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• W2154677006 abstract "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 × 77 × 115Å, are consistent with the previously reported sizes of motor proteins on the surface of OHCs. 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 × 77 × 115Å, are consistent with the previously reported sizes of motor proteins on the surface of OHCs. The mammalian ear has specialized function in collecting sound signals and transmitting them to the nerve. Several amplifying systems are developed for sound collection. The pinna and ear canal funnel have suitable shapes for effective sound collection and condensation. The bones of the middle ear (malleus, incus, and stapes) convert the sound energy in the eardrum to the pressure waves in the fluid of the cochlea with 20-40-fold amplification. In the inner ear, a positive feedback loop generates synchronous force by sensing the vibrations within the organ of Corti, which amplifies gained sound signals more than 100 times, although the mechanisms are not clearly understood (1Robles L. Ruggero M.A. Physiol. Rev. 2001; 81: 1305-1352Crossref PubMed Scopus (1111) Google Scholar, 2Geleoc G.S. Holt J.R. Trends Neurosci. 2003; 26: 115-117Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). One of the hypotheses is that voltage-dependent changes in the length of the outer hair cells (OHCs) 2The abbreviations used are:3Dthree-dimensionalAFMatomic force microscopyDDMn-dodecyl β-d-maltosideFSCFourier shell correlationGNGgrowing neural gas networkMRAmultireference alignmentNNneural networkOHCsouter hair cellsRSStokes radiusSAsimulated annealingSECsize exclusion chromatographyTBSTris-buffered saline. generate the lateral membrane motility and amplify the sound (3Brownell W.E. Bader C.R. Bertrand D. de Ribaupierre Y. Science. 1985; 227: 194-196Crossref PubMed Scopus (1479) Google Scholar, 4Kachar B. Brownell W.E. Altschuler R. Fex J. Nature. 1986; 322: 365-368Crossref PubMed Scopus (281) Google Scholar, 5Dallos P. J. Neurosci. 1992; 12: 4575-4585Crossref PubMed Google Scholar). These changes are proposed to be caused by prestin (SLC26A5) (6Zheng J. Shen W. He D.Z. Long K.B. Madison L.D. Dallos P. Nature. 2000; 405: 149-155Crossref PubMed Scopus (994) Google Scholar, 7Ludwig J. Oliver D. Frank G. Klocker N. Gummer A.W. Fakler B. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 4178-4183Crossref PubMed Scopus (130) Google Scholar), which is a membrane integral protein and is categorized to the solute linked carrier (SLC) 26 family of anion transporters. It has been also reported that prestin responds to the membrane potential by harboring cytoplasmic anions (Cl- or HCO-3), as extrinsic voltage sensors, and changes its structure (8Oliver D. He D.Z. Klocker N. Ludwig J. Schulte U. Waldegger S. Ruppersberg J.P. Dallos P. Fakler B. Science. 2001; 292: 2340-2343Crossref PubMed Scopus (364) Google Scholar). Because prestin is densely embedded in the plasma membrane of the OHCs, the total length of the cell also changes as much as 5% as follows: decreases by depolarization and increases by hyperpolarization (9Ashmore J.F. J. Physiol. (Lond.). 1987; 388: 323-347Crossref Scopus (673) Google Scholar, 10Santos-Sacchi J. Dilger J.P. Hear. Res. 1988; 35: 143-150Crossref PubMed Scopus (266) Google Scholar). A voltage-dependent structural change was also observed in heterologously expressed cells, which strongly supports that prestin is a motor protein (6Zheng J. Shen W. He D.Z. Long K.B. Madison L.D. Dallos P. Nature. 2000; 405: 149-155Crossref PubMed Scopus (994) Google Scholar). three-dimensional atomic force microscopy n-dodecyl β-d-maltoside Fourier shell correlation growing neural gas network multireference alignment neural network outer hair cells Stokes radius simulated annealing size exclusion chromatography Tris-buffered saline. The prestin gene (81.4 kDa, 744 amino acids) was first identified using subtractive cloning between the motile OHC cDNA library and the nonmotile inner hair cell library (the other type of hair cell in the organ of Corti) (6Zheng J. Shen W. He D.Z. Long K.B. Madison L.D. Dallos P. Nature. 2000; 405: 149-155Crossref PubMed Scopus (994) Google Scholar). Hydropathic analysis of the sequence and antibody recognition experiments on each side of the membrane suggested the prestin has 10 (11Navaratnam D. Bai J.P. Samaranayake H. Santos-Sacchi J. Biophys. J. 2005; 89: 3345-3352Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar) or 12 (8Oliver D. He D.Z. Klocker N. Ludwig J. Schulte U. Waldegger S. Ruppersberg J.P. Dallos P. Fakler B. Science. 2001; 292: 2340-2343Crossref PubMed Scopus (364) Google Scholar, 12Zheng J. Long K.B. Shen W. Madison L.D. Dallos P. Neuroreport. 2001; 12: 1929-1935Crossref PubMed Scopus (85) Google Scholar) transmembrane segments with relatively large N and C termini in the cytoplasm (7Ludwig J. Oliver D. Frank G. Klocker N. Gummer A.W. Fakler B. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 4178-4183Crossref PubMed Scopus (130) Google Scholar, 12Zheng J. Long K.B. Shen W. Madison L.D. Dallos P. Neuroreport. 2001; 12: 1929-1935Crossref PubMed Scopus (85) Google Scholar). Zheng et al. (13Zheng J. Du G.G. Anderson C.T. Keller J.P. Orem A. Dallos P. Cheatham M. J. Biol. Chem. 2006; 281: 19916-19924Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar) concluded prestin as a tetramer by chemical cross-linking experiment and perfluoro-octanoate electrophoresis, using an affinity-purified specimen. They also revealed that the tetramer is constituted of the dimer unit, and the component subunits are connected by disulfide bonds. An oligomeric structure of prestin was also supported by FRET analysis (11Navaratnam D. Bai J.P. Samaranayake H. Santos-Sacchi J. Biophys. J. 2005; 89: 3345-3352Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 14Greeson J.N. Organ L.E. Pereira F.A. Raphael R.M. Brain Res. 2006; 1091: 140-150Crossref PubMed Scopus (31) Google Scholar). Mutation in the prestin gene was reported to cause autosomal, recessive, nonsyndromic deafness (DFNB14 (15Liu X.Z. Ouyang X.M. Xia X.J. Zheng J. Pandya A. Li F. Du L.L. Welch K.O. Petit C. Smith R.J. Webb B.T. Yan D. Arnos K.S. Corey D. Dallos P. Nance W.E. Chen Z.Y. Hum. Mol. Genet. 2003; 12: 1155-1162Crossref PubMed Scopus (156) Google Scholar)). Homozygous mutant mice have a loss in OHC electromotility in vitro and a hundredfold loss of cochlear sensitivity in vivo, without disrupting mechanoelectrical transduction in OHCs. In heterozygotes, the electromotility was halved, and a significant elevation in cochlear thresholds was observed (16Liberman M.C. Gao J. He D.Z. Wu X. Jia S. Zuo J. Nature. 2002; 419: 300-304Crossref PubMed Scopus (683) Google Scholar). Further study demonstrated that the prestin knock-out mice lose frequency selectivity in the compound action potential tuning curve, supporting the hypothesis of contributing prestin and OHC electromotility in the cochlear amplification (17Cheatham M.A. Huynh K.H. Gao J. Zuo J. Dallos P. J. Physiol. (Lond.). 2004; 560: 821-830Crossref Scopus (128) Google Scholar). In addition, salicylate disrupts voltage-dependent length changes of the OHCs in vitro, through the competitive inhibition of anion incorporation to the prestin (8Oliver D. He D.Z. Klocker N. Ludwig J. Schulte U. Waldegger S. Ruppersberg J.P. Dallos P. Fakler B. Science. 2001; 292: 2340-2343Crossref PubMed Scopus (364) Google Scholar). Therefore, the disruption of prestin is considered as a cause of hearing loss and subjective tinnitus by administration of large doses of aspirin (18Cazals Y. Prog. Neurobiol. 2000; 62: 583-631Crossref PubMed Scopus (202) Google Scholar). Prestin locates on the lateral membrane of the OHC in high densities (19Belyantseva I.A. Adler H.J. Curi R. Frolenkov G.I. Kachar B. J. Neurosci. 2000; 20 (RC1116 (1-5))Crossref Google Scholar, 20Weber T. Zimmermann U. Winter H. Mack A. Kopschall I. Rohbock K. Zenner H.P. Knipper M. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 2901-2906Crossref PubMed Scopus (95) Google Scholar) and in much lower densities on the rest of the membrane, including the basal membrane (21Yu N. Zhu M.L. Zhao H.B. Brain Res. 2006; 1095: 51-58Crossref PubMed Scopus (48) Google Scholar). It has been observed as highly accumulated membrane integral proteins (2,500 particles/μm2) about 11 nm in diameter by using the freeze-fracture technique (22Kalinec F. Holley M.C. Iwasa K.H. Lim D.J. Kachar B. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 8671-8675Crossref PubMed Scopus (214) Google Scholar) and atomic force microscopy (AFM) (23Le Grimellec C. Giocondi M.C. Lenoir M. Vater M. Sposito G. Pujol R. J. Comp. Neurol. 2002; 451: 62-69Crossref PubMed Scopus (25) Google Scholar). Recent AFM observation of Chinese hamster ovary cells expressing recombinant prestin showed similar particles of diameters between 8 and 12 nm on the cell surface (24Murakoshi M. Gomi T. Iida K. Kumano S. Tsumoto K. Kumagai I. Ikeda K. Kobayashi T. Wada H. J. Assoc. Res. Otolaryngol. 2006; 7: 267-278Crossref PubMed Scopus (18) Google Scholar), suggesting that reported particles on the surface of OHCs are the motor protein prestin. The change in OHC length occurs in microseconds (as fast as 20 kHz) (7Ludwig J. Oliver D. Frank G. Klocker N. Gummer A.W. Fakler B. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 4178-4183Crossref PubMed Scopus (130) Google Scholar), the fastest movement among known motor proteins. Structural information of prestin is necessary for understanding the generation of the force and its regulation mechanisms. To address this issue, we purified recombinant prestin protein from baculovirus-infected Sf9 cells, observed the negatively stained molecules using EM, and reconstructed the 3D structure by single particle analysis (25Frank J. Three-dimensional Electron Microscopy of Macromolecular Assemblies: Visualization of Biological Molecules in Their Native State. Oxford University Press, NY2006Crossref Scopus (373) Google Scholar, 26van Heel M. Gowen B. Matadeen R. Orlova E.V. Finn R. Pape T. Cohen D. Stark H. Schmidt R. Schatz M. Patwardhan A. Q. Rev. Biophys. 2000; 33: 307-369Crossref PubMed Scopus (470) Google Scholar, 27Henderson R. Q. Rev. Biophys. 2004; 37: 3-13Crossref PubMed Scopus (178) Google Scholar). Molecular Biology and Infection of Sf9 Cells—Rat prestin cDNA with an hemagglutinin tag at its N terminus (a gift from Dr. Fakler) was used in this study (7Ludwig J. Oliver D. Frank G. Klocker N. Gummer A.W. Fakler B. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 4178-4183Crossref PubMed Scopus (130) Google Scholar). The entire insert (SpcI-HindIII fragment) was subcloned to a pBluescript SK(-) vector. The original hemagglutinin tag was removed by replacing the 5′ end of the NotI (vector)-NruI (insert) with a PCR product starting with GCG GCC GCC ATG. The FLAG tag was inserted at the C-terminal end of prestin by replacing the 3′ end (AccI (insert)-XhoI (vector)) with a PCR product that has a FLAG tag immediately after the prestin coding region in-frame and an XhoI site. The sequence was confirmed by DNA sequencing. The prestin cDNA tagged with the FLAG sequence was further subcloned to the pFastBac donor plasmid (Invitrogen) and used to transform the DH10Bac Escherichia coli strain to obtain the Bacmid DNA containing FLAG-tagged prestin. The Sf9 cells were infected with the recombinant Bacmid. After 96 h of infection, the supernatant, including the virus particles, was collected and applied to the next culture. By repeating this step five times, a high titer virus stock was obtained. For protein isolation, cells were harvested after 96 h of infection using Teflon cell scrapers, collected by centrifuge, immediately frozen, and stored at -80 °C until use (28Mio K. Kubo Y. Ogura T. Yamamoto T. Sato C. Biochem. Biophys. Res. Commun. 2005; 337: 998-1005Crossref PubMed Scopus (40) Google Scholar). Frozen cells were homogenized in 10 volumes (v/w) of TBS (20 mm Tris-HCl, pH 7.4, at 4 °C, 150 mm NaCl) with a Teflon homogenizer. Homogenates were first centrifuged for 15 min at 10,000 × g to remove debris, and the supernatant was further centrifuged at 100,000 × g for 60 min to obtain membrane fraction. All the procedures were performed on ice or at 4 °C. Protein Preparation—The membrane fraction was homogenized with a Teflon homogenizer in 4 ml of TBS (pH 7.4 at 4 °C) containing 50 mm n-dodecyl β-d-maltoside (DDM) (Sigma), protease inhibitor mixture tablets (EDTA-free; Roche Diagnostics), and 0.02% sodium azide. After centrifuging for 10 min at 15,000 × g, the supernatant containing the solubilized FLAG-tagged prestin was loaded onto a column containing 1 ml of anti-FLAG affinity gel (Sigma) pre-equilibrated with the same buffer. The column was then washed with 20 ml of wash buffer (TBS containing 5 mm DDM, 300 mm MgCl2, 0.02% sodium azide), and the bound protein was eluted with elution buffer containing 100 μg/ml FLAG peptide (Sigma). The elution was analyzed by silver staining and by Western blotting, and the mixture of two peak fractions (400 μl) was concentrated to 50 μl with a Microcon centrifuge filter unit YM-50 (Millipore). It was further purified by Superdex 200 (3.2/30) size exclusion chromatography (SEC) in a SMART system (GE Healthcare) with a TBS containing 5 mm DDM, 300 mm MgCl2, and 0.02% sodium azide. The elution of protein was monitored by absorbance at 280 nm and collected 20-μl fractions with a flow rate of 40 μl/min. Protein concentrations were determined using the BCA method (29Smith P.K. Krohn R.I. Hermanson G.T. Mallia A.K. Gartner F.H. Provenzano M.D. Fujimoto E.K. Goeke N.M. Olson B.J. Klenk D.C. Anal. Biochem. 1985; 150: 76-85Crossref PubMed Scopus (18647) Google Scholar). SDS and Native Gel Electrophoresis—The standard method of Laemmli (30Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207231) Google Scholar) was applied for the SDS-PAGE. Samples were mixed with a sample buffer containing 62.5 mm Tris-HCl, pH 6.8, 2% SDS, 25% glycerol, 0.04 m dithiothreitol, and 0.01% bromphenol blue and then incubated at 95 °C for 3 min. Proteins were separated in a 2-15% gradient acrylamide gel with an electrophoresis buffer containing SDS and visualized by silver staining. For Western blotting, proteins electrophoresed in the gel were transferred to a polyvinylidene difluoride membrane and detected with an anti-FLAG antibody (Sigma) and with a secondary antibody for chemiluminescent staining. Chemiluminescent images were obtained using a LAS-3000 mini image analyzer (Fujifilm). In the native gel electrophoresis (31Davis B.J. Ann. N. Y. Acad. Sci. 1964; 121: 404-427Crossref PubMed Scopus (15957) Google Scholar), protein samples were combined with an equal volume of sample buffer (62.5 mm Tris-HCl, pH 6.8, 25% glycerol, and 0.01% bromphenol blue), and electrophoresed for 5 h at 60 V in a 7.5% acrylamide gel containing 0.5 mm DDM or in a 2-15% gradient acrylamide gel that did not contain DDM. The running buffer (25 mm Tris-HCl, pH 8.4, and 192 mm glycine) was supplemented with 0.1 mm DDM for protein stabilization. The separated proteins were compared with molecular standards as follows: thyroglobulin (669 kDa), ferritin (440 kDa), catalase (232 kDa), lactate dehydrogenase (140 kDa), and bovine serum albumin (67 kDa). Estimation of Molecular Weight and Stokes Radius by SEC—To calculate the molecular weight of the FLAG-tagged prestin, high molecular weight standard proteins (GE Healthcare) were used for column calibration (32Laurent T.C. Killander J. J. Chromatogr. 1964; 14: 317-330Crossref Google Scholar, 33Mio K. Ogura T. Hara Y. Mori Y. Sato C. Biochem. Biophys. Res. Commun. 2005; 333: 768-777Crossref PubMed Scopus (40) Google Scholar). The distribution coefficient, Kav, was calculated from the equation Kav = (Ve - V0)/(Vt - V0), where Ve is the elution volume of each reference protein or prestin protein. Column void volume (V0) was measured with blue dextran 2000, and Vt represents total bed volume. All the standards and the prestin were solubilized in the same buffer for prestin purification. Prestin elution was repeated three times. The prestin data is presented by average ± S.D. The same data set was applied to determine the Stokes radius (RS) of FLAG-tagged prestin using a calibration curve, which was constructed by plotting RS of reference proteins versus (-log Kav)1/2 according to the relationship (-log Kav)1/2 = α (β + RS) (32Laurent T.C. Killander J. J. Chromatogr. 1964; 14: 317-330Crossref Google Scholar). Table 1 lists the molecular weight, RS, and obtained elution volume of each standard protein.TABLE 1Molecular mass, Stokes radii, and SEC data of standard proteins and purified prestinMolecular massStokes radiusElution volumeDistribution coefficientkDaÅVeKavAldolase15848.11.2820.275Catalase23252.21.2690.267Ferritin44061.01.1080.162Thyroglobulin66985.00.9550.063Prestin (n = 3)275.6 ± 4.8 (Calculated)56.0 ± 0.3 (Calculated)1.190 ± 0.0040.215 ± 0.003 Open table in a new tab Transmission Electron Microscopy—The solubilized prestin of ∼50 μg/ml was adsorbed by thin carbon films rendered hydrophilic by glow-discharge in low air pressure and supported by copper mesh grids. Samples were washed with 5 drops of double-distilled water, negatively stained two times with 2% uranyl acetate solution for 30 s, blotted, and dried in air. Micrographs of negatively stained particles were recorded in a JEOL 100CX transmission electron microscope at ×55,000 magnification with 100-kV acceleration voltages. Images were recorded on SO-163 films (Eastman Kodak Co.), developed with a D19 developer (Kodak), and digitized with a Scitex Leaf-scan 45 scanner (Leaf Systems Inc) at a pixel size of 1.82 Å at the specimen level. Automated Particle Selection and 3D Reconstruction—Primary selection of prestin projections was performed automatically using the auto-accumulation method with simulated annealing (SA) (34Ogura T. Sato C. J. Struct. Biol. 2004; 146: 344-358Crossref PubMed Scopus (34) Google Scholar). Three hundred particles were extracted into 140 × 140 pixel subframes and used to train the auto pickup system of a three-layer pyramidal-type neural network (NN) (35Ogura T. Sato C. J. Struct. Biol. 2001; 136: 227-238Crossref PubMed Scopus (43) Google Scholar, 36Ogura T. Sato C. J. Struct. Biol. 2004; 145: 63-75Crossref PubMed Scopus (48) Google Scholar, 37Sato C. Hamada K. Ogura T. Miyazawa A. Iwasaki K. Hiroaki Y. Tani K. Terauchi A. Fujiyoshi Y. Mikoshiba K. J. Mol. Biol. 2004; 336: 155-164Crossref PubMed Scopus (78) Google Scholar). Using the trained NN, a total of 14,346 particles was automatically selected from digitized EM films. Particles touching neighboring particles were discarded manually. In total, 13,149 images were analyzed after subtracting the uneven background. The selected particles were aligned rotationally and translationally by multireference alignment (MRA) (25Frank J. Three-dimensional Electron Microscopy of Macromolecular Assemblies: Visualization of Biological Molecules in Their Native State. Oxford University Press, NY2006Crossref Scopus (373) Google Scholar, 26van Heel M. Gowen B. Matadeen R. Orlova E.V. Finn R. Pape T. Cohen D. Stark H. Schmidt R. Schatz M. Patwardhan A. Q. Rev. Biophys. 2000; 33: 307-369Crossref PubMed Scopus (470) Google Scholar). To avoid introducing bias in reconstruction, reference-free alignment procedures are preferred for the first reference images (26van Heel M. Gowen B. Matadeen R. Orlova E.V. Finn R. Pape T. Cohen D. Stark H. Schmidt R. Schatz M. Patwardhan A. Q. Rev. Biophys. 2000; 33: 307-369Crossref PubMed Scopus (470) Google Scholar). In this study, the references at the first cycle were created automatically only by the NN program (36Ogura T. Sato C. J. Struct. Biol. 2004; 145: 63-75Crossref PubMed Scopus (48) Google Scholar), which includes neither subjective procedures by human beings nor any symmetry imposition. The aligned images were subgrouped into 300 classes by the modified growing neural gas network (GNG) method (38Ogura T. Iwasaki K. Sato C. J. Struct. Biol. 2003; 143: 185-200Crossref PubMed Scopus (105) Google Scholar). The class averages were used as new references, and this cycle was repeated 22 times. The orientational Euler angles of the class averages were determined by the echo-correlated 3D reconstruction method using SA (39Ogura T. Sato C. J. Struct. Biol. 2006; 156: 371-386Crossref PubMed Scopus (45) Google Scholar), assuming a 4-fold symmetry. These were used to calculate a 3D structure by the simultaneous iterative reconstruction technique method (40Penczek P. Radermacher M. Frank J. Ultramicroscopy. 1992; 40: 33-53Crossref PubMed Scopus (400) Google Scholar). The reprojections from the initial volume were employed as references for MRA. Each image in the library was aligned and clustered, providing improved class averages. The 3D map was further refined until convergence by the projection matching method (41Penczek P.A. Grassucci R.A. Frank J. Ultramicroscopy. 1994; 53: 251-270Crossref PubMed Scopus (359) Google Scholar) together with intermittent MRA-GNG-averaging cycles. Particle images that correlated poorly with the 3D projections were rejected automatically using the cross-correlation function. Final reconstruction included 98.1% of all the selected images. The Fourier shell correlation (FSC) function was used to assess the resolution of the final 3D map at the threshold of 0.5 (42Harauz G. van Heel M. Optik. 1986; 73: 146-156Google Scholar). Formation of the Prestin-Antibody Complex—Purified prestin and anti-FLAG monoclonal antibody (Sigma) were mixed for 30 min at 4 °C, and excessive antibodies were removed by SEC. Fab fragment of anti-FLAG antibody was generated by papain digestion using a commercial kit (Pierce). The Fab fragments were conjugated with colloidal gold particles (BB International), and the conjugate was separated from nonreacted Fab molecules by 10-30% glycerol gradient centrifugation. The Fab-gold conjugates were mixed with purified prestin, left on ice for 30 min, and negatively stained. In obtaining averages of prestin-antibody complex, 353 prestin-antibody complexes were manually picked up from 32 EM films, averaged, and classified into 15 groups by modified GNG method using preexistent prestin two-dimensional averages without antibody as templates. Expression and Purification of the Prestin Protein—Natural expression of prestin is limited to the outer hair cells of the inner ear and is too scarce for purification. To address this, we constructed a baculovirus containing FLAG-tagged prestin cDNA and infected Sf9 cells. We first expressed and purified either N- or C-terminally tagged prestin. As C-terminally tagged prestin expressed at higher efficiency, we have chosen this construct for further study (data not shown). Expression of C-terminally FLAG-tagged prestin was detected in Sf9 cells using an anti-FLAG antibody. More than 90% of the cells were positive in immunological detection with the anti-FLAG antibody, and about 10% of the cells expressed the fused protein at a much higher level (Fig. 1). Recombinant prestin was strongly localized at the plasma membrane, suggesting that infected cells expressed the FLAG-tagged prestin well, and that the product was correctly integrated into the plasma membrane (Fig. 1). Prestin proteins were concentrated from solubilized membrane fractions using FLAG affinity chromatography. Most of the remaining contaminants, especially those of smaller size, were eliminated by further purification using SEC (Fig. 2A). Three absorbance peaks were observed in SEC analysis as follows: a large asymmetrical peak at 0.87 ml elution, a medium sized peak at 1.17 ml elution, and a small rise at 1.46 ml elution, besides a peak of FLAG peptides at 2.0 ml of elution (Fig. 2B).FIGURE 2Purification of the prestin protein from baculovirus-infected Sf9 cells.A, FLAG-tagged prestin was solubilized from the cell membrane and then purified with immunoaffinity chromatography followed by SEC. M, molecular standards; lane 1, silver staining of the total membrane proteins of the infected cells; lane 2, pass-through from the anti-FLAG affinity column; lane 3, prestin-rich eluate from the affinity gel obtained by adding 100 μg/ml of FLAG peptide; and lane 4, purified prestin obtained by SEC, used for analysis in this study. B, Superdex 200 SEC. Eluate from immunoaffinity gel was concentrated using a Microcon YM-50. The proteins are separated into fractions by Superdex 200 column. Large aggregated protein complexes (inset, bar represents 200 Å) are observed at the 0.87-ml peak. The elution of the prestin protein used for this analysis appears as a small bump at the elution volume of 1.17 ml (arrow). A peak at 2.0 ml is an absorbance of FLAG peptide applied to elute FLAG-fused protein. The elution of proteins was monitored at 280 nm absorbance. C, fractions obtained by SEC were electrophoresed in SDS gel under reducing conditions and visualized by silver staining. Purified prestin is detected at ∼80 kDa, as indicated by an arrowhead. Elution volumes and sizes of molecular standards are indicated at the top and the right, respectively. D, Western blotting using anti-FLAG antibody confirms that the purified protein is the FLAG-tagged prestin.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Aliquots from each SEC fraction were separated in SDS-PAGE and detected by silver staining (Fig. 2C) and Western blotting using an anti-FLAG antibody (Fig. 2D). In both detections, predominant bands were observed at the size estimated from the amino acid composition of prestin (81.4 kDa), suggesting that the prestin protein is correctly expressed on the Sf9 membrane (Fig. 2, C and D). The band intensity is strongest at the elution of 1.16-1.24 ml, which corresponds to the second peak in SEC (Fig. 2B). The 81.4-kDa band is also observed in the fractions at 0.84 and 0.92 ml, together with the higher molecular weight bands, corresponding to the first asymmetrical high peak (Fig. 2, C and D). In the fractions of the first peak, variously sized amorphous aggregations were observed using EM (Fig. 2B, inset), which appeared just after the void volume. Despite attempts at solubilization using various detergents and various mechanical methods (such as ultrasonication), formation of the large complexes could not be avoided. Indeed, these large complexes are tightly bound and could not be dissociated even by boiling in the reducing conditions for SDS-PAGE, as shown in the early fractions (Fig. 2, C and D). In the fractions of the third peak at 1.46 ml of elution, FLAG-fused protein was very low by Western blotting. For further analysis of prestin molecule, we used an SEC fraction with a 1.17-ml peak (a small bump indicated by an arrow in Fig. 2B). Estimation of the Molecular Weight and Diameter of Purified Prestin—SEC was used to estimate the molecular weight and diameters of prestin in aqueous solution (32Laurent T.C. Killander J. J. Chromatogr. 1964; 14: 317-330Crossref Google Scholar, 33Mio K. Ogura T. Hara Y. Mori Y. Sato C. Biochem. Biophys. Res. Commun. 2005; 333: 768-777Crossref PubMed Scopus (40) Google Scholar)." @default.
• W2154677006 created "2016-06-24" @default.
• W2154677006 creator A5005137433 @default.
• W2154677006 creator A5034273012 @default.
• W2154677006 creator A5057625483 @default.
• W2154677006 creator A5081883958 @default.
• W2154677006 creator A5088049227 @default.
• W2154677006 creator A5091640228 @default.
• W2154677006 date "2008-01-01" @default.
• W2154677006 modified "2023-10-12" @default.
• W2154677006 title "The Motor Protein Prestin Is a Bullet-shaped Molecule with Inner Cavities" @default.
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