THE 26S PROTEASOME OF THE YEAST SACCHAROMYCES-CEREVISIAE

Proteasomes are large multicatalytic proteinase complexes found in all eukaryotic organisms investigated so far. They have been shown to pla y a central role in cytosolic and nuclear proteolysis. According to th eir sedimentation coefficients two types of these particles can be dis tinguished: 20S proteasomes and 26S proteasomes. In contrast to 20S pr oteasomes, which were mainly characterized on the basis of their abili ty to cleave small chromogenic peptide substrates and certain proteins in an ATP-independent manner, 26S proteasomes degrade ubiquitinylated proteins in an ATP-dependent reaction. 20S proteasomes have been foun d in all eukaryotes from yeast to man. So far 26S proteasomes have onl y been discovered in higher eukaryotes. We now report the existence of the 26S proteasome in a lower eukaryote, the yeast Saccharomyces cere visiae. Formation of the 26S proteasome could most effectively be indu ced in crude extracts of heat stressed yeast cells by incubation with ATP and Mg2+ ions. This treatment yielded a protein complex, which elu ted from gel filtration columns at molecular masses higher than 1500 k Da. Besides chromogenic peptide substrates, this complex cleaves ubiqu itinylated proteins in an ATP-dependent fashion. In non-denaturing-PAG E, the purified 26S proteasome disintegrated and migrated as four prot ein bands. One of these bands could be identified as the 20S proteasom e. On SDS-PAGE, the 26S proteasome showed a complex pattern of subunit bands with molecular masses between 15 and 100 kDa. Further evidence for the 20S proteasome being the proteolytically active core of the 26 S proteasome was obtained by following peptide cleaving activities in extracts of yeast strains carrying mutations in various subunits of th e 20S proteasome.