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Molecular and Cellular Biology, October 1999, p. 6575-6584, Vol. 19, No. 10
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Rpn9 Is Required for Efficient Assembly of the Yeast 26S Proteasome

Junko Takeuchi,1 Masahiro Fujimuro,2 Hideyosi Yokosawa,2 Keiji Tanaka,3 and Akio Toh-e1,*

Department of Biological Sciences, Graduate School of Science, University of Tokyo, Hongo, Tokyo 113-0033,1 Department of Biochemistry, Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo 060-0812,2 and Metropolitan Institute of Medical Science, Honkomagome, Tokyo 113-0021,3 Japan

Received 8 March 1999/Returned for modification 13 April 1999/Accepted 29 June 1999

We have isolated the RPN9 gene by two-hybrid screening with, as bait, RPN10 (formerly SUN1), which encodes a multiubiquitin chain receptor residing in the regulatory particle of the 26S proteasome. Rpn9 is a nonessential subunit of the regulatory particle of the 26S proteasome, but the deletion of this gene results in temperature-sensitive growth. At the restrictive temperature, the Delta rpn9 strain accumulated multiubiquitinated proteins, indicating that the RPN9 function is needed for the 26S proteasome activity at a higher temperature. We analyzed the proteasome fractions separated by glycerol density gradient centrifugation by native polyacrylamide gel electrophoresis and found that a smaller amount of the 26S proteasome was produced in the Delta rpn9 cells and that the 26S proteasome was shifted to lighter fractions than expected. The incomplete proteasome complexes were found to accumulate in the Delta rpn9 cells. Furthermore, Rpn10 was not detected in the fractions containing proteasomes of the Delta rpn9 cells. These results indicate that Rpn9 is needed for incorporating Rpn10 into the 26S proteasome and that Rpn9 participates in the assembly and/or stability of the 26S proteasome.

* Corresponding author. Mailing address: Department of Biological Sciences, Graduate School of Science, University of Tokyo, Hongo, Tokyo 113-0033, Japan. Phone and Fax: 81-3-5684-9420. E-mail: toh-e{at}biol.s.u-tokyo.ac.jp.

Molecular and Cellular Biology, October 1999, p. 6575-6584, Vol. 19, No. 10
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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