Analysis of polyubiquitin conjugates reveals that the Rpn10 substrate receptor contributes to the turnover of multiple proteasome targets
Analysis of polyubiquitin conjugates reveals that the Rpn10 substrate receptor contributes to the turnover of multiple proteasome targets
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DOI：
10.1074/mcp.m400220-mcp200
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发表时间：
2005-06-01
影响因子：
7
通讯作者：
Deshaies, RJ
中科院分区：
文献类型：
--
作者：
Mayor, T;Lipford, JR;Deshaies, RJ
多聚泛素受体Rpn 10将泛素化的Sic 1靶向26 S蛋白酶体进行降解。相反，至少一种泛素-蛋白酶体系统（UPS）底物CPY* 的周转不受RPN 10缺失的影响。为了区分RPN 10是否只参与一小部分细胞周期调节因子（包括Sic 1）的周转，或者在UPS中发挥更普遍的作用，我们试图开发一种通用方法，使我们能够调查选择性地在RPN 10 Delta细胞中积累的泛素化蛋白质的光谱。来自表达六组氨酸标记的泛素（H-6-泛素）的酵母细胞的多聚泛素缀合物首先在多聚泛素结合蛋白亲和树脂上富集。然后将该材料变性并进行IMAC以回收H-6-遍在蛋白和它可能共价连接的蛋白质。使用这种方法，我们确定了127个蛋白质是26 S蛋白酶体的候选底物。然后，我们对缺乏Rpn 10（rpn 10 Delta）的细胞的泛素缀合物进行测序，并鉴定了54种从rpn 10 Delta细胞中独特回收的蛋白质。这些包括UPS的两个已知靶点，细胞周期调节因子Sic 1和转录激活因子Gcn 4。我们的方法比较野生型和突变体细胞中的泛素缀合物蛋白质组具有分辨能力，以确定即使是一个非常不丰富的转录调控蛋白，并应普遍适用于映射酶底物网络的UPS。
The polyubiquitin receptor Rpn10 targets ubiquitylated Sic1 to the 26S proteasome for degradation. In contrast, turnover of at least one ubiquitin-proteasome system ( UPS) substrate, CPY*, is impervious to deletion of RPN10. To distinguish whether RPN10 is involved in the turnover of only a small set of cell cycle regulators that includes Sic1 or plays a more general role in the UPS, we sought to develop a general method that would allow us to survey the spectrum of ubiquitylated proteins that selectively accumulate in rpn10 Delta cells. Polyubiquitin conjugates from yeast cells that express hexahistidine-tagged ubiquitin (H-6-ubiquitin) were first enriched on a polyubiquitin binding protein affinity resin. This material was then denatured and subjected to IMAC to retrieve H-6-ubiquitin and proteins to which it may be covalently linked. Using this approach, we identified 127 proteins that are candidate substrates for the 26S proteasome. We then sequenced ubiquitin conjugates from cells lacking Rpn10 (rpn10 Delta) and identified 54 proteins that were uniquely recovered from rpn10 Delta cells. These include two known targets of the UPS, the cell cycle regulator Sic1 and the transcriptional activator Gcn4. Our approach of comparing the ubiquitin conjugate proteome in wild-type and mutant cells has the resolving power to identify even an extremely inabundant transcriptional regulatory protein and should be generally applicable to mapping enzyme substrate networks in the UPS.