Rpn7 Is required for the structural integrity of the 26 S proteasome of Saccharomyces cerevisiae

J Biol Chem. 2004 Jun 25;279(26):27168-76. doi: 10.1074/jbc.M314231200. Epub 2004 Apr 21.

Abstract

Rpn7 is one of the lid subunits of the 26 S proteasome regulatory particle. The RPN7 gene is known to be essential, but its function remains to be elucidated. To explore the function of Rpn7, we isolated and characterized temperature-sensitive rpn7 mutants. All of the rpn7 mutants obtained accumulated poly-ubiquitinated proteins when grown at the restrictive temperature. The N-end rule substrate (Ub-Arg-beta-galactosidase), the UFD pathway substrate (Ub-Pro-beta-galactosidase), and cell cycle regulators (Pds1 and Clb2) were found to be stabilized in experiments using one of the rpn7 mutants termed rpn7-3 at the restrictive temperature, indicating its defect in the ubiquitin-proteasome pathway. Subsequent analysis of the structure of the 26 S proteasome in rpn7-3 cells suggested that the defect was in the assembly of the 26 S holoenzyme. The most striking characteristic of the proteasome of the rpn7-3 mutant was that a lid subcomplex affinity-purified from the rpn7-3 cells grown at the restrictive temperature contained only 5 of the 8 lid components, a phenomenon that has not been reported in the previously isolated lid mutants. From these results, we concluded that Rpn7 is required for the integrity of the 26 S complex by establishing a correct lid structure.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Substitution
  • Cell Cycle Proteins / metabolism
  • Chromatography, Gel
  • Cyclin B / metabolism
  • Nuclear Proteins / metabolism
  • Peptide Hydrolases / chemistry*
  • Peptide Hydrolases / genetics
  • Peptide Hydrolases / metabolism*
  • Proteasome Endopeptidase Complex*
  • Protein Structure, Tertiary
  • Protein Subunits / genetics
  • Protein Subunits / metabolism*
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Saccharomyces cerevisiae / cytology
  • Saccharomyces cerevisiae / enzymology*
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae / growth & development
  • Saccharomyces cerevisiae Proteins / chemistry
  • Saccharomyces cerevisiae Proteins / genetics
  • Saccharomyces cerevisiae Proteins / metabolism
  • Securin
  • Temperature
  • Two-Hybrid System Techniques

Substances

  • CLB2 protein, S cerevisiae
  • Cell Cycle Proteins
  • Cyclin B
  • Nuclear Proteins
  • PDS1 protein, S cerevisiae
  • Protein Subunits
  • Recombinant Proteins
  • Saccharomyces cerevisiae Proteins
  • Securin
  • Peptide Hydrolases
  • Proteasome Endopeptidase Complex
  • ATP dependent 26S protease