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Originally published In Press as doi:10.1074/jbc.M513854200 on May 8, 2006

J. Biol. Chem., Vol. 281, Issue 29, 19916-19924, July 21, 2006
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Analysis of the Oligomeric Structure of the Motor Protein Prestin*

Jing Zheng{ddagger}1, Guo-Guang Du{ddagger}, Charles T. Anderson{ddagger}, Jacob P. Keller{ddagger}, Alex Orem{ddagger}, Peter Dallos§, and MaryAnn Cheatham{ddagger}

From the {ddagger}Department of Communication Sciences and Disorders, Northwestern University, and §Department of Neurobiology and Physiology, The Neuroscience Institute, Northwestern University, Evanston, Illinois 60208

Received for publication, December 29, 2005 , and in revised form, May 1, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Prestin, a member of the solute carrier family 26, is expressed in the basolateral membrane of outer hair cells. This protein provides the molecular basis for outer hair cell somatic electromotility, which is crucial for the frequency selectivity and sensitivity of mammalian hearing. It has long been known that there are abundantly expressed ~11-nM protein particles present in the basolateral membrane. These particles were hypothesized to be the motor proteins that drive electromotility. Because the calculated size of a prestin monomer is too small to form an ~11-nM particle, the possibility of prestin oligomerization was examined. We investigated possible quaternary structures of prestin by lithium dodecyl sulfate-PAGE, perfluoro-octanoate-PAGE, a membrane-based yeast two-hybrid system, and chemical cross-linking experiments. Prestin, obtained from different host or native cells, is resistant to dissociation by lithium dodecyl sulfate and behaves as a stable oligomer on lithium dodecyl sulfate-PAGE. In the membrane-based yeast two-hybrid system, homo-oligomeric interactions between prestin-bait/prestin-prey suggest that prestin molecules can associate with each other. Chemical cross-linking experiments, perfluoro-octanoate-PAGE/Western blot, and affinity purification experiments all indicate that prestin exists as a higher order oligomer, such as a tetramer, in prestin-expressing yeast, mammalian cell lines and native outer hair cells. Our data from experiments using hydrophobic and hydrophilic reducing reagents suggest that the prestin dimer is connected by a disulfide bond embedded in the prestin hydrophobic core. This stable dimer may act as the building block for producing the higher order oligomers that form the ~11-nM particles in the outer hair cell's basolateral membrane.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Hearing impairment, the most common congenital sensory defect, affects millions of people from newborns to senior citizens, resulting in large hearing-related health care costs (1). Causes of hearing impairment are often associated with damage to outer hair cells (OHCs).2 These sensory receptor cells, located in the mammalian organ of Corti, rapidly change their length (2) and stiffness (3) at acoustic frequencies when their transmembrane voltage is altered. Corresponding to this somatic cell-length change, OHCs exhibit voltage-dependent non-linear capacitance (4). This unique somatic electromotility is thought to provide active mechanical amplification of the cochlear response to sound (5). It has long been known that large (~11-nM diameter) membrane protein particles constitute a substantial portion of the lateral membrane in OHCs (6). It is suspected that these abundantly expressed particles are the "motor proteins" responsible for somatic electromotility (7).

Prestin, the OHC motor protein (8), is located at the same location where the 11-nM protein particles are found, i.e. in the lateral membrane of OHCs (9–11). When prestin is heterologously expressed in several mammalian cell lines, the prestin-expressing cells demonstrate all of the characteristics that are unique to OHCs; that is, voltage-driven non-linear capacitance and shape changes (8), electro-mechanical reciprocality (12, 13), and mechanical force generation with constant amplitude and phase up to a stimulating frequency of at least 20 kHz (12). Such electromotile responses in prestin-transfected cells can also be blocked by salicylate (14), an inhibitor of electromotility in OHCs (15–17). In addition, OHCs from prestin knock-out (KO) mice do not have somatic electromotility (18). Prestin KO mice also lose frequency selectivity and have a 40–60-db reduction in sensitivity (19). In humans mutations in the Prestin gene are also known to cause hearing impairment (20).

Prestin is a glycoprotein (21) belonging to the anion transport family called solute carrier 26 (SLC26) (22). Several members of this family, including prestin, are involved in human diseases including deafness, Pendred syndrome, congenital chloride diarrhea, diastrophic dysplasia, achondro-genesis type IB, and atelosteogenesis type II (for review, see Ref. 23). Although SLC26 proteins play important roles, there is very little known about the relationship between the structure and function of these proteins, particularly their quaternary structures.

Prestin, SLC26A5, has 744 amino acids with a predicted molecular mass of 81.4 kDa. Deglycosylated prestin has an apparent molecular mass of ~70 kDa when observed on a SDS-PAGE/Western blot. The apparent molecular mass of glycosylated prestin ranges from ~80 to ~100 kDa and is cell line-dependent (21). According to the predicted topology map, there are ~400 amino acids that form the ~12 membrane-associated domains (24). Based on the average volume per mass ratio of known proteins, a simple calculation shows that it is highly unlikely that one prestin subunit would inhabit an area as large as that occupied by the 11-nM particles. This raises the question of whether prestin exists as an oligomer in the plasma membrane. Therefore, unveiling prestin's quaternary structure is essential for understanding the mechanisms of the protein function. Hence, the goal of the present paper is to evaluate prestin's quaternary structure using different biological and genetic approaches. Results demonstrate that prestin can form higher order oligomers, probably tetramers, both in OHCs and in heterologous expression systems.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Antibodies
The rabbit anti-C terminus mPres (anti-C-mPres) polyclonal antibody (21) was used in a 1:2000 dilution in immunofluorescence and Western blot experiments. Anti-V5 (Invitrogen) and anti-Xpress (Invitrogen) antibodies were used in a 1:5000 dilution in Western blot. Donkey anti-rabbit IgG-HRP (horseradish peroxidase) and donkey anti-mouse IgG-HRP were purchased from Pierce or Jackson ImmunoResearch.

Prestin-expressing Constructs and Their Hosts
Native Tissue—All surgical and experimental procedures were conducted in accordance with the policies of Northwestern University's Animal Care and Use Committee and the National Institutes of Health. Cochleae from either wild type or prestin KO mice were collected in CelLytic mammalian tissue lysis/extraction reagent (Sigma C3228) supplemented with a combination of inhibiters (100 µg/ml phenylmethylsulfonyl fluoride, 1:50 protease inhibitor mixture (Sigma P8340), 0.01% thimerosal, 10 units/ml DNase). In some case 100 mM iodoacetamide was also added.

Mammalian Cell Expression System—C-tag prestin, gerbil prestin tagged at the C terminus with the hexahistidine and V5 epitope, was described previously (25). The plasmid, pcDNA3.1-CAT (chloramphenicol acetyltransferase), was used as a transfection control. C-tag prestin and pcDNA 3.1-CAT plasmids were transiently expressed in TSA-201 cells, a subclone of human embryonic kidney 293 cells. TSA-201 cells were cultured and transiently transfected with Effectene (Qiagen) as previously described (25). After 48 h of incubation cells were collected in either the Sigma CelLytic cell lysis/extraction reagent or hypotonic lysis buffer (10 mM Tris, pH 7.5, 50 mM sucrose, 1 mM EDTA) supplemented with the inhibitor combination and 100 mM iodoacetamide.

Yeast Expression System—Mouse prestin (from Glu-8 to Ala-744) or the C-terminal domain (from Val-499 to Ala-744) was inserted into the yeast expression vector pYES2/NT-A (Invitrogen) under the control of the GAL1 promoter. Constructs were tagged N-terminally with both the His tag and the Xpress epitope and transformed into Saccharomyces cerevisiae strain INVSc1 (His–, Leu–, Trp–, and Ura–) (Invitrogen) and grown on selective plates or SC medium without uracil according to the manufacturer's instructions (Invitrogen). The selected yeast clone was grown overnight at 30 °C in SC medium without uracil containing 2% glucose with shaking. The yeast were harvested by centrifugation and were then re-grown in induction medium (SC medium without uracil plus 2% galactose) for 24 h. Prestin-expressing yeast were collected by centrifugation and stored at –80 °C. For prestin-protein expression analysis, yeast pellets were resuspended in breaking buffer (50 mM sodium phosphate, pH 7.4, 5% glycerol) supplemented with the inhibiter combination and 100 mM iodoacetamide. Acid-washed glass beads (420–500 µm) were added to yeast to break cell walls. Yeast lysate was then either mixed with lithium dodecyl sulfate (LDS) or perfluoro-octanoate (PFO) loading buffer and run on LDS-PAGE or PFO-PAGE/Western blot.

Immunofluorescence Experiments
After the yeast were grown in induction medium for 6 h, the induced culture was mixed with 3.7% formaldehyde and incubated at room temperature with gentle shaking for 30 min. Yeast were harvested by centrifugation and suspended in 2% formaldehyde/PBS at 4 °C overnight and then rinsed 4 times with PBS. The yeast were then resuspended in protoplast buffer (PBS plus 1.2 M sorbitol, 1% beta-mercaptoethanol, and ~50 µg/ml lyticase) and incubated at 37 °C for 1 h. The resulting cells/spheroplasts were collected by centrifugation at 16,000 x g for 30 s and washed 3 times with sorbitol buffer (PBS plus 1.2 M sorbitol) before being permeabilized in 0.1% Triton X-100/sorbitol buffer (10 min on ice). Suspended yeast were then plated on a multiwell slide (Carlson Scientific, Peotone, IL) coated with 0.1% poly-L-lysine and incubated in PBS containing anti-C-mPres. After washing with PBS, the samples were incubated with fluorescein isothiocyanate-conjugated donkey anti-rabbit IgG. The samples were mounted on glass slides with mounting solution (Fluoromount-G) and observed using a Leica confocal system with a standard configuration DMRXE7 microscope.

LDS-PAGE/Western Blot
After homogenization, low speed centrifugation (800 x g for 10 min) was first applied to the samples to separate the nuclei, unlysed cells, and bone structures. Cell lysates were then mixed with 2x LDS Laemmli sample buffer combined with different concentrations of reducing reagents including either dithiothreitol (DTT), beta-mercaptoethanol, or ethanedithiol before being loaded on a 4–20% Precise gel (Pierce) or a 5% NEXT gel (New Electrophoresis X'PRESS Technology) (Amresco, Solon, OH). LDS was used instead of SDS because the latter precipitates in the cold (49). For NEXT-PAGE, the buffer system was also purchased from Amresco. After separation the gel proteins were electrotransferred onto a nitrocellulose membrane or Immobilon-P transfer membrane (Millipore), blocked with 2% nonfat dry milk and 2% bovine serum albumin, and reacted with primary antibodies (anti-C-mPres, anti-V5, or anti-Xpress) and then with the corresponding secondary antibody (donkey anti-rabbit IgG-horseradish peroxidase or anti-mouse IgG-horse-radish peroxidase). Immunoreactive bands were visualized with the ECL Western blotting detection system (GE Healthcare). The apparent molecular masses were calculated by non-linear curve-fitting of the molecular weight standards indicated at the right of each gel.

PFO-PAGE/Western Blot
After nuclei removal the cell lysates were mixed with various amounts of PFO and incubated for 1 h at room temperature. Lysates were then added to the PFO loading buffer (100 mM Tris, pH 8.0, various amounts of PFO, 20% (v/v) glycerol with 100 mM DTT). Proteins were loaded and fractionated by PFO-PAGE as described (26). 4–20% Precise protein gels (Pierce) were pre-electrophosphoresed in PFO running buffer (25 mM Tris base, 192 mM glycine, 5% (w/v) PFO, pH 8.5) for 5 min before samples were loaded. The subsequent Western blot process was the same as that for the LDS-PAGE/Western blot described above.

Yeast Two-hybridization System
Full-length mPrestin (from Met-1—Ala-744) was inserted into the bait expression vector pAMBV4 (DUALmembrane Biotech, Switzerland) with CUB-LexA-VP16 downstream of, and in-frame with mPrestin. The bait vector carries the LEU2 gene for auxotrophic selection. The sequence of the prestin-bait vector was confirmed through DNA sequencing. The expression of the mPrestin-Cub-LexA-VP16 fusion protein was further verified by Western blot analysis with anti-C-mPres. The full-length mPrestin was also inserted into the prey-expressing vector pDL2-Nx (DUALmembrane Biotech) with NubG upstream of, and in-frame with prestin. The prey vector carries the TRP1 gene for auxotrophic selection. pMBV-Alg5 is a negative control bait construct that expresses the Cub-LexA-VP16 fusion protein in the correct orientation in the yeast membrane. The prestin-bait construct and the negative control, pMBV-Alg5, were transformed into yeast strain DSY-1 (MATa his3{Delta}200 trp1-901 leu2-3, 112 ade2 LYS2: (lexAop)4-HIS3 URA3::(lexAop)8-lacZ GAL4) (DUALmembrane Biotech) and grown on leucine-selective plates (SD-L). Prestin-prey constructs were transformed into prestin-bait-expressing yeast or pMBV-Alg5-expressing yeast and grown on leucine-tryptophan double-selective plates (SD-LT). Positive interactions were identified by the ability of yeast to grow on leucine-tryptophan-histidine triple-selective plates (SD-LTH) in the presence of 2 mM 3-aminotriazole and by beta-galactosidase expression, indicated by the blue color observed in the presence of X-gal.

Prestin Purification
TSA-201 cells transfected with C-tag prestin (25) were collected 48 h after transfection. The cells were homogenized with a Dounce tissue grinder (Wheaton) in the hypotonic lysis buffer described above. Nuclei were removed by centrifugation at 800 x g for 10 min. The whole-cell lysate was treated with 8% PFO for 24 h at room temperature. The undissolved pellet was separated by centrifugation at 16,100 x g for 90 min. The supernatant was filtered (0.45-µm filter) before application to 1 ml of nickel-nitrilotriacetic acid-agarose resin (Invitrogen). The resin was poured into a column and washed with 1x native binding buffer (50 mM sodium phosphate, 0.3 M NaCl, pH 8.0) containing 4% PFO and, sequentially, either 20 or 80 mM imidazole. Prestin was eluted with 1x native binding buffer containing 300 mM imidazole and 4% PFO. Fractions (1 ml) were collected and analyzed by PFO-PAGE/Western blot.

Chemical Cross-linking
Cochleae were dissected and washed in PBS, pH 8.0. The bony cochlear wall was opened at the helicotrema as well as at both oval and round windows, with minimal disruption to the organ of Corti. The cochleae were perfused and incubated with different concentrations of freshly made chemical cross-linker solutions: bismaleimidohexane (BMH) (Pierce) or bis(sulfosuccinimidyl) suberate (BS3) (Pierce). BMH was dissolved in Me2SO, and BS3 was dissolved in PBS, pH 8.0. Perfused cochleae were agitated on an orbital shaker at room temperature for 30 min. Control cochleae, both wild type and knockout, were treated with the same solution but with no cross-linker added. Reactions were stopped by perfusion of 20 mM Tris-buffer, pH 7.5, for BS3 treatment or 50 mM DTT for BMH treatment and incubated for 15 min at room temperature. The cochleae were washed with PBS and homogenized in Sigma CelLytic mammalian tissue lysis/extraction reagent as described above. The whole-cell lysate was mixed with 2x LDS loading buffer and run on 5% NEXT gel/SDS-PAGE (Amresco) followed by Western blot.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Prestin Is Expressed and Located in the Plasma Membrane of Different Expression Systems—Prestin cDNA was inserted into different expression vectors and transfected into various hosts including yeast and mammalian cell lines. Prestin protein expression was analyzed using both immunofluorescence and LDS-PAGE/Western blot. Our data show that full-length prestin could be synthesized in yeast as demonstrated by both immunofluorescence (Fig. 1b) and Western blot experiments (Fig. 1d). Yeast expressing full-length prestin protein showed distinct annular images when assayed with anti-C-mPres indexed with fluorescein isothiocyanate (Fig. 1b). In contrast, the yeast expressing only the C-terminal domain of prestin showed a diffuse fluorescence throughout the entire cell, a typical distribution pattern for cytoplasmic proteins (Fig. 1c). The negative control, uninduced yeast, showed no staining (Fig. 1a). These data suggest that the full-length protein is located in the membrane of yeast, whereas the C-terminal domain is not.

Prestin-expressing mammalian cells can also synthesize full-length prestin and insert it into the plasma membrane (21). Interestingly, LDS-PAGE/Western blots of yeast transfected with full-length prestin revealed the presence of individual and DTT- and LDS-resistant oligomers; the lowest band had an apparent molecular mass similar to a monomer of deglycosylated prestin (~70 kDa). As shown in Fig. 1d, the second and third bands are approximately two and three times the molecular mass of the monomer, suggesting the existence of prestin monomers and higher order oligomers. The uninduced yeast (–G), used as a control, did not show prestin bands. Cochleae from prestin knock-out mice were also compared with those of wild type mice in an LDS-PAGE/Western blot with anti-C-mPres (Fig. 1e). Two prestin-specific bands corresponding to a prestin monomer (~90 kDa) and a dimer (~200 kDa) appear in wild type mice but not in prestin knock-out mice. These data indicate that prestin may physically bind to itself in its native state to form oligomers. It is also noted that prestin protein with a monomeric calculated mass of 80.5 kDa did not always migrate the same distance. Instead, bands running from ~70 kDa (yeast) to ~100 kDa (OHC) were observed. This variation is probably due to glycosylation and/or other modifications specific to the host cells (21).

Prestin Associates with Itself in Living Cells—Prestin is a highly hydrophobic protein with 12 predicted membrane-associated domains containing more than 50% of the amino acids (24). Proteins with hydrophobic natures often have a propensity to aggregate once cells are lysed and solubilized with detergents. In fact, artificial oligomers are found in some proteins (27–29). Therefore, it needs to be considered that the multiple bands observed on Western blot (Fig. 1) could be artifacts. To further evaluate prestin-prestin self-association, we analyzed prestin-prestin interaction using a membrane-based yeast two-hybrid system based on the split-ubiquitin assay. It is well known that native ubiquitin can be split into N-terminal (Nub) and C-terminal (Cub) domains. Nub and Cub retain their basic affinity for each other and spontaneously reassemble to form functional ubiquitin (30). However, a point mutation (I-> G) in the Nub (NubG) domain can greatly diminish the affinity of the two halves, i.e. NubG and Cub do not refold into functional ubiquitin. However, when NubG and Cub are fused to a "bait" and a "prey" protein respectively, any interaction between bait and prey brings NubG and Cub close together, inducing them to form functional ubiquitin despite their weak affinity. This action results in the release of an artificial transcription factor, LexA-VP16, which enters the nucleus and activates the lacZ and His3 genes. Expression of the His3 gene allows yeast growth on His– selective media, whereas expression of the lacZ gene allows yeast clones to appear blue on X-gal plates. Alternatively, if the prey does not interact with prestin, LexA-VP16 is not released, and no growth or color change is observed.


Figure 1
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  		FIGURE 1.
Prestin expression in yeast. a–c, immunofluorescent images of yeast stained with anti-C-mPres (green). a, pYes2/NT vector control. b, full-length prestin-pYes2/NT. c, the C-fragment of prestin-pYes2/NT. d and e, Western blot with anti-C-mPres. d, whole yeast lysate from full-length prestin-pYes2/NT clone with the inducing reagent (+G) or without (–G). e, cochleae from a wild type (WT) and a prestin knock-out (KO) mouse. The asterisk indicates monomeric, dimeric, and trimeric assemblies of the full-length prestin.

 


Figure 2
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  		FIGURE 2.
Interactions between full-length prestin was analyzed through a membrane-based yeast two-hybrid system. A, LDS-PAGE/Western blot of prestin-bait yeast. Anti-C-mPres was used. Prestin-bait expressing yeast were compared with the negative control vector Alg5-bait. B, prestin-bait and Alg-5-bait transformed with various prey constructs were grown on a double-selective plate (SD-LT). C, the same yeast grown on B were grown on a triple-selective plate (SD-LTH). The name before the slash (/) is the bait construct. The name after the slash is the prey construct, i.e. Prestin/Prestin means Prestin-bait/Prestin-prey, and Prestin/– means prestin-bait alone. Prestin-bait yeast were grown on a SD-LTH plate containing 2 mM 3-aminotriazole. Alg5-bait yeast were grown on a SD-LTH plate without 3-aminotriazole.

 
In these experiments we inserted Prestin cDNA into the bait expression vector, pAMBV4, using an in vivo recombination method. Cub and LexA-VP16 are located downstream of, and in-frame with prestin. This prestin-bait combined protein has 1103 amino acids with a predicted molecular mass of 120.5 kDa. Western blots in Fig. 2A show that a prestin protein band with apparent molecular mass of ~120 kDa is found only in the prestin-bait yeast clone. Because there is no homologous prestin gene in yeast, the absence of a prestin band is expected in the negative control yeast, containing the Alg5-bait (pMBV-Alg5 construct). To ensure that the prestin-bait expressed in yeast can interact with prey proteins, prestin-bait yeast were transformed with the positive prey control plasmid pAlg5-NubI (Dualsystems Biotech). Because Alg5 does not interact with the oligosaccharyltransferase complex (31), it will not interact with a glycoprotein-like prestin. However, if prestin is correctly expressed in yeast, wild type Nub part (NubI) can bind to the Cub part that is attached to the C terminus of prestin, resulting in yeast growth on a His–-selective medium.

A double dropout (SD-LT) medium was used to select yeast expressing bait (Leu–) and prey (Trp–, Fig. 2B), whereas a triple dropout (SD-LTH) medium was used to select yeast expressing both bait and prey as well as interactions between them (Fig. 2C). Because it does not require bait-prey interaction to reconstitute ubiquitin, a NubI-prey (pAlg5-NubI) fusion protein can bind Cub from prestin-bait as long as the Cub-LexA-VP16 moiety of the prestin-bait is present on the cytosolic side of the membrane. Fig. 2C shows that prestin-Cub-LexA-VP16/pAlg5-NubI yeast grows on the selective plate (SD-LTH, Prestin/NubI) and shows a blue color, indicating X-gal degradation by the reporter gene lacZ (data not shown). As a negative control, pAlg5-NubG was co-transformed with prestin-bait. As expected, prestin-Cub-LexA-VP16/pAlg5-NubG did not grow on the triple dropout-selective plate (SD-LTH, Fig. 2C, Prestin/NubG). This observation indicates that no interaction between prestin and Alg5 occurs despite the fact that both prestin-bait and pAlg5-NubI or pAlg5-NubG fusion proteins were co-expressed by yeast as demonstrated on SD-LT shown in Fig. 2B (Prestin/NubI, Prestin/NubG). These data suggest that prestin-bait is correctly expressed in yeast with its Cub-LexA-VP16-tag facing the cytoplasm, allowing interaction between Cub and NubI. Prestinbait (Prestin/–) and Alg5-bait (Alg5/–) alone cannot survive on either the double or triple dropout plates (Fig. 2, B and C). These results demonstrate that prestin-bait can interact with potential prey proteins, but prestin-bait alone does not activate reporter genes to produce false positive reactions.

The interaction between prestin-bait/prestin-prey (prestin/prestin) was then tested. When combined with prestin-bait, prestin-prey produced viable yeast. As shown in Fig. 2C, the yeast co-expressing prestin-bait and prestin-prey proteins (Prestin/Prestin) grew on SD-LTH-selective plates and turned blue when tested for the activation of the lacZ gene (data not shown). As expected, yeast expressing prestin-bait and negative control prey pAlg5-NubG showed no growth on the same plates (Prestin/NubG). We also tested the interaction between control Alg5-bait and prestin-prey. As shown in Fig. 2C, prestin-prey did not interact with Alg5-bait since no growth was seen in Alg5/prestin even though both proteins were synthe-sized as demonstrated in Fig. 2B, Alg5/Prestin. In other words, prestin-bait can interact with prestin-prey (but not the control prey), and prestin-prey can interact with prestin-bait (but not the control bait). Because the interaction between full-length prestin-prey and full-length prestin-bait occurs in living yeast, the data suggest that prestin molecules interact with each other to form oligomers before the cells are lysed. Hence, the multiple bands shown on Fig. 1 are probably physiologically significant relevant.

Prestin Can Exist as a Tetramer in Yeast—To further characterize the oligomeric structure of prestin, we used the method of PFO-PAGE. This is because PFO, a non-denaturing detergent (32), does not break the non-covalent interactions between protein subunits of an oligomer at an appropriate concentration, thereby permitting the extraction and determination of oligomeric states of membrane proteins. PFO-PAGE has been used successfully for studying the oligomeric states of membrane proteins such as the vanilloid receptor, claudin-4, and ABCG2 (33–35).

Prestin-expressing yeast were extracted with various PFO concentrations ranging from 0 to 7%. These PFO-treated yeast lysates were mixed with loading buffer containing various amounts of PFO and loaded on a 4–20% gradient gel using a PFO-PAGE/Western blot. As expected, the pattern of protein bands was dependent on the time and temperature of incubation; longer incubation time and higher temperature resulted in a greater degree of subunit dissociation from the protein complex. As shown in Fig. 3a, 0.5% PFO showed very little prestin extracted from yeast membranes. However, after longer exposure, a prestin tetramer band was visible in 0.5% PFO-treated samples (data not show). At 1% PFO, prestin exists as oligomers including dimers, trimers, and tetramers but not monomers. The low concentration of mild detergent apparently preserved the non-covalent protein-protein interactions. Increasing the detergent concentration increased the degree of dissociation. At the highest PFO concentration (7%), prestin exists as trimers, dimers, and monomers but not as tetramers. At 4% PFO, prestin migrated as four bands, which probably correspond to a prestin monomer (~80 kDa), dimer (~170 kDa), trimer (~270 kDa), and tetramer (~330 kDa), as shown in Fig. 3b. These data suggest that prestin likely exists as a tetramer in yeast.


Figure 3
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  		FIGURE 3.
Prestin exists as oligomers in yeast. a, prestin oligomerization analysis by PFO-PAGE/Western blot. Lysates from prestin-pYes2/NT yeast were extracted using different concentrations of PFO (0–7% (w/v)) at room temperature for 1 h and mixed with loading buffer containing various amount of PFO. The samples were loaded on a 4–20% gradient gel, separated, and blotted with anti-C-mPres. Uninduced prestin-pYes2/NT-yeast (–G) was used as a control. b, the molecular masses (large dots) of monomeric and oligomeric prestin were calculated based on the linear regression (line)of the molecular mass ladder (Bio-Rad) used in the gel. The four bands correspond to a prestin monomer (~80 kDa), dimer (~170 kDa), trimer (~270 kDa), and tetramer (~330 kDa).

 
To exclude the possibility of prestin degradation or cleavage, lysates from prestin-expressing yeast were blotted with anti-Xpress and anti-C-mPres antibodies that bind to epitopes at the ends of the N terminus and C terminus of prestin, respectively. The same multiple prestin bands were consistently observed using either anti-Xpress or anti-C-mPres (data not shown). These data suggest that there is no cleavage at either the N or C terminus of the prestin protein.


Figure 4
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  		FIGURE 4.
Prestin oligomers from prestin-expressing mammalian cells. a, C-tagged prestin extracted by metal affinity chromatography from prestin-expressing TSA-201 cells was separated on PFO-PAGE and reacted with anti-V5. Four bands were observed indicating tetrameric prestin. P, prestin. C, chloramphenicol acetyltransferase) in pcDNA 3.1 was used as a control. Fraction numbers are the order of elution fraction tubes. b, the molecular masses (large dots) of monomeric and oligomeric prestin were calculated based on the linear regression (line) of the molecular mass ladder (Bio-Rad) used in the gel. The four bands correspond to a prestin monomer (~110 kDa), dimer (~200 kDa), trimer (~290 kDa), and tetramer (~380 kDa).

 
Prestin Exists as a Tetramer in Mammalian Cells—Given that prestin can form a tetramer in yeast, it is of interest to determine whether such oligomers exist in mammalian cells, where the function of prestin has been tested (8). Unlike the strong ionic detergents SDS or LDS, PFO does not prevent the binding of a His tag to the nickel column (36). This advantage allowed us to extract prestin protein from prestin-expressing TSA 201 cells with metal affinity chromatography. When the eluted protein was subjected to PFO-PAGE/Western blot, as shown in Fig. 4, four prestin bands were observed, indicating that prestin exists as a tetramer in mammalian cells.

It should be noted that in the above experiments the mild ionic detergent PFO was used to extract prestin from membranes. However, the formation of oligomeric prestin was not due to the use of PFO because the application of other ionic detergents such as LDS also reveal the trimer and the dimer of prestin in yeast and the dimer in OHCs (Fig. 1). Therefore, PFO appears to be able to preserve higher order oligomeric states such as tetramers in both yeast and TSA cells. However, PFO does not extract prestin from OHC membranes even if 8% PFO is used (data not shown). Prestin from OHCs appeared to be solubilized only under the conditions that strong detergents such as SDS and LDS were used. The extremely high protein content and the high level of cholesterol in the OHC plasma membrane (37) may contribute to such phenomena.

Prestin Exists as a Tetramer in the Plasma Membrane of OHCs—To determine whether oligomeric prestin exists in native living cells without interruption by detergents, mouse OHCs were treated with the chemical cross-linking reagents BS3 and BMH before detergents were added to the cochlear samples. According to Pierce, BS3 is a water-soluble, membrane-impermeable homobifunctional N-hydroysuccinimide ester with a space-arm length of 11.4 Å that is used for cell-surface protein cross-linking. In contrast, BMH has a space-arm length of 16.1 Å. and is a water-insoluble, membrane-permeable homobifunctional sulfhydryl-reactive cross-linker that is used for integral membrane-protein cross-linking. According to the topology map of prestin' (24), there are three lysines on extracellular loops that can be cross-linked by BS3 as well as nine cysteine residues that can potentially react with BMH. If prestin in OHCs exists as an oligomer, it may be cross-linked by BS3 and/or BMH. Because the cross-linker forms covalent bonds among protein subunits, the original oligomeric forms can be preserved and analyzed using a 5% NEXT gel, a SDS-PAGE-based system with a different running buffer (Amresco). We chose the NEXT gel instead of the traditional SDS-PAGE because it is a continuous gel electrophoresis system that produces better separation for high molecular mass proteins like prestin. As shown in Fig. 5A, the amount of prestin monomer decreases with increasing concentration of BS3, whereas the amount of the oligomeric forms increases concomitantly. Incubation with 10 mM BS3 for 30 min resulted in the formation of dimer, trimer, and tetramer with dimer as the major band. Cochleae were also treated with BMH that has a different space-arm length and reactive group. As shown in Fig. 5B, four distinguishable bands appeared on the LDS-PAGE/Western blot similar to the BS3-treated cochleae. These data further suggest that prestin exists as a high order oligomer in OHCs, at least as high as a tetramer.

Prestin's Oligomer Is a Stable Structure—Unless they are covalently linked, most proteins dissociate in the presence of SDS. Prestin's dimer, however, is a stable structure that is resistant to dissociation by SDS under almost all conditions. Therefore, we examined the possibility of disulfide linkage and tested the effect of the reducing reagents DTT and iodoacetamide on prestin oligomerization in OHCs. DTT is used to break S-S bonds formed from cysteine residues within or between protein subunits, whereas iodoacetamide is widely used to irreversibly "cap" free SH residues of cysteines (38, 39). The latter reaction prevents formation of nonspecific disulfide bonds during sample preparation. As shown in Fig. 6A, adding 100 mM DTT alone or with 100 mM iodoacetamide and vice versa appears to have little effect on the oligomeric state of prestin as expressed in OHCs, i.e. there are always monomer (~100 kDa) and dimer (~200 kDa) bands in 5% NEXT gel-SDS-PAGE/Western blots. This result suggests two possibilities. First, the reducing reagent DTT might have no effect on the dimer because there are no disulfide bonds, and the dimer is held together by other forces. Alternatively, the disulfide bond between two prestin monomers might be hidden in the hydrophobic core, which the hydrophilic reducing reagent DTT does not reach. Noting that 7 of the nine cysteines in prestin topology map are indeed located in the hydrophobic region, we applied the hydrophobic reducing reagent ethanedithiol to the prestin sample. As shown in Fig. 6B, ethanedithiol nearly abolished the prestin dimer in SDS-PAGE. In addition, increasing the concentration of another reducing reagent, beta-mercaptoethanol (Fig. 6C) also decreased but did not eliminate the dimer. Taken together, these data suggest that disulfide bonds located in the hydrophobic core probably mediate dimerization.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
We inserted prestin into various expression systems including yeast and mammalian cell lines. In all these systems, prestin consistently showed a stable oligomer on LDS-PAGE/Western blot (Fig. 1). Because there was concern as to whether such high molecular mass oligomers were caused by aggregation after cell lysis and solubilization, we tested prestin-prestin interaction in the membrane-based yeast two-hybridization system. The in vivo data (Fig. 2) suggested that prestin already existed in oligomeric form before the cells were lysed and solubilized. To further investigate whether prestin exists in higher order oligomeric states, we used the non-denaturing detergent, PFO. Across a range of prestin-expressing systems, PFO-PAGE/Western blot data consistently reveal discrete prestin bands that are approximate multiples of the smallest observable band. Prestin bands corresponding to monomers, dimers, trimers, and tetramers were consistently found in prestin-transfected yeast (Fig. 3) and in prestin isolated from a mammalian cell line, TSA-201 (Fig. 4). In addition, when prestin was bound chemically in its native cochlear tissue by different cross-linkers and then analyzed in SDS-PAGE, four prestin bands were observed (Fig. 5). Taken together, the evidence demonstrates that prestin is physiologically an oligomer.


Figure 5
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  		FIGURE 5.
Chemical cross-linking of prestin. Mouse cochleae treated with BS3 (A) or with BMH (B) were separated by 5% NEXT-gel/SDS-PAGE followed by Western blot analysis using anti-C-mPres. KO is the cochlea from a prestin knock-out mouse; WT is from a wild type mouse.

 


Figure 6
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  		FIGURE 6.
Effects of reducing reagents on the prestin oligomer. Cochlear lysates were mixed with LDS Laemmli loading buffer supplemented with different reducing reagents. The samples were run on 5% NEXT-Gel/SDS-PAGE, then reacted with anti-C-mPres after Western blot (A). Cochleae were treated with 100 mM DTT and/or 100 mM iodoacetamide (Iodo). The two bands are prestin monomer and dimer, respectively. B, cochleae were treated with different concentrations of the hydrophobic reducing reagent ethanedithiol (EDT). C, cochleae were mixed with different amounts of the hydrophilic reducing reagent beta-mercaptoethanol (beta-Me). WT, wild type.

 
The hydrophobic region of prestin has 12 predicted membrane-interacting domains (14, 24) and contains ~400 of prestin's 744 amino acids with a predicted molecular mass of 42 kDa. We estimated a tetramer of the prestin membrane-interacting domains to be on the order of 11 nM in diameter (10.8 nM). This size is consistent with the protein complex observed in the lateral membrane of OHCs (7, 40). Based on the stoichiometric response to salicylate, Tunstall et al. (16) estimated that each protein complex was likely to be three or four linked subunits, although a later experiment did not agree with their results (17). More recently, Wada and co-workers (41) used atomic force microscopy and reported that 8–13-nM particle-like structures were significantly higher in prestin-expressing Chinese hamster ovary cells than in untransfected cells. Their data support the idea that prestin forms particle-like structures in mammalian cell lines, similar to those in OHCs (42).

Evidence in this report suggests that prestin exists as a tetramer with an estimated diameter of ~11 nM. This dimension is virtually identical to that of the particles found in the OHC lateral membrane. However, this presumed equivalence of particle and motor was questioned by Santos-Sacchi et al. (43). Their data from guinea pig OHCs indicate that voltage-dependent capacitance increases as cell length decreases. Although particle density also increases in short basal OHCs, a quantitative correspondence was not found. In other words the increase in particle density appeared to be insufficient to explain the increase in charge density. For example, the theoretical maximum density of intramembranous particles, ~10,000/µm2, implies a maximum charge of ~10,000 electrons/µm2 assuming that each particle possesses a single charge as indicated by the two-state Boltzmann model. Because electrophysiological measurements demonstrate that short OHCs exhibit a charge density of ~30,000/µm2, the authors concluded that the particles may not represent the motors. Although this may be the simplest conclusion, another possibility must be entertained.

If one assumes that each prestin monomer possesses one electron charge, then a tetramer should represent four electron charges. In other words, each intramembranous particle would be formed from four prestin monomers each with a valence of one and each with independent voltage sensing. This possibility is strengthened by taking the highest particle density reported in the literature, 7155/µm2 (44), and multiplying by 4 to obtain 28,620 electron charges/µm2. This latter figure is very similar to the maximum charge density measured electrophysiologically in short OHCs (43). Hence, evidence that prestin exists as a tetramer, with four independent subunits each with a single electron charge, is consistent with the idea that the particles represent motor molecules.

Our data also suggest that disulfide bonds in the hydrophobic region play a role in connecting prestin monomers together to form dimers. Therefore, we propose that prestin dimers are the basic building blocks connected by disulfide bonds, whereas prestin tetramers, which appear as particles in the OHC lateral membrane, are formed from dimers connected via hydrophobic bonds. Although a tetramer appears to be the most likely choice because of its estimated size, one cannot rule out the possibility that prestin hexamers, which have an estimated size of 13.4 nM, might also contribute to the motor complex. Which cysteines in prestin are involved in the formation of disulfide bonds and how they regulate prestin oligomerization is unknown. We also do not know whether oligomerization is required for prestin function. Hence, further investigations are needed to ascertain the functional relevance of prestin oligomerization.

Although there is no report as yet suggesting that other members of the SLC26 family exist as oligomers, it would not be surprising if an oligomeric form were unique to prestin (SLC26A5) because it is different from other members of the SLC26 family. For example, prestin is abundantly expressed in OHCs, where it is estimated that a large portion of the lateral membrane is occupied by prestin particles (45). In contrast, none of the other SLC26 family members have such high expression levels. Second, the degree to which prestin transports anions remains unclear. This contrasts with all other members of the SLC26A family where anion transport across the plasma membrane is a basic function (22, 46). It should be mentioned, however, that prestin appears to utilize intracellular Cl– ions either for extrinsic voltage sensing (14), or for the allosteric modification required for charge translocation (47, 48) or for anion exchange (48). In this process, Cl– anions or some other charge moiety are partially translocated across the membrane in response to changes in the transmembrane voltage. This translocation is assumed to trigger conformational changes in the molecule, resulting in somatic elongation or contraction of OHCs. Other members of the SLC26A family, such as prestin's closest relatives PDS (Pendred syndrome (pendrin), SLC26A4) and PAT1 (putative anion exchanger 1, SLC26A6), do not show such activity under similar conditions (8, 14). It is, therefore, anticipated that characterization of prestin oligomerization will assist evaluation of other members in this solute carrier family.


   FOOTNOTES
 
* This work was supported by the NIDCD, National Institutes of Health Grants DC006412 and DC00089. 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. Back

1 To whom correspondence should be addressed: Northwestern University, Dept. of Communication Sciences and Disorders, 2240 Campus Dr., Evanston, IL 60208. Tel.: 847-491-2450; Fax: 847-491-2523; E-mail: jzh215{at}northwestern.edu.

2 The abbreviations used are: OHC, outer hair cell; LDS, lithium dodecyl sulfate; PFO, perfluoro-octanoate; SLC26, solute carrier 26; anti-C-mPres, a polyclonal antibody against C terminus of mouse prestin protein; KO, knockout; PBS, phosphate-buffered saline; C-tag prestin, gerbil prestin tagged at the C terminus with the hexahistidine and V5 epitope; CAT, chloramphenicol acetyltransferase; DTT, dithiothreitol; NEXT-PAGE, New Electrophoresis X'PRESS Technology PAGE; BMH, bismaleimidohexane; BS3, bis(sulfosuccinimidyl) suberate; SD-L, SC medium without leucine; SD-LT, SC medium without leucine and tryptophan; SD-LTH, SC medium without leucine, tryptophan, and histidine; Nub, the N-terminal domain of ubiquitin; Cub, the C-terminal domain of ubiquitin; NubG, the mutant form of Nub; X-gal, 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside. Back


   ACKNOWLEDGMENTS
 
We thank Dr. W. Russin at the Biological Imaging Facility of Northwestern University for help in image processing and Dr. J. Zuo of the St. Jude Children's Research Hospital for providing prestin knock-out mice. We also thank Dr. L. D. Madison for suggestions and R. Edge for technical assistance.



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