Abstract
Proteasomes are highly complex proteases responsible for selective protein degradation in the eukaryotic cell. 26 S proteasomes consist of two regulatory 19 S cap complexes and the 20 S proteasome, which acts as the proteolytic core module. We isolated six mutants of the yeast Saccharomyces cerevisiae containing mutations in the 20 S proteasome beta-type subunit Pre3. Three mutations (pre3-2, pre3-3, and pre3-5) which reside at the active site cleft of the Pre3 subunit solely caused reduction of the proteasomal peptidylglutamyl peptide-hydrolyzing activity but did not lead to detectable defects in protein degradation nor to any other phenotype. However, the pre3-2 mutation strengthened phenotypes induced by other 20 S proteasomal mutations, indicating that the peptidylglutamyl peptide-hydrolyzing activity has to fulfill some rescue functions. The other three mutations (pre3-1, pre3-4, and pre3-6) are located at diverse sites of the Pre3 protein and caused multiple defects in proteasomal peptide cleaving activities. pre3-1 and pre3-6 mutants exhibited significant defects in proteasomal protein degradation; they accumulated ubiquitinated proteins and stabilized defined substrate proteins as, e.g. fructose-1,6-bisphosphatase. In addition, pre3-1 and pre3-6 mutant cells exhibited pleiotropic phenotypes as temperature sensitivity and cell cycle-related effects.
Highlights
Proteasomes are large proteinase complexes operating in the cytoplasm and the nucleus of the eukaryotic cell
Three mutations which reside at the active site cleft of the Pre3 subunit solely caused reduction of the proteasomal peptidylglutamyl peptide-hydrolyzing activity but did not lead to detectable defects in protein degradation nor to any other phenotype
Two complexes are found: (i) the cylindrically shaped 20 S proteasome and (ii) the larger 26 S proteasome, which is composed of the 20 S proteasome as the proteolytic core and two additional S cap complexes attached at both ends of the S cylinder [1]
Summary
Proteasomes are large proteinase complexes operating in the cytoplasm and the nucleus of the eukaryotic cell. Three mutations (pre3–2, pre3–3, and pre3–5) which reside at the active site cleft of the Pre3 subunit solely caused reduction of the proteasomal peptidylglutamyl peptide-hydrolyzing activity but did not lead to detectable defects in protein degradation nor to any other phenotype.
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