areas

  • Biochemistry

Cite this

Specificity of the ubiquitin isopeptidase in the PA700 regulatory complex of 26 S proteasomes. / Lam, Y. Amy; DeMartino, George N.; Pickart, Cecile M.; Cohen, Robert E.

In: Journal of Biological Chemistry, Vol. 272, No. 45, 07.11.1997, p. 28438-28446.

Research output: Contribution to journalArticle

Lam, Y. Amy ; DeMartino, George N. ; Pickart, Cecile M. ; Cohen, Robert E. / Specificity of the ubiquitin isopeptidase in the PA700 regulatory complex of 26 S proteasomes. In: Journal of Biological Chemistry. 1997 ; Vol. 272, No. 45. pp. 28438-28446.
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