The lysine 48 and lysine 63 ubiquitin conjugates are processed differently by the 26 s proteasome.
The role of Lys-63 ubiquitin chains in targeting proteins for proteasomal degradation is still obscure. We systematically compared proteasomal processing of Lys-63 ubiquitin chains with that of the canonical proteolytic signal, Lys-48 ubiquitin chains. Quantitative mass spectrometric analysis of ubiquitin chains in HeLa cells determines that the levels of Lys-63 ubiquitin chains are insensitive to short-time proteasome inhibition. Also, the Lys-48/Lys-63 ratio in the 26 S proteasome-bound fraction is 1.7-fold more than that in the cell lysates, likely because some cellular Lys-63 ubiquitin conjugates are sequestered by Lys-63 chain-specific binding proteins. In vitro, Lys-48 and Lys-63 ubiquitin chains bind the 26 S proteasome comparably, whereas Lys-63 chains are deubiquitinated 6-fold faster than Lys-48 chains. Also, Lys-63 tetraubiquitin-conjugated UbcH10 is rapidly deubiquitinated into the monoubiquitinated form, whereas Lys-48 tetraubiquitin targets UbcH10 for degradation. Furthermore, we found that both the ubiquitin aldehyde- and 1,10-phenanthroline-sensitive deubiquitinating activities of the 26 S proteasome contribute to Lys-48- and Lys-63-linkage deubiquitination, albeit the inhibitory extents are different. Together, our findings suggest that compared with Lys-48 chains, cellular Lys-63 chains have less proteasomal accessibility, and proteasome-bound Lys-63 chains are more rapidly deubiquitinated, which could cause inefficient degradation of Lys-63 conjugates.
Department of Biochemistry and Molecular Genetics, University of Colorado Denver School of Medicine, Aurora, Colorado 80045, USA.
Andrew D
AD
Jacobson
Nan-Yan
NY
Zhang
Ping
P
Xu
Ke-Jun
KJ
Han
Seth
S
Noone
Junmin
J
Peng
Chang-Wei
CW
Liu
D000818:Animals; D002417:Cattle; D006367:HeLa Cells; D006801:Humans; D008239:Lysine / Q000737:chemistry* / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex / Q000737:chemistry* / Q000378:metabolism; D025801:Ubiquitin / Q000737:chemistry* / Q000378:metabolism; D054875:Ubiquitination / Q000502:physiology*
D025801:Ubiquitin; D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease; D008239:Lysine