Disassembly of Lys11 and mixed linkage polyubiquitin conjugates provides insights into function of proteasomal deubiquitinases Rpn11 and Ubp6.
Protein homeostasis is largely dependent on proteolysis by the ubiquitin-proteasome system. Diverse polyubiquitin modifications are reported to target cellular proteins to the proteasome. At the proteasome, deubiquitination is an essential preprocessing event that contributes to degradation efficiency. We characterized the specificities of two proteasome-associated deubiquitinases (DUBs), Rpn11 and Ubp6, and explored their impact on overall proteasome DUB activity. This was accomplished by constructing a panel of well defined ubiquitin (Ub) conjugates, including homogeneous linkages of varying lengths as well as a heterogeneously modified target. Rpn11 and Ubp6 processed Lys(11) and Lys(63) linkages with comparable efficiencies that increased with chain length. In contrast, processing of Lys(48) linkages by proteasome was inversely correlated to chain length. Fluorescently labeled tetra-Ub chains revealed endo-chain preference for Ubp6 acting on Lys(48) and random action for Rpn11. Proteasomes were more efficient at deconjugating identical substrates than their constituent DUBs by roughly 2 orders of magnitude. Incorporation into proteasomes significantly enhanced enzymatic efficiency of Rpn11, due in part to alleviation of the autoinhibitory role of its C terminus. The broad specificity of Rpn11 could explain how proteasomes were more effective at disassembling a heterogeneously modified conjugate compared with homogeneous Lys(48)-linked chains. The reduced ability to disassemble homogeneous Lys(48)-linked chains longer than 4 Ub units may prolong residency time on the proteasome.
From the Department of Biology, Technion-Israel Institute of Technology, 32000 Haifa, Israel.
Wissam
W
Mansour
From the Department of Biology, Technion-Israel Institute of Technology, 32000 Haifa, Israel,; the Department of Chemistry and Biochemistry, Center for Biomolecular Structure and Organization, University of Maryland, College Park, Maryland 20742.
Mark A
MA
Nakasone
the Max-Delbrück-Zentrum für Molekulare Medizin, Robert-Rössle-Strasse 10, 13125 Berlin, Germany, and.
Maximilian
M
von Delbrück
From the Department of Biology, Technion-Israel Institute of Technology, 32000 Haifa, Israel.
Zanlin
Z
Yu
From the Department of Biology, Technion-Israel Institute of Technology, 32000 Haifa, Israel.
Daria
D
Krutauz
From the Department of Biology, Technion-Israel Institute of Technology, 32000 Haifa, Israel.
Noa
N
Reis
the Department of Biochemistry and Molecular Biology, Faculty of Medicine, Nursing and Health Sciences, Monash University, Melbourne 3800, Australia.
Oded
O
Kleifeld
the Max-Delbrück-Zentrum für Molekulare Medizin, Robert-Rössle-Strasse 10, 13125 Berlin, Germany, and.
Thomas
T
Sommer
the Department of Chemistry and Biochemistry, Center for Biomolecular Structure and Organization, University of Maryland, College Park, Maryland 20742.
David
D
Fushman
From the Department of Biology, Technion-Israel Institute of Technology, 32000 Haifa, Israel,. Electronic address: glickman@tx.technion.ac.il.
Michael H
MH
Glickman
D010450:Endopeptidases / Q000235:genetics / Q000378:metabolism*; D008239:Lysine / Q000235:genetics / Q000378:metabolism; D025821:Polyubiquitin / Q000235:genetics / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D059748:Proteolysis; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*
C115451:RPN11 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D025821:Polyubiquitin; D010450:Endopeptidases; D046988:Proteasome Endopeptidase Complex; C109028:UBP6 protein, S cerevisiae; D008239:Lysine
Cysteine Protease; Deubiquitination; Metalloprotease; Polyubiquitin; Proteasome; Rpn11; Ubiquitin; Ubp6