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Sequential processivity and CAND1 regulate SCF ubiquitin ligases

Citation

Pierce, Nathan William (2012) Sequential processivity and CAND1 regulate SCF ubiquitin ligases. Dissertation (Ph.D.), California Institute of Technology. http://resolver.caltech.edu/CaltechTHESIS:11282011-135435821

Abstract

The modular design of the multi-subunit SCF ubiquitin ligases allows for recognition of a diverse set of target proteins. However, the speed and complexity of the SCF ubiquitylation reaction have precluded direct experimental tests to understand how SCF complex formation is regulated and the pathway by which ubiquitin chains are generated. Herein we introduce new theoretical and experimental methodologies to address both limitations. First, a quantitative framework based on product distribution predicts that the really interesting new gene (RING) E3s SCFCdc4 and SCFβ-TrCP work with the E2 Cdc34 to build polyubiquitin chains on substrates by sequential transfers of single ubiquitins. Measurements with millisecond time resolution directly demonstrate that substrate polyubiquitylation proceeds sequentially. Second, we present a novel FRET assay that enables real-time measurements of binding dynamics of the SCFFbxw7 complex. We find that the Cul1-associated protein CAND1 is able to actively remove Fbxw7/Skp1 from Cul1/Rbx1 by changing the dissociation rate of the complex a million-fold, yet CAND1 does not affect the assembly rate of SCFFbxw7. This activity is abolished when Cul1 is neddylated. Experiments show that CAND1 accelerates the rate at which multiple SCF complexes can form. Thus, CAND1 appears to function as an exchange factor. Lastly, several measurements reveal an extra step in the ubiquitylation pathway for yeast SCF that implies a substrate induced conformational change exists for Fbox proteins. These results present an unprecedented glimpse into the mechanism of RING ubiquitin ligases and their regulation by CAND1.

Item Type:Thesis (Dissertation (Ph.D.))
Subject Keywords:Ubiquitin Ligases, SCF, CAND1
Degree Grantor:California Institute of Technology
Division:Biology
Major Option:Biology
Awards:Lawrence L. and Audrey W. Ferguson Prize, 2012
Thesis Availability:Public (worldwide access)
Research Advisor(s):
  • Deshaies, Raymond Joseph
Thesis Committee:
  • Dunphy, William G. (chair)
  • Shan, Shu-ou
  • Sternberg, Paul W.
  • Deshaies, Raymond Joseph
Defense Date:10 November 2011
Record Number:CaltechTHESIS:11282011-135435821
Persistent URL:http://resolver.caltech.edu/CaltechTHESIS:11282011-135435821
Related URLs:
URLURL TypeDescription
http://dx.doi.org/10.1038/nature08595DOIUNSPECIFIED
Default Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:6739
Collection:CaltechTHESIS
Deposited By: Nathan Pierce
Deposited On:06 Jan 2012 22:30
Last Modified:14 Feb 2013 18:45

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