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 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 Mg2+ 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 ATP-dependent fashion. In non-denaturing-PAGE, the purified 26S proteasome disintegrated and migrated as four protein bands. One of these bands could be identified 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.
Fischer
M
M
Institut für Biochemie, Universität Stuttgart, Universität Stuttgart, Germany.
Hilt
W
W
Richter-Ruoff
B
B
Gonen
H
H
Ciechanover
A
A
Wolf
D H
DH
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008274:Magnesium / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D009842:Oligopeptides / Q000378:metabolism; D017354:Point Mutation; D046988:Proteasome Endopeptidase Complex; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013696:Temperature; D014452:Ubiquitins / Q000378:metabolism
D009097:Multienzyme Complexes; D009842:Oligopeptides; D014452:Ubiquitins; D000255:Adenosine Triphosphate; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D008274:Magnesium