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.
Mio
Kazuhiro
K
Neuroscience Research Institute and Biological Information Research Center, National Institute of Advanced Industrial Science and Technology, Umezono 1-1-4, Tsukuba, Ibaraki 305-8568, Japan.
Kubo
Yoshihiro
Y
Ogura
Toshihiko
T
Yamamoto
Tomomi
T
Arisaka
Fumio
F
Sato
Chikara
C
D000818:Animals; D027321:Anion Transport Proteins; D016367:Baculoviridae / Q000235:genetics; D002462:Cell Membrane / Q000502:physiology; D018076:DNA, Complementary; D004591:Electrophoresis, Polyacrylamide Gel; D005822:Genetic Vectors; D018072:Hair Cells, Auditory, Outer / Q000502:physiology*; D007091:Image Processing, Computer-Assisted; D008854:Microscopy, Electron; D008958:Models, Molecular; D008970:Molecular Weight; D010957:Plasmids; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry; D018411:Spodoptera; D000076988:Sulfate Transporters; D014158:Transcription, Genetic
D027321:Anion Transport Proteins; D018076:DNA, Complementary; D011506:Proteins; D011994:Recombinant Proteins; C409207:Slc26a5 protein, rat; D000076988:Sulfate Transporters