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Using In Vitro Fluorescence Resonance Energy Transfer to Study the Dynamics Of Protein Complexes at a Millisecond Time Scale
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Cand1 promotes assembly of new SCF complexes through dynamic exchange of F box proteins

Nathan W Pierce1, J Eugene Lee, Xing Liu

  • 1Division of Biology, MC 156-29, California Institute of Technology, 1200 East California Boulevard, Pasadena, CA 91125, USA.

Cell
|March 5, 2013
PubMed

Insights

The protein Cand1 acts as a catalyst, dramatically increasing the dissociation of SCF (Skp1, cullin, and F box) complexes. This regulation is crucial for maintaining the cellular diversity of these essential ubiquitin ligases.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Cellular Biology

Background:

  • The Skp1-cullin-F-box (SCF) complex is a modular ubiquitin ligase essential for targeted protein degradation.
  • SCF complexes utilize diverse F-box proteins as substrate receptors, but mechanisms sustaining their cellular repertoire are unknown.

Purpose of the Study:

  • To investigate the regulatory mechanisms governing the stability and composition of SCF complexes.
  • To elucidate the role of the cullin-binding protein Cand1 in SCF complex dynamics.

Main Methods:

  • Real-time measurements of SCF complex formation and disassembly.
  • In vitro binding and ubiquitylation assays.
  • Cellular depletion of Cand1 and subsequent analysis of SCF complex composition.

Main Results:

  • SCF(Fbxw7) complexes are highly stable but dissociate rapidly in the presence of Cand1 when deconjugated from Nedd8.
  • Cand1 functions as a protein exchange factor, accelerating the equilibration of Cul1-Rbx1 with various F-box protein-Skp1 modules.
  • Cand1 depletion impairs the recruitment of new F-box proteins to Cul1, altering the cellular landscape of SCF complexes.

Conclusions:

  • Catalyzed protein exchange, mediated by factors like Cand1, is proposed as a general mechanism for regulating dynamic macromolecular machines.
  • A model is presented where substrates, Nedd8, and Cand1 collaborate to control the cellular repertoire of SCF complexes.

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