Structure of the Blm10-20 S Proteasome Complex by Cryo-Electron Microscopy. Insights Into the Mechanism of Activation of Mature Yeast Proteasomes

J Mol Biol. 2006 Oct 27;363(3):648-59. doi: 10.1016/j.jmb.2006.08.010. Epub 2006 Aug 9.

Abstract

The 20 S proteasome is regulated at multiple levels including association with endogenous activators. Two activators have been described for the yeast 20 S proteasome: the 19 S regulatory particle and the Blm10 protein. The sequence of Blm10 is 20% identical to the mammalian PA200 protein. Recent studies have shown that the sequences of Blm10 and PA200 each contain multiple HEAT-repeats and that each binds to the ends of mature proteasomes, suggesting a common structural and biochemical function. In order to advance structural studies, we have developed an efficient purification method that produces high yields of stoichiometric Blm10-mature yeast 20 S proteasome complexes and we constructed a three-dimensional (3D) model of the Blm10-20 S complex from cryo-electron microscopy images. This reconstruction shows that Blm10 binds in a defined orientation to both ends of the 20 S particle and contacts all the proteasome alpha subunits. Blm10 displays the solenoid folding predicted by the presence of multiple HEAT-like repeats and the axial gates on the alpha rings of the proteasome appear to be open in the complex. We also performed a genetic analysis in an effort to identify the physiological role of Blm10. These experiments, however, did not reveal a robust phenotype upon gene deletion, overexpression, or in a screen for synthetic effects. This leaves the physiological role of Blm10 unresolved, but challenges earlier findings of a role in DNA repair.

Publication types

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

MeSH terms

  • Animals
  • Cattle
  • Cryoelectron Microscopy
  • DNA Damage
  • Enzyme Activation
  • Models, Molecular
  • Multienzyme Complexes
  • Proteasome Endopeptidase Complex / chemistry*
  • Proteasome Endopeptidase Complex / metabolism
  • Protein Conformation*
  • Protein Subunits / chemistry
  • Protein Subunits / metabolism
  • Saccharomyces cerevisiae / enzymology
  • Saccharomyces cerevisiae Proteins / chemistry*
  • Saccharomyces cerevisiae Proteins / genetics
  • Saccharomyces cerevisiae Proteins / metabolism

Substances

  • Blm10 protein, S cerevisiae
  • Multienzyme Complexes
  • Protein Subunits
  • Saccharomyces cerevisiae Proteins
  • Proteasome Endopeptidase Complex