Analysis of activator-binding sites on the APC/C supports a cooperative substrate-binding mechanism

Mol Cell. 2009 Apr 10;34(1):68-80. doi: 10.1016/j.molcel.2009.02.027.

Abstract

The anaphase-promoting complex or cyclosome (APC/C) is a ubiquitin ligase essential for the completion of mitosis in all eukaryotic cells. Substrates are recruited to the APC/C by activator proteins (Cdc20 or Cdh1), but it is not known where substrates are bound during catalysis. We explored this problem by analyzing mutations in the tetratricopeptide-repeat-containing APC/C subunits. We identified residues in Cdc23 and Cdc27 that are required for APC/C binding to Cdc20 and Cdh1 and for APC/C function in vivo. Mutation of these sites increased the rate of activator dissociation from the APC/C but did not affect reaction processivity, suggesting that the mutations have little effect on substrate dissociation from the active site. Further studies revealed that activator dissociation from the APC/C is inhibited by substrate, and that substrates are not bound solely to activator during catalysis but interact bivalently with an additional binding site on the APC/C core.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Amino Acid Motifs
  • Amino Acid Sequence
  • Anaphase-Promoting Complex-Cyclosome
  • Apc8 Subunit, Anaphase-Promoting Complex-Cyclosome
  • Binding Sites
  • Cdc20 Proteins
  • Cdh1 Proteins
  • Cell Cycle Proteins / chemistry
  • Cell Cycle Proteins / genetics
  • Cell Cycle Proteins / metabolism
  • Enzyme Activation
  • Models, Biological
  • Molecular Sequence Data
  • Mutation
  • Protein Binding / physiology
  • Saccharomyces cerevisiae / enzymology*
  • Saccharomyces cerevisiae / metabolism
  • Saccharomyces cerevisiae Proteins / chemistry
  • Saccharomyces cerevisiae Proteins / genetics
  • Saccharomyces cerevisiae Proteins / metabolism
  • Sequence Analysis, Protein
  • Ubiquitin-Protein Ligase Complexes / chemistry
  • Ubiquitin-Protein Ligase Complexes / metabolism*

Substances

  • Apc8 Subunit, Anaphase-Promoting Complex-Cyclosome
  • CDC20 protein, S cerevisiae
  • CDC23 protein, S cerevisiae
  • CDH1 protein, S cerevisiae
  • Cdc20 Proteins
  • Cdh1 Proteins
  • Cell Cycle Proteins
  • Saccharomyces cerevisiae Proteins
  • Ubiquitin-Protein Ligase Complexes
  • Anaphase-Promoting Complex-Cyclosome