The motor protein prestin is a bullet-shaped molecule with inner cavities
The motor protein prestin is a bullet-shaped molecule with inner cavities
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DOI：
10.1074/jbc.m702681200
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发表时间：
2008-01-11
影响因子：
4.8
通讯作者：
Sato, Chikara
中科院分区：
文献类型：
--
作者：
Mio, Kazuhiro;Kubo, Yoshihiro;Sato, Chikara
普雷斯廷是位于哺乳动物内耳外毛细胞（OHC）的跨膜马达蛋白。普雷斯廷的电压依赖性构象变化产生OHC长度的变化。据报道，普雷斯廷功能的丧失会引起严重的听觉缺陷，这表明OHC长度的普雷斯丁依赖性变化可能至少是耳蜗放大的一部分。在此，我们在Sf 9细胞中表达了重组FLAG融合的普雷斯廷蛋白，并在EM中纯化成均匀大小的颗粒。纯化的普雷斯廷的正方形顶视图、多种抗FLAG抗体与每个普雷斯廷颗粒的结合、天然PAGE分析和从尺寸排阻色谱法获得的比单体的估计大得多的分子量都支持普雷斯廷是四聚体（Zheng，J.，杜，G. G.，安德森角T.，Keller，J. P.，Orem，A.，Dallos，P.，和Cheatham，M.（2006）J.Biol.Chem.281，19916-19924）。从负染色的普雷斯廷颗粒，在2 nm分辨率下重建三维结构，假设4重对称。普雷斯廷被证明是一个子弹形的颗粒与一个大的胞质结构域。表面表示显示分子上的压痕，切片图像显示稀疏密度的内腔。尺寸为77 × 77 × 115埃，与先前报道的外毛细胞表面马达蛋白的尺寸一致。
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 115 angstrom, are consistent with the previously reported sizes of motor proteins on the surface of OHCs.