The PRE4 gene codes for a subunit of the yeast proteasome necessary for peptidylglutamyl-peptide-hydrolyzing activity. Mutations link the proteasome to stress- and ubiquitin-dependent proteolysis.
Proteinase yscE, the yeast proteasome, is a member of the nonlysosomal, high molecular mass (approximately 700 kDa) multifunctional proteinase complexes that are highly conserved from yeast to man. We have isolated mutants defective in one of the three proteolytic activities of the enzyme complex, i.e. in cleavage of peptide bonds after acidic amino acids. Using one of these mutants (pre4-1), we cloned the PRE4 gene and uncovered an open reading frame with 266 amino acids coding for a predicted protein of 29.4 kDa. The protein proved to be a subunit of proteinase yscE. The Pre4 amino acid sequence shows strong homology to the beta-subunit of the Xenopus laevis proteasome. Chromosomal deletion of the PRE4 gene is lethal. The pre4-1 mutant allele was cloned and sequenced. The mutant protein is shortened by 15 amino acids at the carboxyl terminus. Mutations (pre1-1, pre2-2) in the chymotrypsin-like activity of proteinase yscE uncovered the enzyme to be involved in ubiquitin-linked and stress-dependent proteolytic pathways. In contrast to these mutants, pre4-1 mutants did not exhibit any apparent stress-dependent phenotypes. However, pre1-1 pre4-1 double mutants showed enhanced canavanine sensitivity and increased accumulation of ubiquitin protein conjugates, as compared with pre1-1 single mutants.
Hilt
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Universität Stuttgart, Institut für Biochemie, Germany.
Enenkel
C
C
Gruhler
A
A
Singer
T
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Wolf
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DH
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002172:Canavanine / Q000494:pharmacology; D003001:Cloning, Molecular; D003546:Cysteine Endopeptidases / Q000235:genetics* / Q000378:metabolism*; D017353:Gene Deletion; D005800:Genes, Fungal; D005804:Genes, Lethal; D005816:Genetic Complementation Test; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000235:genetics* / Q000378:metabolism*; D016296:Mutagenesis; D046988:Proteasome Endopeptidase Complex; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000254:growth & development; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013696:Temperature; D014452:Ubiquitins / Q000378:metabolism*; D014982:Xenopus laevis
D046911:Macromolecular Substances; D009097:Multienzyme Complexes; D014452:Ubiquitins; D002172:Canavanine; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex