No abstract

Rapid degradation of specific regulatory proteins plays a role in a wide range of cellular phenomena, including cell cycle progression and the regulation of cell growth and differentiation. A major mechanism of selective protein turnover in vivo involves a large multi-subunit protease known as the proteasome or multi-catalytic proteinase. At the same time, the degradation of many cellular proteins requires their covalent ligation to the polypeptide ubiquitin. Here we show that the yeast S. cerevisiae MATa repressor, which is known to be ubiquitinylated in vivo, requires the proteasome for its rapid intracellular proteolysis.

Akatraet Proteasomes are large multicatalytic proteinase complexes found in all eukaryotic organisms investigated so far. They have been shown to play a central role in cytosolic and nuclear proteolysis. According to their sedimentation coefficients two types of these particles can be distinguished: 20s proteasomes and 26s proteasomes. In contrast to 20s proteasomes, which were mainly characterized on the basis of their ability 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 found in all eukaryotes from yeast to man. So far 26s proteasomes have only been discovered in higher eukaryotes. We now report the existence of the 26s proteasome in a lower eukaryote, the yeast Saccharomyces cerevisiae. Formation of the 26s proteasome could most effectively be induced in crude extracts of heat stressed yeast cells by incubation with ATP and M$ ions. This treatment yielded a protein complex, which eluted from gel filtration columns at molecular masses higher than 1500 kDa. Besides chromogenic peptide substrates, this complex cleaves ubiquitinylated proteins in an ATE-dependent fashion. In non-denaturing-PAGE, the purified 26s proteasome disintegrated and migrated as four protein bands. One of these bands could be ident&d as the 20s proteasome. 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 26s proteasome was obtained by following peptide cleaving activities in extracts of yeast strains carrying mutations in various subunits of the 20s proteasome.

The cell cycle of eukaryotic cells is strictly regulated. This regulation is performed by a serine/threonine kinase. The different functions of this kinase in the cell cycle are modulated by different cyclins, which fluctuate in concentration ('cycle') during the different stages of the cell cycle. Using yeast as a model organism we show here that the activity of the multifunctional proteinase, the proteasome, is directly connected to the function of the mitotic cyclin Clb2. Our studies indicate that the proteasome is the proteolytic regulator of this cyclin and thus a central regulator of the cell cycle.