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Recombinant expression, reconstitution and structure of human anaphase-promoting complex (APC/C).

Mechanistic and structural studies of large multi-subunit assemblies are greatly facilitated by their reconstitution in heterologous recombinant systems. In the present paper, we describe the generation of recombinant human APC/C (anaphase-promoting complex/cyclosome), an E3 ubiquitin ligase that regulates cell-cycle progression. Human APC/C is composed of 14 distinct proteins that assemble into a complex of at least 19 subunits with a combined molecular mass of ~1.2 MDa. We show that recombinant human APC/C is correctly assembled, as judged by its capacity to ubiquitinate the budding yeast APC/C substrate Hsl1 (histone synthetic lethal 1) dependent on the APC/C co-activator Cdh1 [Cdc (cell division cycle) 20 homologue 1], and its three-dimensional reconstruction by electron microscopy and single-particle analysis. Successful reconstitution validates the subunit composition of human APC/C. The structure of human APC/C is compatible with the Saccharomyces cerevisiae APC/C homology model, and in contrast with endogenous human APC/C, no evidence for conformational flexibility of the TPR (tetratricopeptide repeat) lobe is observed. Additional density present in the human APC/C structure, proximal to Apc3/Cdc27 of the TPR lobe, is assigned to the TPR subunit Apc7, a subunit specific to vertebrate APC/C.

Pubmed ID: 23078409

Authors

  • Zhang Z
  • Yang J
  • Kong EH
  • Chao WC
  • Morris EP
  • da Fonseca PC
  • Barford D

Journal

The Biochemical journal

Publication Data

January 15, 2013

Associated Grants

  • Agency: Cancer Research UK, Id: 14109
  • Agency: Cancer Research UK, Id: C576/A14109

Mesh Terms

  • Anaphase-Promoting Complex-Cyclosome
  • Animals
  • Apc3 Subunit, Anaphase-Promoting Complex-Cyclosome
  • Apc7 Subunit, Anaphase-Promoting Complex-Cyclosome
  • Cell Cycle Proteins
  • Cell Line
  • Humans
  • Microscopy, Electron
  • Models, Molecular
  • Multiprotein Complexes
  • Protein Multimerization
  • Protein Structure, Quaternary
  • Protein Subunits
  • Recombinant Proteins
  • Substrate Specificity
  • Ubiquitin-Protein Ligase Complexes
  • Ubiquitin-Protein Ligases
  • Ubiquitination