Rpn7 Is required for the structural integrity of the 26 S proteasome of Saccharomyces cerevisiae.
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.
Isono
Erika
E
Department of Biological Sciences, Graduate School of Science, University of Tokyo, Hongo, Tokyo 113-0033, Japan.
Saeki
Yasushi
Y
Yokosawa
Hideyoshi
H
Toh-e
Akio
A
D019943:Amino Acid Substitution; D018797:Cell Cycle Proteins / Q000378:metabolism; D002850:Chromatography, Gel; D019926:Cyclin B / Q000378:metabolism; D009687:Nuclear Proteins / Q000378:metabolism; D010447:Peptide Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D064249:Securin; D013696:Temperature; D020798:Two-Hybrid System Techniques
C093749:CLB2 protein, S cerevisiae; D018797:Cell Cycle Proteins; D019926:Cyclin B; D009687:Nuclear Proteins; C578004:PDS1 protein, S cerevisiae; D021122:Protein Subunits; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D064249:Securin; D010447:Peptide Hydrolases; D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease