Analysis of a gene encoding Rpn10 of the fission yeast proteasome reveals that the polyubiquitin-binding site of this subunit is essential when Rpn12/Mts3 activity is compromised.
Substrates are targeted for proteolysis by the ubiquitin pathway by the addition of a polyubiquitin chain before being degraded by the 26 S proteasome. Previously, a subunit of the proteasome, S5a, was identified that was able to bind to polyubiquitin in vitro and thus proposed to act as a substrate recognition component. Deletion of the corresponding Saccharomyces cerevisiae gene, MCB1/RPN10, rendered cells viable indicating that other proteasomal polyubiquitin receptors must exist. In this study, we describe pus1(+), the fission yeast homologue of RPN10. This gene is also not required for cell viability; however, the Deltapus1 mutant is synthetically lethal with mutations in other proteasomal component-encoding genes, namely mts3, pad1, and mts4 (RPN12, RPN11, and RPN1). Overexpression of pus1(+) is able to rescue mts3-1 at 32 degrees C but overexpression of a cDNA encoding a version of Pus1 that does not bind to polyubiquitin cannot and leads to greatly reduced viability when used to rescue the mts3-1Deltapus1 double mutant. The Mts3 protein was unable to bind to polyubiquitin in vitro, but the Pus1 and Mts3 proteins were found to bind to one another in vitro, which taken together with the genetic data suggests that they are also closely associated in vivo.
Wilkinson
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CR
MRC Human Genetics Unit, Western General Hospital, Crewe Road, Edinburgh, EH4 2XU, Scotland, United Kingdom.
Ferrell
K
K
Penney
M
M
Wallace
M
M
Dubiel
W
W
Gordon
C
C
D000595:Amino Acid Sequence; D000818:Animals; D017360:Arabidopsis / Q000235:genetics; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000235:genetics; D010450:Endopeptidases / Q000378:metabolism; D005656:Fungal Proteins / Q000378:metabolism*; D017353:Gene Deletion; D005800:Genes, Fungal; D006801:Humans; D006836:Hydro-Lyases / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D010447:Peptide Hydrolases / Q000235:genetics / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex; D016601:RNA-Binding Proteins; D012441:Saccharomyces cerevisiae / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D012568:Schizosaccharomyces / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism*; D029702:Schizosaccharomyces pombe Proteins; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D002352:Carrier Proteins; D005656:Fungal Proteins; C498506:PSMD4 protein, human; D016601:RNA-Binding Proteins; C074096:RPN12 protein, S cerevisiae; C098743:RPN12 protein, S pombe; D029701:Saccharomyces cerevisiae Proteins; D029702:Schizosaccharomyces pombe Proteins; C498507:pus1 protein, S pombe; D010450:Endopeptidases; D010447:Peptide Hydrolases; D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease; D006836:Hydro-Lyases; C022479:pseudouridylate synthetase