Mutations in the yeast proteasome beta-type subunit Pre3 uncover position-dependent effects on proteasomal peptidase activity and in vivo function.
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.
Gueckel
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R
Institut für Biochemie, Universität Stuttgart, Pfaffenwaldring 55, 70569 Stuttgart, Germany.
Enenkel
C
C
Wolf
D H
DH
Hilt
W
W
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002453:Cell Cycle / Q000235:genetics; D002455:Cell Division / Q000235:genetics; D048429:Cell Size / Q000235:genetics; D003001:Cloning, Molecular; D016213:Cyclins / Q000502:physiology; D003546:Cysteine Endopeptidases / Q000737:chemistry*; D004252:DNA Mutational Analysis; D010450:Endopeptidases / Q000235:genetics*; D004795:Enzyme Stability; D005656:Fungal Proteins / Q000737:chemistry; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000737:chemistry*; D009154:Mutation / Q000235:genetics; D010641:Phenotype; D046988:Proteasome Endopeptidase Complex; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D013696:Temperature
D016213:Cyclins; D005656:Fungal Proteins; D009097:Multienzyme Complexes; D010450:Endopeptidases; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex