Chouduri Aktar Uzzaman, Tokumoto Toshinobu, Dohra Hideo, Ushimaru Takashi, Yamada Shinpei
Department of Biology, Faculty of Science, National University Corporation, Shizuoka University, 836 Oya, Suruga-ku, Shizuoka 422-8529, Japan.
BMC Biochem. 2008 Jul 21;9:20. doi: 10.1186/1471-2091-9-20.
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.
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.
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.
Rpt6 - 1是一种温度敏感型酵母突变体，其编码26S蛋白酶体调节亚基RPT6的基因缺失，该突变体能够在25℃生长，但不能在37℃生长。在本研究中，对从rpt6 - 1突变体中纯化的20S蛋白酶体的肽酶活性、激活谱和亚基组成进行了表征。
从rpt6 - 1中纯化的20S蛋白酶体在没有激活剂的情况下表现出低水平的肽酶活性，但在有激活剂的情况下具有几乎相同的激活活性，这表明蛋白酶体通道存在门控缺陷。通过二维凝胶电泳分离所有亚基，然后使用基质辅助激光解吸电离飞行时间质谱（MALDI - TOF - MS）鉴定每个亚基，对20S蛋白酶体的组成进行详细分析，结果显示两个亚基α1和α7在电泳迁移率和pI值方面与野生型细胞的亚基不同。这两个α亚基的变化在允许温度下很明显，但在限制温度下的应激反应过程中消失。有趣的是，随着这些变化的消失，rpt6 - 1突变体中20S蛋白酶体的肽酶活性水平恢复到与野生型相同。这些结果表明，α1和α7这两种不同形式的α亚基在rpt6 - 1突变体中阻断了蛋白酶体通道。
rpt6 - 1突变体中20S蛋白酶体的两个α亚基（α1和α7）与其野生型对应亚基不同，并且发现该突变体中的肽酶活性低于野生型菌株。
