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J. Biol. Chem., Vol. 283, Issue 2, 1137-1145, January 11, 2008

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The Motor Protein Prestin Is a Bullet-shaped Molecule with Inner Cavities^*

Kazuhiro Mio, Yoshihiro Kubo^¶||, Toshihiko Ogura^**, Tomomi Yamamoto, Fumio Arisaka, and Chikara Sato¹

From the Neuroscience Research Institute and Biological Information Research Center, National Institute of Advanced Industrial Science and Technology, Umezono 1-1-4, Tsukuba, Ibaraki 305-8568, the Division of Biophysics and Neurobiology, Department of Molecular Physiology, National Institute for Physiological Sciences, Myodaiji, Okazaki, Aichi 444-8585, the ^¶COE Program for Brain Integration and Its Disorders, Tokyo Medical and Dental University Graduate School, Yushima 1-5-45, Bunkyo, Tokyo 113-8519, ^||SORST, Japan Science and Technology Corporation, Kawaguchi, Saitama 332-0012, ^**PRESTO, Japan Science and Technology Agency, 4-1-8 Honcho Kawaguchi, Saitama 332-0012, and the Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Yokohama 226-8501, Japan

Prestin is a transmembrane motor protein localized at the outer hair cells (OHCs) of the mammalian inner ear. Voltage-dependent conformational changes in prestin generate changes in the length of OHCs. A loss of prestin function is reported to induce severe auditory deficiencies, suggesting prestin-dependent changes of OHC length may be at least a part of cochlear amplification. Here we expressed the recombinant FLAG-fused prestin proteins in Sf9 cells and purified to particles of a uniform size in EM. The square-shaped top view of purified prestin, the binding of multiple anti-FLAG antibodies to each prestin particle, the native-PAGE analysis, and the much larger molecular weight obtained from size exclusion chromatography than the estimation for the monomer all support that prestin is a tetramer (Zheng, J., Du, G. G., Anderson, C. T., Keller, J. P., Orem, A., Dallos, P., and Cheatham, M. (2006) J. Biol. Chem. 281, 19916-19924). From negatively stained prestin particles, the three-dimensional structure was reconstructed at 2 nm resolution assuming 4-fold symmetry. Prestin is shown to be a bullet-shaped particle with a large cytoplasmic domain. The surface representation demonstrates indentations on the molecule, and the slice images indicate the inner cavities of sparse densities. The dimensions, 77 x 77 x 115Å_, are consistent with the previously reported sizes of motor proteins on the surface of OHCs.

Received for publication, March 28, 2007 , and in revised form, August 3, 2007.

^* This work was supported by a grant-in-aid for Scientific Research on Priority Areas, Structure of Biological Macromolecular Assemblies, a grant from the Japan New Energy and Industrial Technology Development Organization (to C. S.), a grant from Precursory Research for Embryonic Science and Technology (PRESTO) of the Japan Science and Technology Agency (to T. O.), by research grants from the Ministry of Education, Sciences, Sports, Culture and Technology of Japan, and by a grant from the Japan Society for Promotion of Sciences (to Y. K.). 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.

¹ To whom correspondence should be addressed. Tel.: 81-29-861-5562; Fax: 81-29-861-6478; E-mail: ti-sato{at}aist.go.jp.

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