Laboratory of Protein Metabolism, Tokyo Metropolitan Institute of Medical Science, Setagaya-ku, Tokyo 156-8506, Japan.
Mol Cell. 2011 Jun 10;42(5):637-49. doi: 10.1016/j.molcel.2011.04.021.
The 26S proteasome is a 2.5 MDa macromolecular machine responsible for targeted protein degradation. Recently, four chaperones were identified that promote the assembly of the 19S regulatory particle (RP). Here, we probe the dynamic architecture of the proteasome by applying quantitative proteomics and mass spectrometry (MS) of intact complexes to provide a detailed characterization of how Ubp6 assists this assembly process. Our MS data demonstrate stoichiometric binding of chaperones and Ubp6 to the basal part of the RP. Genetic interactions of Ubp6 with Hsm3, but not with the other chaperones, indicate a functional overlay with Hsm3. Our biochemical data identified Ubp6 as an additional member of the Hsm3 module. Deletions of ubp6 with hsm3 perturb 26S proteasome assembly, which we attribute to an accumulation of ubiquitylated substrates on these assembly precursors. We therefore propose that Ubp6 facilitates proteasomal assembly by clearing ubiquitylated substrates from assembly precursors by its deubiquitylating activity.
26S 蛋白酶体是一种 2.5MDa 的大分子机器，负责靶向蛋白质降解。最近，鉴定出了四种伴侣蛋白，它们促进 19S 调节颗粒 (RP) 的组装。在这里，我们通过应用定量蛋白质组学和完整复合物的质谱 (MS) 来探测蛋白酶体的动态结构，为 Ubp6 如何协助这个组装过程提供了详细的特征描述。我们的 MS 数据表明伴侣蛋白和 Ubp6 与 RP 的基底部分呈化学计量结合。Ubp6 与 Hsm3 的遗传相互作用，但与其他伴侣蛋白没有相互作用，表明与 Hsm3 有功能重叠。我们的生化数据将 Ubp6 鉴定为 Hsm3 模块的另一个成员。ubp6 与 hsm3 的缺失会破坏 26S 蛋白酶体的组装，我们将其归因于这些组装前体上积累了泛素化的底物。因此，我们提出 Ubp6 通过其去泛素化活性从组装前体中清除泛素化的底物，从而促进蛋白酶体的组装。
