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Vol. 18, Issue 2, 569-580, February 2007

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The Assembly Pathway of the 19S Regulatory Particle of the Yeast 26S Proteasome

Erika Isono^*,, Kiyoshi Nishihara^*,, Yasushi Saeki^*,, Hideki Yashiroda^||, Naoko Kamata^*, Liying Ge^*,¶, Takashi Ueda^*, Yoshiko Kikuchi^*, Keiji Tanaka^||, Akihiko Nakano^*,#, and Akio Toh-e^*,

*Department of Biological Sciences, Graduate School of Science, University of Tokyo, Tokyo 113-0033, Japan; Entwicklungsgenetik, ZMBP, University of Tübingen, D-72076 Tübingen, Germany; ^||Tokyo Metropolitan Institute of Medical Science, Tokyo 113-8613, Japan; and ^#RIKEN Discovery Research Institute, Saitama 351-0198, Japan

Submitted July 26, 2006; Revised November 13, 2006; Accepted November 20, 2006
Monitoring Editor: William Tansey

The 26S proteasome consists of the 20S proteasome (core particle) and the 19S regulatory particle made of the base and lid substructures, and it is mainly localized in the nucleus in yeast. To examine how and where this huge enzyme complex is assembled, we performed biochemical and microscopic characterization of proteasomes produced in two lid mutants, rpn5-1 and rpn7-3, and a base mutant N rpn2, of the yeast Saccharomyces cerevisiae. We found that, although lid formation was abolished in rpn5-1 mutant cells at the restrictive temperature, an apparently intact base was produced and localized in the nucleus. In contrast, in N rpn2 cells, a free lid was formed and localized in the nucleus even at the restrictive temperature. These results indicate that the modules of the 26S proteasome, namely, the core particle, base, and lid, can be formed and imported into the nucleus independently of each other. Based on these observations, we propose a model for the assembly process of the yeast 26S proteasome.

The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org).

Present addresses: Graduate School of Frontier Sciences, University of Tokyo, Chiba 277-8561, Japan;

Tokyo Metropolitan Institute of Medical Sciences, Tokyo 113-8613, Japan;

^¶ Graduate School of Frontier Sciences, University of Tokyo, Tokyo 108-8639, Japan.

Address correspondence to: Akio Toh-e (toh-e{at}rinshoken.or.jp)

Abbreviations used: CBB, Coomassie brilliant blue; CP, core particle; FRAP, fluorescence recovery after photobleaching; GFP, green fluorescent protein; MCA, methycoumaryl-7-amide; NLS, nuclear localization signal; PCI, proteasome/COP9/Initiation factor; mRFP, monomeric red fluorescent protein; RP, regulatory particle; Rpn, regulatory particle non-ATPase; Rpt, regulatory particle triple A ATPase; Suc-LLVY, succinyl-leucyl-leucyl-valyl-tyrosyl; Ub, ubiquitin; UFD, ubiquitin fusion degradation; UPS, ubiquitin–proteasome system.

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