| Research Abstract |
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.
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