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 degrees C but not at 37 degrees 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, alpha1 and alpha7, differed from those of wild-type cells in both electrophoretic mobility and pI values. The changes in these two alpha-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 alpha-subunits, alpha1 and alpha7, block the proteasome channel in the rpt6-1 mutant.


CONCLUSIONS
Two alpha-subunits (alpha1 and alpha7) 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.
Chouduri
Aktar Uzzaman
AU
Department of Biology, Faculty of Science, National University Corporation, Shizuoka University, 836 Oya, Suruga-ku, Shizuoka 422-8529, Japan. auchow5@yahoo.com
Tokumoto
Toshinobu
T
Dohra
Hideo
H
Ushimaru
Takashi
T
Yamada
Shinpei
S
D000251:Adenosine Triphosphatases; D000818:Animals; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D017353:Gene Deletion; D006863:Hydrogen-Ion Concentration; D013058:Mass Spectrometry; D010447:Peptide Hydrolases / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex / Q000235:genetics* / Q000378:metabolism*; D011817:Rabbits; D012097:Repressor Proteins / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics*; D012680:Sensitivity and Specificity; D013696:Temperature
D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D010447:Peptide Hydrolases; D046988:Proteasome Endopeptidase Complex; D000251:Adenosine Triphosphatases; C069670:RPT6 protein, S cerevisiae