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J. Biol. Chem., Vol. 279, Issue 22, 22799-22802, May 28, 2004

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A Single-headed Recombinant Fragment of Dictyostelium Cytoplasmic Dynein Can Drive the Robust Sliding of Microtubules^*

Masaya Nishiura, Takahide Kon, Katsuyuki Shiroguchi, Reiko Ohkura, Tomohiro Shima, Yoko Y. Toyoshima, and Kazuo Sutoh

From the Department of Life Sciences, University of Tokyo, Komaba 3–8-1, Meguro-ku, Tokyo 153–8902 and Okazaki Institute for Integrative Bioscience, National Institutes of Natural Sciences, Higashiyama 5–1, Myodaiji, Okazaki 444–8787, Japan

A cytoplasmic dynein is a microtubule-based motor protein involved in diverse cellular functions, such as organelle transport and chromosome segregation. The dynein has two ring-shaped heads that contain six repeats of the AAA domain responsible for ATP hydrolysis. It has been proposed that the ATPase-dependent swing of a stalk and a stem emerging from each of the heads generates the power stroke (Burgess, S.A. (2003) Nature 421, 715–718). To understand the molecular mechanism of the dynein power stroke, it is essential to establish an easy and reproducible method to express and purify the recombinant dynein with full motor activities. Here we report the expression and purification of the C-terminal 380-kDa fragment of the Dictyostelium cytoplasmic dynein heavy-chain fused with an affinity tag and green fluorescent protein. The purified single-headed recombinant protein drove the robust minus-end-directed sliding of microtubules at a velocity of 1.2 µm/s. This recombinant protein had a high basal ATPase activity (4s^{– 1}), which was further activated by >15-fold on the addition of 40 µM microtubules. These results show that the 380-kDa recombinant fragment retains all the structures required for motor functions, i.e. the ATPase activity highly stimulated by microtubules and the robust motility.

Received for publication, December 8, 2003 , and in revised form, March 1, 2004.

^* This work was supported by a grant-in-aid for scientific research (B) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (to K. S.) and by a Core Research for Evolution Science and Technology (CREST) program grant from the Japan Science and Technology Cooperation (to K. S. and Y. Y. T.) 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.

To whom correspondence should be addressed. Tel. and Fax: 81-3-5454-6751; E-mail: sutoh{at}bio.c.u-tokyo.ac.jp.

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