| Research Abstract |
The yeast 26S proteasome, a ca. 200kDa protein complex, consists of 20S proteasome and the 19S regulatory particle. This protein complex abundantly exists in the yeast cells, but because of its large size, it was difficult to purify holoenzyme intact. During this project, we devised a quick and easy method to purify the 26S proeasome; we constructed yeast strains, one of whose proteasome subunits, Rpnl, Rpnl 1, and Prel, had been tagged with 3xFlag and proteasomes produced in these strains were pulled down with anti-Hag antibody. By exploiting this purification method, we characterized proteasomes produced by rpnZ temperature-sensitive mutants and found that a new subcomplex existed in the extract of the rpn7-3 mutant cells. A major obstacle for biochemical study of the 26S proteasome is the absence of proper substrate, polyubiquitinated protein. In this study, we developed a method to produce polyubiquitinated Sici by in vitro reaction by using Rps5 as an E3 enzyme. A key idea is to introduce a PY motif, that attract Rsp5, into the Sici sequence. We confirmed that polyLibiquitinated Sici is a very good substrate of' the 26S proteasome.
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