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Published May 2011 | Supplemental Material + Published
Journal Article Open

The steady-state repertoire of human SCF Ubiquitin ligase complexes does not require ongoing Nedd8 conjugation


The human genome encodes 69 different F-box proteins (FBPs), each of which can potentially assemble with Skp1-Cul1-RING to serve as the substrate specificity subunit of an SCF ubiquitin ligase complex. SCF activity is switched on by conjugation of the ubiquitin- like protein Nedd8 to Cul1. Cycles of Nedd8 conjugation and deconjugation acting in conjunction with the Cul1-sequestering factor Cand1 are thought to control dynamic cycles of SCF assembly and disassembly, which would enable a dynamic equilibrium between the Cul1- RING catalytic core of SCF and the cellular repertoire of FBPs. To test this hypothesis, we determined the cellular composition of SCF complexes and evaluated the impact of Nedd8 conjugation on this steady-state. At least 42 FBPs assembled with Cul1 in HEK 293 cells, and the levels of Cul1-bound FBPs varied by over two orders of magnitude. Unexpectedly, quantitative mass spectrometry revealed that blockade of Nedd8 conjugation led to a modest increase, rather than a decrease, in the overall level of most SCF complexes. We suggest that multiple mechanisms including FBP dissociation and turnover cooperate to maintain the cellular pool of SCF ubiquitin ligases.

Additional Information

© 2011 The American Society for Biochemistry and Molecular Biology, Inc. Received November 16, 2010. Accepted December 17, 2010. J. E. Lee was supported by the Ruth L. Kirschstein NRSA fellowship (CA138126) and the Proteome Exploration lab was supported by the Beckman Institute at Caltech and an award from the Gordon and Betty Moore Foundation. R. J. D. is an Investigator of the Howard Hughes Medical Institute and this work was supported in part by HHMI and an NIH grant (GM065997) to R. J. D. Acknowledgments—We thank P. Kaiser for the HTBH tag and J. Wade Harper for generously sharing results prior to publication. MLN4924 was a generous gift from Millennium: The Takeda Oncology Company.

Attached Files

Published - Lee2011p13889Mol_Cell_Proteomics.pdf

Supplemental Material - mcp.M110.006460-1.xls

Supplemental Material - mcp.M110.006460-2.xls

Supplemental Material - mcp.M110.006460-3.xls

Supplemental Material - mcp.M110.006460-4.xls

Supplemental Material - mcp.M110.006460-5.xls

Supplemental Material - mcp.M110.006460-6.pdf


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