C Termini of Proteasomal ATPases Play Nonequivalent Roles in Cellular Assembly of Mammalian 26 S Proteasome
C Termini of Proteasomal ATPases Play Nonequivalent Roles in Cellular Assembly of Mammalian 26 S Proteasome
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DOI：
10.1074/jbc.m111.246793
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发表时间：
2011-07-29
影响因子：
4.8
通讯作者：
DeMartino, George N.
中科院分区：
文献类型：
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作者：
Kim, Young-Chan;DeMartino, George N.
26 S蛋白酶体由2个亚基组成：20 S蛋白酶体和PA 700/19 S调节颗粒。这些亚复合物组装成26 S蛋白酶体的细胞机制和控制组装过程的分子决定因素尚不清楚。在这里，我们证明了在26 S蛋白酶体组装在哺乳动物细胞中的PA 700的六个AAA亚基（Rpt 1-Rpt 6）的C末端的非等效作用。Rpt 3和Rpt 5的C-末端HbYX基序（其中Hb是疏水残基，Y是酪氨酸，X是任何氨基酸）对26 S蛋白酶体的组装是必不可少的，但第三个亚基Rpt 2的C-末端HbYX基序则不是。三个非HbYX基序Rpt亚基的C末端都不是细胞26 S蛋白酶体组装所必需的，尽管Rpt 6的最后三个残基的缺失使20 S-PA 700相互作用不稳定。由于C-末端截短而不能组装成26 S蛋白酶体的Rpt亚基被整合到完整的PA 700中。此外，完整的PA 700积累作为一个孤立的亚复合物时，细胞20 S蛋白酶体含量减少的RNAi。这些结果表明，20 S蛋白酶体不是组装PA 700的必需模板。总的来说，这些结果确定了26 S蛋白酶体组装所需的两个Rpt亚基的特定结构元件，证明PA 700可以独立于20 S蛋白酶体组装，并表明完整的PA 700是26 S蛋白酶体组装的细胞途径中的直接中间体。
The 26 S proteasome comprises two multisubunit subcomplexes as follows: 20 S proteasome and PA700/19 S regulatory particle. The cellular mechanisms by which these subcomplexes assemble into 26 S proteasome and the molecular determinants that govern the assembly process are poorly defined. Here, we demonstrate the nonequivalent roles of the C termini of six AAA subunits (Rpt1-Rpt6) of PA700 in 26 S proteasome assembly in mammalian cells. The C-terminal HbYX motif (where Hb is a hydrophobic residue, Y is tyrosine, and X is any amino acid) of each of two subunits, Rpt3 and Rpt5, but not that of a third subunit Rpt2, was essential for assembly of 26 S proteasome. The C termini of none of the three non-HbYX motif Rpt subunits were essential for cellular 26 S proteasome assembly, although deletion of the last three residues of Rpt6 destabilized the 20 S-PA700 interaction. Rpt subunits defective for assembly into 26 S proteasome due to C-terminal truncations were incorporated into intact PA700. Moreover, intact PA700 accumulated as an isolated subcomplex when cellular 20 S proteasome content was reduced by RNAi. These results indicate that 20 S proteasome is not an obligatory template for assembly of PA700. Collectively, these results identify specific structural elements of two Rpt subunits required for 26 S proteasome assembly, demonstrate that PA700 can be assembled independently of the 20 S proteasome, and suggest that intact PA700 is a direct intermediate in the cellular pathway of 26 S proteasome assembly.