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Functional and biochemical characterization of the 20S proteasome in a yeast temperature-sensitive mutant, rpt6-1
BACKGROUND: Rpt6-1 is a thermosensitive yeast mutant with a deletion of a gene encoding a regulatory subunit of the 26S proteasome, RPT6, which is able to grow at 25°C but not at 37°C. In this study, peptidase activities, activation profiles, and the subunit composition of the 20S proteasome purifie...
Autores principales: | , , , , |
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Formato: | Texto |
Lenguaje: | English |
Publicado: |
BioMed Central
2008
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2515314/ https://www.ncbi.nlm.nih.gov/pubmed/18644121 http://dx.doi.org/10.1186/1471-2091-9-20 |
Sumario: | BACKGROUND: Rpt6-1 is a thermosensitive yeast mutant with a deletion of a gene encoding a regulatory subunit of the 26S proteasome, RPT6, which is able to grow at 25°C but not at 37°C. In this study, peptidase activities, activation profiles, and the subunit composition of the 20S proteasome purified from the rpt6-1 mutant was characterized. RESULTS: The 20S proteasome purified from rpt6-1 exhibited low levels of peptidase activities in the absence of activators, but nearly same activated activities in the presence of activators, suggesting a gating defect in the proteasome channel. Detailed analyses of the composition of the 20S proteasome through separation of all subunits by two-dimensional gel electrophoresis followed by identification of each subunit using MALDI-TOF-MS revealed that two subunits, α1 and α7, differed from those of wild-type cells in both electrophoretic mobility and pI values. The changes in these two α-subunits were apparent at the permissive temperature, but disappeared during stress response at the restrictive temperature. Interestingly, upon disappearance of these changes, the levels of peptidase activity of the 20S proteasome in the rpt6-1 mutant were restored as the wild-type. These results suggest that two different forms of the α-subunits, α1 and α7, block the proteasome channel in the rpt6-1 mutant. CONCLUSION: Two α-subunits (α1 and α7) of the 20S proteasome in the rpt6-1 mutant differed from their wild-type counterparts and peptidase activities were found to be lower in the mutant than in the wild-type strain. |
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