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J. Biol. Chem., Vol. 280, Issue 38, 32586-32593, September 23, 2005

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Interaction of a Small Heat Shock Protein of the Fission Yeast, Schizosaccharomyces pombe, with a Denatured Protein at Elevated Temperature^*

Maya Hirose, Hideki Tohda, Yuko Giga-Hama, Reiko Tsushima, Tamotsu Zako, Ryo Iizuka, Changi Pack¶, Masataka Kinjo¶, Noriyuki Ishii||, and Masafumi Yohda1

From the Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, 2-24-16, Naka-cho, Koganei-shi, Tokyo 184-8588, ASPEX Division, Asahi Glass Co., Ltd., 1150 Hazawa-cho, Kanagawa-ku, Yokohama-shi, Kanagawa 221-8755, ^¶Laboratory of Supramolecular Biophysics, Research Institute for Electronic Science, Hokkaido University, N12W6, Kita-ku, Sapporo, Hokkaido 060-0812, and the ^||Biological Information Research Center, National Institute of Advanced Industrial Science and Technology, Tsukuba Central 6, 1-1-1, Higashi, Tsukuba-shi, Ibaraki, 305-8566, Japan

We have expressed, purified, and characterized one small heat shock protein of the fission yeast Schizosaccharomyces pombe, SpHsp16.0. SpHsp16.0 was able to protect citrate synthase from thermal aggregation at 45 °C with high efficiency. It existed as a hexadecameric globular oligomer near the physiological growth temperature. At elevated temperatures, the oligomer dissociated into small species, probably dimers. The dissociation was completely reversible, and the original oligomer reformed immediately after the temperature dropped. Large complexes of SpHsp16.0 and denatured citrate synthase were observed by size exclusion chromatography and electron microscopy following incubation at 45 °C and then cooling. However, such large complexes did not elute from the size exclusion column incubated at 45 °C. The denatured citrate synthase protected from aggregation was trapped by a GroEL trap mutant at 45 °C. These results suggest that the complex of SpHsp16.0 and denatured citrate synthase at elevated temperatures is in the transient state and has a hydrophobic nature. Analyses of the interaction between SpHsp16.0 and denatured citrate synthase by fluorescence cross-correlation spectrometry have also shown that the characteristics of SpHsp16.0-denatured citrate synthase complex at the elevated temperature are different from those of the large complex obtained after the shift to lowered temperatures.

Received for publication, April 15, 2005 , and in revised form, July 25, 2005.

^* This work was supported by the Ministry of Education, Science, Sports, Culture, and Technology through Tokyo University of Agriculture and Technology as part of the 21st Century Center of Excellence program of the "Future Nano-Materials" research and education project. This work was also supported by Grants-in-aid for Scientific Research on Priority Areas 15032212 and 17028013 and by a grant of the National Project on Protein Structural and Functional Analyses from the Ministry of Education, Science, Sports and Culture of Japan (to M. Y.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1.

¹ To whom correspondence should be addressed: Dept. of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, 2-24-16, Naka-cho, Koganei-shi, Tokyo 184-8588, Japan. Tel. and Fax: 81-42-388-7479; E-mail: yohda{at}cc.tuat.ac.jp.

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