The motor protein prestin is a bullet-shaped molecule with inner cavities

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 A, are consistent with the previously reported sizes of motor proteins on the surface of OHCs.