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.
Mesh Terms:
Amino Acid Substitution, Cell Cycle Proteins, Chromatography, Gel, Cyclin B, Nuclear Proteins, Peptide Hydrolases, Proteasome Endopeptidase Complex, Protein Structure, Tertiary, Protein Subunits, Recombinant Proteins, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Temperature, Two-Hybrid System Techniques
J. Biol. Chem.
Date: Jun. 25, 2004
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