Rpn7 is required for the structural integrity of the 26 S proteasome of Saccharomyces cerevisiae
Rpn7 is required for the structural integrity of the 26 S proteasome of Saccharomyces cerevisiae
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DOI：
10.1074/jbc.m314231200
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发表时间：
2004-06-25
影响因子：
4.8
通讯作者：
Toh-e, A
中科院分区：
文献类型：
--
作者：
Isono, E;Saeki, Y;Toh-e, A
Rpn 7是26 S蛋白酶体调节颗粒的lid亚基之一。已知RPN 7基因是必需的，但其功能仍有待阐明。为了探索Rpn 7的功能，我们分离并鉴定了温度敏感的Rpn 7突变体。所有的rpn 7突变体获得积累的多聚泛素化蛋白时，在限制性温度下生长。在限制性温度下，发现使用称为rpn 7 -3的rpn 7突变体之一的实验中，N端规则底物（Ub-Arg-β-半乳糖苷酶）、UFD途径底物（Ub-Pro-β-半乳糖苷酶）和细胞周期调节剂（Pds 1和Clb 2）是稳定的，表明其在泛素-蛋白酶体途径中的缺陷。随后对rpn 7 -3细胞中26 S蛋白酶体结构的分析表明，缺陷在于26 S全酶的组装。最显着的特点的蛋白酶体的rpn 7 -3突变体是，一个盖子亚复合物亲和纯化的rpn 7 -3细胞生长在限制性温度只包含5的8个盖子组件，一种现象，还没有被报道在以前分离的盖子突变体。根据这些结果，我们得出结论，Rpn 7是通过建立正确的盖结构来保持26 S复合物完整性所必需的。
Rpn7 is one of the lid subunits of the 26 S proteasome regulatory particle. The RPN7 gene is known to be essential, but its function remains to be elucidated. To explore the function of Rpn7, we isolated and characterized temperature-sensitive rpn7 mutants. All of the rpn7 mutants obtained accumulated poly-ubiquitinated proteins when grown at the restrictive temperature. The N-end rule substrate (Ub-Arg-beta-galactosidase), the UFD pathway substrate ( Ub-Pro-beta-galactosidase), and cell cycle regulators (Pds1 and Clb2) were found to be stabilized in experiments using one of the rpn7 mutants termed rpn7-3 at the restrictive temperature, indicating its defect in the ubiquitin-proteasome pathway. Subsequent analysis of the structure of the 26 S proteasome in rpn7-3 cells suggested that the defect was in the assembly of the 26 S holoenzyme. The most striking characteristic of the proteasome of the rpn7-3 mutant was that a lid subcomplex affinity-purified from the rpn7-3 cells grown at the restrictive temperature contained only 5 of the 8 lid components, a phenomenon that has not been reported in the previously isolated lid mutants. From these results, we concluded that Rpn7 is required for the integrity of the 26 S complex by establishing a correct lid structure.