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 pro tein degradation in the eukaryotic cell. 26 S proteasomes consist of t wo regulatory 19 S cap complexes and the 20 S proteasome, which acts a s the proteolytic core module. We isolated six mutants of the yeast Sa ccharomyces cerevisiae containing mutations in the 20 S proteasome P-t ype subunit Pre3. Three mutations (pre3-2, pre3-3, and pre3-5) which r eside at the active site cleft of the Pre3 subunit solely caused reduc tion of the proteasomal peptidylglutamyl peptide-hydrolyzing activity but did not lead to detectable defects in protein degradation nor to a ny other phenotype. However, the pre3-2 mutation strengthened phenotyp es induced by other 20 S proteasomal mutations, indicating that the pe ptidylglutamyl peptide-hydrolyzing activity has to fulfill some rescue functions. The other three mutations (pre3-1,pre3-4 and pre3-6) are l ocated at diverse sites of the Pre3 protein and caused multiple defect s in proteasomal peptide cleaving activities. pre3-1 and pre3-6 mutant s exhibited significant defects in proteasomal protein degradation; th ey accumulated ubiquitinated proteins and stabilized defined substrate proteins as, e.g. fructose-1,6-bisphosphatase. In addition, pre3-1 an d pre3-6 mutant cells exhibited pleiotropic phenotypes as temperature sensitivity and cell cycle-related effects.