Isolation of the Schizosaccharomyces pombe proteasome subunit Rpn7 and a structure-function study of the proteasome-COP9-initiation factor domain

J Biol Chem. 2007 Nov 2;282(44):32414-23. doi: 10.1074/jbc.M706276200. Epub 2007 Aug 29.


Proper assembly of the 26 S proteasome is required to efficiently degrade polyubiquitinated proteins. Many proteasome subunits contain the proteasome-COP9-initiation factor (PCI) domain, thus raising the possibility that the PCI domain may play a role in mediating proteasome assembly. We have previously characterized the PCI protein Yin6, a fission yeast ortholog of the mammalian Int6 that has been implicated in breast oncogenesis, and demonstrated that it binds and regulates the assembly of the proteasome. In this study, we isolated another PCI proteasome subunit, Rpn7, as a high copy suppressor that rescued the proteasome defects in yin6 null cells. To better define the function of the PCI domain, we aligned protein sequences to identify a conserved leucine residue that is present in nearly all known PCI domains. Replacing it with aspartate in yeast Rpn7, Yin6, and Rpn5 inactivated these proteins, and mutant human Int6 mislocalized in HeLa cells. Rpn7 and Rpn5 bind Rpn9 with high affinity, but their mutant versions do not. Our data suggest that this leucine may interact with several hydrophobic amino acid residues to influence the spatial arrangement either within the N-terminal tandem alpha-helical repeats or between these repeats and the more C-terminal winged helix subdomain. Disruption of such an arrangement in the PCI domain may substantially inactivate many PCI proteins and block their binding to other proteins.

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

  • Amino Acid Sequence
  • COP9 Signalosome Complex
  • Carrier Proteins / chemistry
  • Carrier Proteins / genetics
  • Carrier Proteins / metabolism*
  • HeLa Cells
  • Humans
  • Molecular Sequence Data
  • Multiprotein Complexes / metabolism
  • Peptide Hydrolases / metabolism
  • Proteasome Endopeptidase Complex / chemistry
  • Proteasome Endopeptidase Complex / isolation & purification*
  • Proteasome Endopeptidase Complex / metabolism
  • Protein Structure, Tertiary
  • Protein Subunits / metabolism
  • Schizosaccharomyces / chemistry
  • Schizosaccharomyces / metabolism
  • Schizosaccharomyces pombe Proteins / chemistry
  • Schizosaccharomyces pombe Proteins / genetics
  • Schizosaccharomyces pombe Proteins / isolation & purification*
  • Schizosaccharomyces pombe Proteins / metabolism
  • Sequence Alignment
  • Structure-Activity Relationship


  • Carrier Proteins
  • Multiprotein Complexes
  • Protein Subunits
  • Schizosaccharomyces pombe Proteins
  • int6 protein, S pombe
  • rpn501 protein, S pombe
  • Peptide Hydrolases
  • COP9 Signalosome Complex
  • Proteasome Endopeptidase Complex