Proteinase yscE, the yeast proteasome/multicatalytic-multifunctional proteinase: mutants unravel its function in stress induced proteolysis and uncover its necessity for cell survival.

Abstract

Proteinase yscE is the yeast equivalent of the proteasome, a multicatalytic‐multifunctional proteinase found in higher eukaryotic cells. We have isolated three mutants affecting the proteolytic activity of proteinase yscE. The mutants show a specific reduction in the activity of the complex against peptide substrates with hydrophobic amino acids at the cleavage site and define two complementation groups, PRE1 and PRE2. The PRE1 gene was cloned and shown to be essential. The deduced amino acid sequence encoded by the PRE1 gene reveals weak, but significant similarities to proteasome subunits of other organisms. Two‐dimensional gel electrophoresis identified the yeast proteasome to be composed of 14 different subunits. Comparison of these 14 subunits with the translation product obtained from PRE1 mRNA synthesized in vitro demonstrated that PRE1 encodes the 22.6 kd subunit (numbered 11) of the yeast proteasome. Diploids homozygous for pre1–1 are defective in sporulation. Strains carrying the pre1–1 mutation show enhanced sensitivity to stresses such as incorporation of the amino acid analogue canavanine into proteins or a combination of poor growth medium and elevated temperature. Under these stress conditions pre1–1 mutant cells exhibit decreased protein degradation and accumulate ubiquitin‐protein conjugates.

Rapid purification of a functionally active plant sucrose carrier from transgenic yeast using a bacterial biotin acceptor domain

“…The proteinase-deficient S. cerevisiae strain c13-ABYS-86 [14] was used for all expression studies to avoid potential problems with yeast inherent proteinase activities. For cloning in E. coli the strain DH5~ was used.…”

Molecular cloning of an essential yeast gene encoding a proteasomal subunit

“…Nudeotide sequence determination of this DNA fragment defined in close proximity to the GCN5 gene, which is involved in the general control of amino acid biosynthesis in yeast [35,36], a second open reading frame with the coding capacity of 252 carboxy-40 terminal amino acids located at the very end of the cloned piece (Fig, IA), By using an overlapping clone and a synthetic primer homologous to the end of the Recently the sequence of additional proteasome subunits has been determined from both these species [20][21][22][23][24][25] as well as from yeast [26][27][28][29], and we have found, by doing multiple alignments, that they all share a high degree of simi. !arity.…”

“…The proteasome catalyses the cleavage of peptide bonds on the carboxyl side of basic, neutral and acidic residues [7][8][9] but its exact function in the cell is still unknown. It has been implicated in playing a role in the degradation of proteins via the ATP-dependent ubiquitin-mediated proteolysis [10,11] and it was reported recently that prel-I yeast cells (mutations in the PREI gene encoding a proteasomal subunit affecting the proteolytic activity of the proteasome) exhibit decreased protein degradation and accumulate ubiquitin-protein conjugates [28]. The 20 S particles have been proposed to be also involved in tRNA processing and mRNA translation [12,13].…”

“…Cloning of a number of proteasomal subunits from rat [18][19][20][21][22][23], D. melanogaster [l S,24,2~ and yeast [26][27][28][29] has revealed another interesting property of these proteins. They belong to a unique family of proteins which share extensive inter-and intra-species similarities.…”

“…This suggests that the proteasome is organi~d from multiple similar subunits which are evolutionarily related. Very recently the molecular cloning of six yeast proteasomal subunits revealed that these similar subunits might serve different functions since only five of them are essential for cell viability [26][27][28][29]. In this report we present the sequence of a yeast gene encoding a proteasomal protein which is also essential for cellular survival.…”