The 26S proteasome of the yeast Saccharomyces cerevisiae

Abstract

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.

Isolation and Characterization of SUG2

“…In this regard we note that while under our conditions almost all of the 19S/PA700 proteins can be found in association with the 20 S proteasome, an earlier report (46) found that unusual growth conditions were required to stabilize the 19S-20 S interaction. Given the dependence of the stability of 26 S to extract and growth conditions, and the possibility that other activators of the 20 S proteasome may compete with 19S/ PA700 for binding to the 20 S, we feel that the extent to which yeast 19S/PA700 is associated with the 20 S proteasome in vivo is still not clear.…”

Importance of the ATP-Ubiquitin-Proteasome Pathway in the Degradation of Soluble and Myofibrillar Proteins in Rabbit Muscle Extracts

“…First, most of the proteasomes were removed from the crude extracts by ultracentrifugation at 100,000 ϫ g for 6 h (40). To confirm that the proteasome content was in fact reduced, we assayed the hydrolysis of Suc-Leu-Leu-Val-Tyr-MCA, a preferred substrate of the proteasome (41). This activity was 60 -70% lower after ultracentrifugation than in the whole extracts before centrifugation.…”

Proteasomes in the archaea: from structure to function

“…In addition, yeast genomic analysis using DNA chip technology reveals a 5 to 10 fold increase in mRNA transcripts of not only ubiquitin but also the Rpn1 (non-ATPase) and beta1 subunits of the 26S proteasome after exposure to a DNA-damaging agent (23). Furthermore, energy-dependent 26S proteasome assembly is enhanced in cell lysate prepared from nutrient depleted, heat-stressed yeast cells versus non-stressed cells (293). In some cancer cell lines, the proteasome is localized to the nucleus after stress (e.g.…”