REGULATORY MECHANISMS OF THE 26S PROTEASOME ASSEMBLY

在本研究中，我们研究了26S蛋白酶体组装的调控机制，得到了以下结果。1. 我们建立了从出芽酵母中快速分离19S调节复合体、盖子复合体和26S蛋白酶体底复合体的方法。Rpn10是一个多泛素链结合亚基，被发现是lid复合物的一个组成部分。2. 接下来，我们研究了Rpn10在26S蛋白酶体组装中的作用。我们利用不同的RPN10缺失突变体，分析了它们的生长、26S蛋白酶体的数量以及它们对rpn12-1菌株温度敏感性的抑制作用，发现RPN10的n端区域在26S蛋白酶体的组装中起重要作用。3. 除Rpn10外，研究还发现泛素样蛋白Rad23和Dsk2作为多泛素链结合蛋白，将多泛素化蛋白招募到26S蛋白酶体。4. 以Rpnl2为诱饵，通过双杂交筛选，分离到一个新的蛋白Nob1。在生长阶段检测到该蛋白，而在静止阶段未检测到该蛋白。在前一阶段，它与26S蛋白酶体结合。我们提出Nob1是一个候选蛋白，以生长依赖的方式在26S蛋白酶体组装中起作用。

In this study, we investigated the regulatory mechanisms of the 26S proteasome assembly and obtained the following results. 1. We established methods for rapid isolation of the 19S regulatory complex, the lid complex, and the base complex of the 26S proteasome from budding yeasts. Rpn10, a multiubiquitin chain-binding subunit, was found to be as a component of the lid complex. 2. We next investigated the role of Rpn10 in the 26S proteasome assembly. Using, various deletion mutants of RPN10, we analyzed their growth, the amounts of the 26S proteasome, and their suppressive effects on the temperature-sensitivity of rpn12-1 strain, and found that the N-terminal region of Rpn10 plays an important role in the 26S proteasome assembly. 3. In addition to Rpn10, Rad23 and Dsk2, ubiquitin-like proteins, were found to act as multiubiquitin chain-binding proteins to recruit polyubiquitinated proteins to the 26S proteasome. 4. By two-hybrid screening using Rpnl2 as bait, a novel protein, Nob1, was isolated. This protein was detected in the growing stage but not in the stationary stage. In the former stage, it was bound to the 26S proteasome. We proposed that Nob1 is a candidate protein functioning in the 26S proteasome assembly in a growth-dependent manner.