Quantitative analysis of in vitro ubiquitinated cyclin B1 reveals complex chain topology.
Protein ubiquitination regulates many cellular processes, including protein degradation, signal transduction, DNA repair and cell division. In the classical model, a uniform polyubiquitin chain that is linked through Lys 48 is required for recognition and degradation by the 26S proteasome. Here, we used a reconstituted system and quantitative mass spectrometry to demonstrate that cyclin B1 is modified by ubiquitin chains of complex topology, rather than by homogeneous Lys 48-linked chains. The anaphase-promoting complex was found to attach monoubiquitin to multiple lysine residues on cyclin B1, followed by poly-ubiquitin chain extensions linked through multiple lysine residues of ubiquitin (Lys 63, Lys 11 and Lys 48). These heterogeneous ubiquitin chains were sufficient for binding to ubiquitin receptors, as well as for degradation by the 26S proteasome, even when they were synthesized with mutant ubiquitin that lacked Lys 48. Together, our observations expand the context of what can be considered to be a sufficient degradation signal and provide unique insights into the mechanisms of substrate ubiquitination.
Kirkpatrick
Donald S
DS
Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA.
Hathaway
Nathaniel A
NA
Hanna
John
J
Elsasser
Suzanne
S
Rush
John
J
Finley
Daniel
D
King
Randall W
RW
Gygi
Steven P
SP
D000595:Amino Acid Sequence; D064173:Anaphase-Promoting Complex-Cyclosome; D001665:Binding Sites / Q000502:physiology; D019926:Cyclin B / Q000032:analysis* / Q000737:chemistry* / Q000378:metabolism; D056744:Cyclin B1; D066298:In Vitro Techniques; D008239:Lysine / Q000378:metabolism; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D046988:Proteasome Endopeptidase Complex / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding / Q000502:physiology; D020836:Protein Structure, Quaternary / Q000502:physiology; D015398:Signal Transduction / Q000502:physiology; D025801:Ubiquitin / Q000032:analysis* / Q000737:chemistry* / Q000378:metabolism; D043743:Ubiquitin-Protein Ligase Complexes / Q000737:chemistry / Q000378:metabolism; D015003:Yeasts / Q000378:metabolism
D019926:Cyclin B; D056744:Cyclin B1; D025801:Ubiquitin; D043743:Ubiquitin-Protein Ligase Complexes; D064173:Anaphase-Promoting Complex-Cyclosome; D046988:Proteasome Endopeptidase Complex; D008239:Lysine