Active site mutants in the six regulatory particle ATPases reveal multiple roles for ATP in the proteasome
Active site mutants in the six regulatory particle ATPases reveal multiple roles for ATP in the proteasome
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DOI：
10.1093/emboj/17.17.4909
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发表时间：
1998-09-01
期刊：
影响因子：
11.4
通讯作者：
Finley, D
中科院分区：
文献类型：
--
作者：
Rubin, DM;Glickman, MH;Finley, D
ATP酶家族位于蛋白酶体的调节颗粒内。已经提出这些蛋白质（Rpt 1-Rpt 6）介导底物解折叠，这可能是底物通过从调节颗粒进入蛋白水解核心颗粒的通道易位所需的。为了分析ATP水解在蛋白质分解中的作用，在单个ATP酶的水平上，我们将等同的定点突变引入每个RPT基因的ATP结合基序中。活性位点赖氨酸的非保守取代在六种情况下的四种是致命的，并在两种情况下赋予强烈的生长缺陷。因此，尽管ATP酶具有多样性和序列相似性，但它们在功能上不是冗余的。特定底物的降解可以通过在许多Rpt蛋白中的ATP结合位点取代来抑制，这表明它们在单个底物的降解中合作。不同的rpt突变体的表型缺陷是惊人的变化。最不同的表型是RPT 1突变体，这是强烈的生长缺陷，尽管没有显示出一般的蛋白质周转缺陷。此外，rpt 1是唯一的rpt突变体中显示G（1）细胞周期缺陷。从RPT 2突变体纯化的蛋白酶体显示出对肽酶活性的显著抑制，表明蛋白酶体通道的门控缺陷。总之，ATP通过多种机制促进蛋白酶体的蛋白质分解，如rpt突变体的不同表型所反映的。
A family of ATPases resides within the regulatory particle of the proteasome. These proteins (Rpt1-Rpt6) have been proposed to mediate substrate unfolding, which may be required for translocation of substrates through the channel that leads from the regulatory particle into the proteolytic core particle. To analyze the role of ATP hydrolysis in protein breakdown at the level of the individual ATPase, we have introduced equivalent site-directed mutations into the ATP-binding motif of each RPT gene. Non-conservative substitutions of the active-site lysine were lethal in four of six cases, and conferred a strong growth defect in two cases. Thus, the ATPases are not functionally redundant, despite their multiplicity and sequence similarity. Degradation of a specific substrate can be inhibited by ATP-binding-site substitutions in many of the Rpt proteins, indicating that they co-operate in the degradation of individual substrates. The phenotypic defects of the different rpt mutants were strikingly varied. The most divergent phenotype was that of the rpt1 mutant, which was strongly growth defective despite showing no general defect in protein turnover. In addition, rpt1 was unique among the rpt mutants in displaying a G(1) cell-cycle defect. Proteasomes purified from an rpt2 mutant showed a dramatic inhibition of peptidase activity, suggesting a defect in gating of the proteasome channel. In summary, ATP promotes protein breakdown by the proteasome through multiple mechanisms, as reflected by the diverse phenotypes of the rpt mutants.