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 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.
Lam
Y A
YA
Department of Biochemistry, University of Iowa, Iowa City, Iowa 52242, USA.
DeMartino
G N
GN
Pickart
C M
CM
Cohen
R E
RE
D000818:Animals; D002417:Cattle; D018360:Crystallography, X-Ray; D010450:Endopeptidases / Q000737:chemistry / Q000378:metabolism*; D007532:Isoleucine / Q000378:metabolism; D007930:Leucine / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D010447:Peptide Hydrolases / Q000737:chemistry / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*; D011817:Rabbits; D013379:Substrate Specificity
C085834:PA700 proteasome activator; D011506:Proteins; D007532:Isoleucine; D010450:Endopeptidases; D010447:Peptide Hydrolases; D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease; C091939:ubiquitin isopeptidase; D007930:Leucine