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Originally published In Press as doi:10.1074/jbc.M704924200 on December 11, 2007

J. Biol. Chem., Vol. 283, Issue 7, 4177-4188, February 15, 2008
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Conserved Dimeric Subunit Stoichiometry of SLC26 Multifunctional Anion Exchangers*Formula

Silvia Detro-Dassen{ddagger}1, Michael Schänzler§1, Heike Lauks{ddagger}, Ina Martin§, Sonja Meyer zu Berstenhorst§, Doreen Nothmann§, Delany Torres-Salazar§, Patricia Hidalgo§, Günther Schmalzing{ddagger}24, and Christoph Fahlke§34

From the {ddagger}Abteilung Molekulare Pharmakologie, Rheinisch-Westfälische Technische Hochschule Aachen University, Wendlingweg 2, Aachen 52074, the §Institut für Neurophysiologie, Medizinische Hochschule Hannover, Carl-Neuberg-Strasse 1, Hannover 30625, and the Zentrum für Systemische Neurowissenschaften Hannover 30625, Hannover, Germany

Received for publication, June 14, 2007 , and in revised form, December 3, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The SLC26 gene family encodes multifunctional transport proteins in numerous tissues and organs. Some paralogs function as anion exchangers, others as anion channels, and one, prestin (SLC26A5), represents a membrane-bound motor protein in outer hair cells of the inner ear. At present, little is known about the molecular basis of this functional diversity. We studied the subunit stoichiometry of one bacterial, one teleost, and two mammalian SLC26 isoforms expressed in Xenopus laevis oocytes or in mammalian cells using blue native PAGE and chemical cross-linking. All tested SLC26s are assembled as dimers composed of two identical subunits. Co-expression of two mutant prestins with distinct voltage-dependent capacitances results in motor proteins with novel electrical properties, indicating that the two subunits do not function independently. Our results indicate that an evolutionarily conserved dimeric quaternary structure represents the native and functional state of SLC26 transporters.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The solute carrier 26 (SLC26)5 gene family was defined ten years ago by expression cloning of a Na+-independent Formula transporter from rat liver (1). Subsequently, many homologs were found in mammals, non-mammals, plants, fungi, and bacteria (2). The functional importance of this family is highlighted by several inherited human diseases caused by mutations in SLC26 genes (3, 4), such as diastrophic dysplasia (SLC26A2), congenital chloride diarrhea (SLC26A3), Pendred syndrome (a syndrome comprising sensorineural deafness and enlarged thyroid (SCL26A4)), and inner ear deafness (SLC26A5 or pres). The symptomatic variety of these diseases illustrates the diversity of cellular functions performed by this class of transport proteins.

Mammalian SLC26s are the members of this family that have been functionally studied the best. Most of them operate as anion exchangers, and distinct isoforms differ significantly in anion specificity. Some transport monovalent and divalent anions, whereas others transport only monovalent anions (1, 5-9). There are also family members that do not function as anion exchangers, most notably, SLC26A5 or prestin, the motor protein in the outer hair cells (10). Outer hair cells in the mammalian cochlea change length in response to acoustic signals, and this mechanical amplification enhances the hearing sensitivity by >40 db. The basis for these length changes are voltage-dependent conformational changes of prestin that are transformed into somatic length changes of outer hair cells (11). Moreover, recent work suggested that SLC26A7 and SLC26A9 are not anion carriers, but anion channels (12, 13). The SLC26 family thus exhibits an amazing variety of functions, yet the molecular basis of this diversity is poorly understood. As a fundamental step to understand structure-function relationships, we decided to study the subunit stoichiometry of various SLC26 homologs from humans, rat, zebrafish, and Pseudomonas aeruginosa. We demonstrate that all tested isoforms exhibit a dimeric subunit stoichiometry.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Expression of His- or YFP Fusion Proteins in Xenopus Oocytes or in Mammalian Cells—pGEMHE-hSLC26A3-HisCT (polyhistidine tag at the carboxyl-terminal end) encoding the human SLC26A3 (GenBankTM accession number NM_000111 [GenBank] ) (14), pGEMHE-rprestin-HisCT encoding the rat prestin (GenBankTM accession number NM_030840), pGEMHE-zfprestin-HisCT encoding prestin from Danio rerio (zebrafish) (GenBankTM accession number BX571796) (15), and pGEMHE-HisNT-hClC-1 (polyhistidine tag at the amino-terminal end; GenBankTM accession number NM_000083) plasmids were generated by adding a cDNA fragment encoding six histidine residues carboxyl- or amino-terminal to the coding region by PCR and subcloning into pGEMHE vector (16). A construct encoding a rat prestin concatamer was generated by covalently linking two prestin copies in a single open reading frame by a cDNA sequence containing four repeats of the tripeptide AGS (17). PASulP (GenBankTM accession number NP_250338 [GenBank] ) was identified by searches for rat prestin homologues using BLAST searches at the National Center for Biotechnology Information (NCBI). It was amplified by PCR from genomic P. aeruginosa DNA and subcloned into pGEMHE using BamHI and HindIII restriction sites. Transcription of cRNAs and handling of oocytes was performed as described (18).

To create YFP fusion proteins, the YFP cDNA was excised from pEYFP-N1/pECFP-N1 (Clontech) and inserted into pcDNA3.1 (Invitrogen) connected to the coding sequences of rat and zebrafish prestin and human SLC26A3 by short linker sequences in a single open reading frame. SLC26 proteins were expressed in tsA201 cells as described (19). For some experiments, stable inducible cell lines, generated by selecting Flp-In T-REx cells (Invitrogen) transfected with pcDNA5/FRT/TO-YFP-rprestin, were used after 24-h incubation with 1 µg/ml tetracycline. Point mutations were introduced using the QuikChangeTM method. All constructs were verified by restriction analysis and DNA sequencing. Supplemental Fig. S1 gives an alignment of the SLC26 isoforms characterized in this study.

Functional Characterization of SLC26A3 and Prestin—Radioactive chloride uptake into oocytes was studied 4-5 days after cRNA injection. Oocytes were preincubated for 10 min in chloride-free medium (98 mM potassium gluconate, 1.8 mM calcium gluconate, 5 mM HEPES-Tris, pH 7.5) and then transferred to the uptake solution (100 mM potassium gluconate, 5 mM HEPES-Tris, 3 mM 36Cl, pH 7.5) above 300 µl of mineral oil. After various time periods, 36Cl uptake was terminated by centrifuging the oocytes into the mineral oil layer. Scintillation counting was performed after lysis of the oocytes in scintillation counting tubes containing 100 µl of 0.5% SDS. For each incubation period, uptake was also determined for at least two uninjected oocytes. These control values were averaged and subtracted from radioactive uptake levels measured on injected oocytes. Additional control experiments were performed with oocytes expressing SLC26A3 transporters incubated in the uptake solution supplemented by 100 µM DIDS. DIDS was dissolved in Me2SO at a concentration of 10 mM and diluted into the uptake solution.

Transfected HEK293 or tsA201 cells were studied through whole cell patch clamping using an Axopatch 200B amplifier. In experiments measuring voltage-dependent capacitances, the external solution contained (in mM): 140 NaCl, 4 KCl, 2 CaCl2, 1 MgCl2, 5 HEPES, at pH 7.4, and the intracellular solution contained (in mM): 120 NaCl, 2 MgCl2, 5 EGTA, 10 HEPES, at pH 7.4. The initial pipette resistances were 1-2.5 megohms. Stray pipette capacitance was neutralized before establishing the whole cell configuration. All data acquisition and analysis were performed with the Windows-based patch clamp program, jClamp (SciSoft, Ridgefield, CT). Non-linear charge movement was calculated using a continuous high resolution (2.56-ms sampling) two-sine stimulus protocol (10-mV peaks at both 390.6 and 781.2 Hz) superimposed onto voltage steps (150-ms duration) from either -200 to +80 mV or -160 to +120 mV (20). The voltage dependence of the non-linear capacitance was fit to the first derivative of a two-state Boltzmann function (21).

Formula(Eq. 1)
where β = z(eo{delta}/kBT). In addition, Qmax is the maximum charge transferred across the membrane; V1/2 is the potential at half-maximal charge transfer; z is the number of elementary charges, eo, displaced across a fraction, {delta}, of the membrane dielectric; kB is the Boltzmann constant, and T is the absolute temperature.

In co-transfected cells, the voltage dependence of the nonlinear capacitance was fit with,

Formula(Eq. 2)
where pD154N is the percentage of subunits carrying the D154N mutation. CD154N, CD342Q, and CHeterodimer are voltage-dependent capacitances of homogenous populations of homodimeric mutant prestin or of heterodimeric prestin. Qmax was assumed to be identical for each non-linear capacitance term.

To measure Cl-/Formula exchange by zebrafish prestin, external solutions containing (in mM) 135 NaCl, 5 Na2SO4, 4 KCl, 2 CaCl2, 1 MgCl2, 5 HEPES, pH 7.4, and internal solutions (in mM) containing: 30 Na2SO4, 90 NaCl, 2 MgCl2, 5 EGTA, 10 HEPES, pH 7.4, were used.

Purification of [35S]Methionine-labeled Protein from Xenopus Oocytes—cRNA-injected and non-injected control oocytes were incubated for the indicated time with RevidueTM L-[35S]methionine (>37 TBq/mmol, GE Healthcare Biosciences) at ~25 Mbq/ml (~0.1 MBq/oocyte) in frog Ringer's solution at 19 °C for metabolic labeling. Either immediately after the pulse or after an additional chase period, the radiolabeled oocytes were extracted with digitonin (1.0%) in 0.1 M sodium phosphate buffer, pH 8.0. His-tagged proteins were isolated by metal affinity chromatography using Ni2+-NTA-agarose (Qiagen), as detailed previously (18), with the modification that iodoacetamide was routinely included at 10 mM and 1 mM in the lysis and washing buffers, respectively (22). In some experiments, 1% perfluoro-octanoic acid, ammonium salt (PFO, Sigma-Aldrich) was used as detergent for membrane protein extraction and purification in 0.1 M sodium phosphate buffer, pH 8.0, with and without 10 mM iodoacetamide as indicated. Proteins were eluted from Ni2+-NTA-agarose with PFO elution buffer consisting of 250 mM imidazole/HCl and 1% PFO at pH 7.4 and stored at 0 °C until analysis later on the same day.

Chemical Cross-linking—His-tagged SLC26 protein bound to Ni2+-NTA beads was washed in triplicate with imidazole-free sodium phosphate buffer (pH 8.0) supplemented with 0.2% digitonin. The Ni2+-NTA beads (packed volume, ~15 µl) were resuspended in 50 µl of 0.2 M triethanolamine/HCl (pH 8.5), 0.5% digitonin. The cross-linking reaction was initiated by adding glutardialdehyde (Roth Chemicals) or dimethyl adipimidate (Pierce) from freshly prepared solutions in distilled water or 0.2 M triethanolamine/HCl (pH 8.5), respectively. After 30 min at room temperature, the cross-linking reaction was terminated by washing the Ni2+-NTA-agarose beads twice with imidazole-free sodium phosphate buffer, 0.2% digitonin. Proteins were eluted from Ni2+-NTA-agarose with non-denaturing elution buffer, consisting of 250 mM imidazole/HCl and 0.5% digitonin at pH 7.4, and then stored at 0 °C until analysis later on the same day.


Figure 1
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  		FIGURE 1.
His and YFP tags leave function of SLC26A3 and prestin unaltered. A, time dependence of 36Cl- uptake into oocytes expressing human SLC26A3 and His-SLC26A3. To test the DIDS sensitivity of the hSLC26A3 transporters, 100 µM DIDS was added to the uptake solution. B, normalized non-linear charge movement associated with rat prestin, His-rprestin, and YFP-rprestin heterologously expressed in tsA201 cells. C, normalized current-voltage relationships from tsA201 cells expressing zebrafish prestin, His-zfprestin, and YFP-zfprestin. Salicylate was applied by moving cells into the stream of a perfusion solution containing 10 mM salicylate.

 
Purification of YFP Fusion Proteins from Mammalian Cells—tsA201 or HEK293 cell dishes were washed with cold phosphate-buffered saline and lysed with 800 µl of 0.1 M sodium phosphate buffer containing 1% digitonin, protease inhibitors, and 50 mM iodoacetamide. For lysis, the whole dish was incubated 15 min on ice, followed by complete transfer of the cell lysate into a microtube and subsequent quadruplicate vortexing during incubation on ice for another 15 min. SLC26 proteins were isolated by metal affinity chromatography using Ni2+-NTA-agarose (Qiagen). In some experiments, 1% PFO in PFO extraction buffer was used for cell lysis.

SDS-PAGE, BN-PAGE, and PFO-PAGE—[35S]Methionine-labeled proteins were denatured for 10 min at 56 °C with SDS sample buffer containing 20 mM dithiothreitol (DTT) and electrophoresed in parallel with 14C-labeled molecular mass markers (Rainbow, GE Healthcare Biosciences, Freiberg, Germany) on linear SDS-polyacrylamide gels. BN-PAGE was performed as described (18) immediately after protein purification. We loaded the protein purified from the equivalent of 0.5 oocytes in each lane. Molecular masses were determined by comparison with the defined membrane protein complexes generated by partial denaturing of the homopentameric {alpha}1 GlyR (23). PFO-PAGE was performed as described using freshly poured Tris-glycine (Laemmli) gels without SDS and Tris-glycine running buffer supplemented with 0.5% (w/v) PFO instead of SDS (24, 25). The homopentameric {alpha}1 GlyR was used as a mass marker as above.

YFP-tagged proteins were visualized by scanning the wet PAGE gels with a fluorescence scanner (Typhoon, GE Healthcare Biosciences). PAGE gels with radioactive proteins were fixed and dried, exposed onto a phosphorscreen, and scanned with a Storm 820 PhosphorImager (GE Healthcare Biosciences). Individual bands were quantified with the ImageQuaNT software. To investigate the glycosylation state of the proteins, samples were treated for 2 h with either endoglycosidase H (EndoH) or PNGase F (New England Biolabs, Beverly, MA) in the presence of reducing SDS sample buffer and 1% (w/v) Nonidet P-40 to counteract SDS inactivation of PNGase F.

Data Analysis—Protein intensities were quantified with the ImageQuaNT software (GE Healthcare Biosciences). Each experiment was performed at least in triplicate. Data are given as mean ± S.E.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
His and YFP Tags Leave Function of SLC26A3 and Prestin Unaltered—We added carboxyl-terminal hexahistidine tags to various SLC26 isoforms to purify the proteins from Xenopus oocytes by a single Ni2+-metal affinity chromatography step. Moreover, SLC26 isoforms were expressed as YFP fusion proteins in mammalian cells, to visualize protein bands using a fluorescence scanner. To test whether these sequence alterations affect functional properties, we compared chloride transport by WT and His-tagged SLC26A3, non-linear charge movement by WT and His- or YFP-tagged rat prestin, and Cl-/Formula antiport by WT and His- or YFP-tagged zebrafish prestin.

SLC26A3 was initially identified as a potential tumor suppressor that was down-regulated in adenoma and abundant in normal colonic mucosa (14). It was demonstrated to be a DIDS-sensitive anion exchanger (27, 28). We expressed tagged and untagged SLC26A3 in Xenopus oocytes and measured radioactive chloride uptake with and without added DIDS (27). Oocytes expressing WT or His-tagged SLC26A3 accumulated chloride with similar time courses and magnitudes (Fig. 1A). In both cases, 36Cl uptake was significantly higher in oocytes expressing SLC26A3 than in control oocytes, and incubation with 100 µM DIDS caused a reduction of the chloride uptake to ~50%.

Rat prestin performs voltage-induced conformational changes that can be monitored as voltage-dependent capacitance changes (10, 29). We expressed WT and His- or YFP-tagged rat prestin in tsA201 cells and measured voltage-dependent capacitance using a software-based lock-in technique (phase tracking) in the whole cell patch clamp technique. Neither the absolute amplitude of the non-linear capacitance (data not shown) nor the voltage dependence of rat prestin (Fig. 1B) was affected by any of the added tags. Rat prestin charge movement is not affected by 100 µM DIDS (data not shown). However, non-linear capacitances by WT and tagged prestin were eliminated by salicylate (29) (Fig. 1B).


Figure 2
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  		FIGURE 2.
Biochemical characterization of human SLC26A3 and rat prestin. A-C, human SLC26A3 and rat prestin purified by Ni2+-affinity chromatography after an overnight [35S]methionine pulse and a 24-h chase from X. laevis oocytes were resolved on a reducing SDS-PAGE urea gel (6% acrylamide/8 M urea) in parallel with 14C-labeled molecular mass markers (positions indicated in kDa at the left margins). Mutation of single or multiple asparagine glycosyl acceptors to glutamine combined with deglycosylation analysis as indicated reveals that SLC26A3 and prestin carry three (B) or two (C) high mannose type N-glycans, respectively. Glycosylated and non-glycosylated forms are indicated by black and white dots, respectively. Black dots indicate the number of glycans of the respective polypeptide.

 
Zebrafish prestin is functionally different from rat prestin. In addition to mediating voltage-dependent charge movements (30), it functions as Cl-/Formula exchanger with 1:1 stoichiometry (31). We measured currents in cells expressing WT or YFP or His fusion proteins of zebrafish prestin. The three tested proteins displayed anion currents with identical reversal potential and therefore unchanged transport stoichiometry (Fig. 1C). Mean current amplitudes were comparable (data not shown), and 10 mM salicylate (31) reduced current amplitudes to ~50% in all cases (Fig. 1C). The addition of the His tag or YFP tag did not affect the function of the tested SLC26 isoforms. We conclude that none of the tags prevents oligomerization processes of SLC26 that are of functional importance.

SDS-PAGE Analysis of Mammalian SLC26 Isoforms Heterologously Expressed in Xenopus Oocytes—Expression of SLC26 isoforms in Xenopus oocytes resulted in protein amounts that are below the resolution limit of Coomassie or silver staining. We therefore used metabolic labeling with [35S]methionine for signal enhancement to visualize analytical quantities of recombinant proteins.

His-tagged SLC26A3 and rat prestin were expressed and metabolically labeled in Xenopus oocytes. After extraction with 1% (w/v) digitonin, proteins were purified by metal affinity chromatography. Both proteins express well in Xenopus oocytes and are metabolically stable during a sustained chase. When resolved by reducing SDS-PAGE with a low percentage of acrylamide, as in Fig. 2A, the SLC26A3 and prestin polypeptides migrated at 15-20% lower masses than calculated from the amino acid sequences (85 kDa for His-SLC26A3 and 82 kDa for His-prestin). Both polypeptides shifted to higher apparent molecular masses at higher acrylamide concentrations (results not shown). This indicates that the mass deviations are due to anomalous migration in SDS-PAGE gels and not to post-translational processing of the polypeptide chains. Anomalous fast electrophoretic mobility has frequently been observed with highly hydrophobic membrane proteins (32).

Human SLC26A3 harbors four sequons for N-glycosylation, 153NAT, 161NNS, 164NNS, and 165NSS, in the predicted second ectodomain loop, which is flanked by the transmembrane segments TM3 and TM4. Deglycosylation with either EndoH or PNGase F reduced the molecular mass by ~8 kDa (Fig. 2B, lanes 1-3), suggesting the presence of three N-linked high mannose-type oligosaccharides of 2-3 kDa each. Substitution of Asn153 by glutamine resulted in a di-glycosylated polypeptide (lanes 4-6), and additional glutamine substitution of Asn161, Asn164, and Asn165 resulted in a non-glycosylated polypeptide (lanes 10-12). Thus, the three sequons that are in close proximity to one another acquire only two oligosaccharides, indicating that either Asn164 or Asn165 is sterically inaccessible for the oligosaccharyltransferase (32, 33).

Rat prestin carries five potential glycosylation sites, with two adjacent sequons - 163NAT and 166NGT - located in the second ectodomain. Deglycosylation produced a ~5-kDa mass shift (Fig. 2C, lanes 2-3), suggesting that two sites carry N-glycans. Glutamine substitution of Asn163 led to a monoglycosylated polypeptide (lanes 4-6), and no mass shift by PNGase F was observed when both sites, Asn163 and Asn166, were replaced by glutamines (lanes 7-9). The three remaining N-glycosylation sequons, 589NATV, 603NATK, and 736NATP, were not used.

SLC26A3 and Rat Prestin Migrate as Discrete Dimers in BN-PAGE Gels—To examine whether SLC26 transporters assemble into stable higher order structures, we natively purified human SLC26A3 or rat prestin from Xenopus oocytes and resolved it on BN-PAGE gels. The non-denatured hSLC26A3 exchanger migrated as a distinct band of ~200 kDa (Fig. 3A, lane 1). The size of the native SLC26A3 oligomer was assessed by comparing it to a molecular weight standard produced by partial dissociation of the homopentameric {alpha}1 GlyR receptor (Fig. 3A, lane 8). This procedure results in the occurrence of oligomers consisting of one to five GlyR {alpha}1 subunits (molecular mass ~52 kDa), representing a reliable size standard for membrane proteins of up to 260 kDa (32). To display the number of subunits incorporated in the SLC26A3 transporter complex, we weakened non-covalent subunit interactions by treating the natively purified protein with increasing concentrations of SDS. Incubation of the SLC26A3 exchanger with 0.01% SDS resulted in the complete disappearance of the 200-kDa band in favor of one faster migrating band of ~100 kDa (Fig. 3A, lane 4). The similarity with the calculated mass (91 kDa including the 6-kDa mass contributed by two N-linked glycans) and the absence of a faster migrating band in the presence of a 10-fold higher SDS concentration (Fig. 3A, lane 6) indicate that the 100-kDa band represents the monomeric state of SLC26A3, and the 200-kDa band represents the dimeric state. In the absence of the denaturant SDS, the 200-kDa band was resistant to 1-h incubation at 37 °C with Coomassie dye (Fig. 3A, lane 2) or Coomassie dye plus the strong reductant DTT (Fig. 3A, lane 3). Non-glycosylated SLC26A3 transporters also existed exclusively as dimers (results not shown). Other multimerization states were virtually absent (Fig. 3). Natively purified rat prestin migrated at three positions equal to ~97, ~190 kDa, and ~250 kDa in BN-PAGE gels (Fig. 3B, lane 3). Denaturing by SDS resulted in a complete dissociation of the oligomeric prestin into the ~90-kDa form (Fig. 3B, lanes 5 and 6), closely similar to the calculated mass of the rat prestin monomer of 87 kDa (protein core of 82 kDa including His tag plus 5 kDa of N-linked carbohydrate). No faster migrating protein species was observed, indicating that the 90-kDa band represents the monomer and the ~180-kDa band the dimer of rat prestin.


Figure 3
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  		FIGURE 3.
Oligomeric state of human SLC26A3 and rat prestin in Xenopus oocytes as determined by BN-PAGE. A and B, autoradiography of BN-PAGE gels of [35S]methionine-labeled, recombinant SLC26A3 (A) and prestin (B) proteins purified from X. laevis oocytes. An additional band that most likely represents the complex-glycosylated prestin dimer is given as *, and monomeric by-products of the prestin concatamer as #. The treatment of the samples before loading is given in the tables below each figure. Recombinant{alpha}1 GlyR was purified from X. laevis oocytes to serve as a molecular mass standard. The ellipses schematically illustrate dimeric and monomeric states. C, SDS-PAGE analysis of monomeric and concatenated prestin-prestin as resolved in a reducing SDS-PAGE urea gel (10% acrylamide). D, SDS-PAGE analysis of SLC26A3 and prestin under non-reducing and reducing conditions. Doublet bands in C and D represent di- and mono-glycosylated forms of rprestin. E, comparative BN-PAGE analysis of the oligomeric state of hClC-1 and SLC26A3. Where indicated, the samples were denatured with SDS for 1 h at 37 °C before loading onto the gel.

 
The ~250-kDa band (indicated by a star) was consistently observed when non-denatured prestin was resolved in BN-PAGE gels. Its intensity was about 10-30% of that of the ~190-kDa band, as determined by quantitative PhosphorImager scanning. The non-glycosylated 163Q166Q-prestin mutant migrated only at two positions in the BN-PAGE gel corresponding to ~90 kDa and ~190 kDa (Figs. 3B, lane 7). This suggests that the ~250-kDa band is related to the presence of N-linked glycans, and that both the ~190-kDa band and the ~250-kDa band represent dimeric states. Quantification of the relative intensity of the different bands revealed that dimers represented 52% of the total prestin in this experiment.

To rule out the possibility that the lowest molecular band corresponds to an unusually stable dimer and the higher molecular mass band to a tetrameric conformation, we engineered a concatenated rat prestin construct by linking two coding regions in a single open reading frame. After expression in Xenopus oocytes, two major bands were observed in BN-PAGE (Fig. 3B, compare lanes 9 and 3), corresponding to (prestin-prestin) and (prestin-prestin)2. Both conformations are stable and occur with comparable probability, further corroborating the dimeric subunit stoichiometry of prestin. Under denaturing conditions, the ~190-kDa band of the covalently linked dimer became more prominent (Fig. 3B, lane 10), obviously due to the dissolution of the dimerized tandem dimer and higher mass aggregates. In addition, a faint band appeared that migrated at the same position as the prestin monomer (Fig. 3B, indicated by a cross in lane 10). This monomeric form arises most likely from a proteolytic cleavage in the linker region of the concatenated prestin dimer, as has been observed previously with all other concatamers studies in our laboratories (17, 32). Reducing SDS-PAGE resolved the full-length prestin concatamer as a 140-kDa polypeptide corresponding approximately to twice the apparent 63 kDa of the prestin monomer (Fig. 3C). In addition, proteolysis-derived monomers are visible as weak bands (indicated by a cross).

Our conclusions are in conflict with a recent report that postulated that gerbil prestin exists as a tetramer formed by association of two disulfide-linked dimers (34). To identify possible reasons for these differences in experimental results, we performed three experiments. We tested the effect of reducing agents on the oligomeric state for all studied SLC26 isoforms (Figs. 3D, 5C, and 5G). Next, we demonstrated that BN-PAGE correctly displays the dimeric structure of ClC channels, a protein family firmly established to form dimers (Fig. 3E). Lastly, we repeated some experiments on rat prestin using PFO as detergent (34) (see Fig. 7).


Figure 4
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  		FIGURE 4.
Oligomeric state of human SLC26A3 and rat prestin in Xenopus oocytes as determined by chemical cross-linking. A, autoradiography of SDS-PAGE urea gels of rat prestin after purification and incubation with cross-linkers as indicated. B, BN-PAGE analysis of the same samples as in A in the non-denatured and SDS-denatured state. Numbers in A and B indicate the percent of dimers relative to total prestin protein. C, SDS-PAGE analysis of SLC26A3, rat prestin, and the concatenated prestin-prestin tandem dimer. After elution with non-denaturing buffer, samples were supplemented with reducing SDS sample buffer and resolved in SDS-PAGE urea gels (4-10% acrylamide gradient). D, BN-PAGE analysis of the same samples as in C were resolved in the non-denatured and SDS-denatured state as indicated. The ellipses schematically illustrate dimeric and monomeric states.

 
Prestin Multimers Are Not Covalently Linked by Disulfide Bonds—Recombinant rat prestin migrated entirely as monomers in BN-PAGE gels after denaturing with low concentrations of SDS in the absence of added reductant (Fig. 3B, lanes 4-6). Comparative resolution of non-reduced and DTT-reduced prestin on SDS-PAGE gels did not provide evidence for disulfide-linked multimers (Fig. 3D, lanes 3 and 4). Similar results were obtained with SLC26A3 (Fig. 3D, lanes 1 and 2) and zebrafish prestin (see Fig. 5C). We conclude that SLC26 dimers are held together by non-covalent forces rather than by covalent disulfide bonds.

BN-PAGE Displays the Dimeric Structure of the Human Muscle Chloride Channel hClC-1—The BN-PAGE technique has been shown to correctly display the quaternary state of various ion channels and transporters, including the pentameric structure of Cys-loop receptors (23, 35, 36) and the trimeric structure of glutamate transporters (32, 37, 38) and P2X receptors (18). To validate this experimental approach for a dimeric membrane protein, we expressed a member of the ClC family of voltage-gated chloride channels, the human muscle chloride channel (hClC-1), in Xenopus laevis oocytes (39, 40).

Non-denatured recombinant hClC-1 channels purified by metal affinity chromatography from [35S]methionine-labeled oocytes migrated as a single sharp band in the BN-PAGE gel (Fig. 3E, lane 1) at the same position as the non-denatured 260 kDa {alpha}1 GlyR. SDS led to a concentration-dependent appearance of a faster-migrating protein band of ~130 kDa (Fig. 3E, lane 2), closely similar to the calculated mass of the hClC-1 monomer (including a single 3-kDa oligosaccharide side chain at Asn430 (41)). BN-PAGE is therefore able to display correctly the quaternary structure of a dimeric membrane protein. The identity of the oligomeric states of both classes of proteins can be illustrated by directly comparing hClC-1 and SLC26A3 on the same BN-PAGE gel (Fig. 3E).

Cross-linking Generates Covalently Linked SLC26 Dimers—Next we used chemical cross-linking as an alternative approach to determine the oligomeric state of SLC26 proteins. Glutardialdehyde efficiently cross-linked natively purified rat prestin to dimers (Fig. 4A). Quantification of the protein bands showed that 66-71% of prestin migrated as covalently linked dimers in the reducing SDS-PAGE gel when incubated at ≥1 mM glutardialdehyde. The non-glycosylated prestin mutant was cross-linked to dimers with the same effectiveness as WT prestin (results not shown).


Figure 5
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  		FIGURE 5.
Glycosylation and oligomeric states of zebrafish prestin and a SLC26 paralog from P. aeruginosa. A, autoradiography of zebrafish prestin (zfprestin) purified directly after an overnight [35S]methionine pulse and after an additional 24-h chase interval. Samples were deglycosylated with EndoH or PNGase F as indicated. B, SDS-PAGE analysis of WT and glycosylation-deficient mutant zfprestin, purified after an overnight pulse and 24-h chase, treated with EndoH or PNGase F, and resolved by reducing SDS-PAGE. C, SDS-PAGE of the same samples as in B, resolved under non-reducing and reducing conditions. D, BN-PAGE analysis of zfprestin. Samples were treated as indicated to induce dissociation into lower order intermediates, including monomers. E, oligomeric state of zfprestin as determined by glutardialdehyde (GA) cross-linking followed by reducing SDS-PAGE. F, BN-PAGE analysis of the same samples as in E were resolved in non-denatured states. The ellipses schematically illustrate dimeric and monomeric states. G, autoradiography of a BN-PAGE gel containing [35S]methionine-labeled PASulP purified from X. laevis oocytes. Samples were treated as indicated before loading onto the gel.

 
Cross-linking by glutardialdehyde increased the fraction of dimeric proteins from 60% (no cross-linker, Fig. 4B, lane 1) to up to 80% in BN-PAGE gel (10 mM glutardialdehyde, Fig. 4B, lane 8). Thus, a certain fraction of non-covalently linked prestin dimers dissociates into monomers during overnight BN-PAGE electrophoreses. The imidoester dimethyl adipimidate, which has a longer spacer arm than glutardialdehyde (8.6 Å versus ~5 Å), was much less efficient in generating covalently linked dimers, as visualized by reducing SDS-PAGE (Fig. 4A, lanes 6-8) and BN-PAGE (Fig. 4B, lanes 10-13).

SLC26A3 (Fig. 4C, lanes 1 and 2) could also be cross-linked to dimers. Glutardialdehyde cross-linking did not affect the migration of the SLC26 dimers in the BN-PAGE gel (Fig. 4D). However, cross-linking completely prevented the SDS-induced dissociation of the dimers into monomers (compare lanes 2 and 4). The co-analyzed concatenated rat prestin dimer migrated at exactly the same position as the glutardialdehyde-cross-linked prestin on the BN-PAGE gel (lanes 9 and 10). No adducts larger than dimers were observed for any tested SLC26 isoform.

Non-mammalian Eukaryotic and Prokaryotic SLC26 Paralogs Are Also Assembled as Dimers—The SLC26 family contains paralogs in mammals, non-mammals, plants, fungi, and bacteria (2). To assess whether the oligomeric state is evolutionarily conserved in non-mammalian eukaryotes and prokaryotes, we studied the subunit stoichiometry of zebrafish (zf) prestin (15) and a bacterial isoform from P. aeruginosa (PASulP).

SLC26 paralogs were expressed as His fusion proteins in Xenopus oocytes and metabolically labeled. The zebrafish paralog acquired EndoH-resistant complex-type carbohydrates during a sustained pulse (Fig. 5A). Complex-glycosylation resulted in an ~25 kDa increase in the apparent molecular mass (indicated by a star). Elimination of two putative N-glycosylation sequons, 161NGT and 164NSS, by glutamine substitution of Asn161 and Asn164 prevented N-glycosylation and thus complex glycosylation (Fig. 5B). Comparative analysis under non-reducing and reducing conditions provided no evidence for the existence of disulfide-bonded zebrafish prestin oligomers (Fig. 5C).

In BN-PAGE, the non-denatured zebrafish prestin migrated predominantly as a dimer (~240 kDa) (Fig. 5D, lane 1) that dissociated into the monomeric ~120 kDa form when treated with SDS (Fig. 5D, lanes 2 and 3). The existence of dimeric prestin was further verified by cross-linking with glutardialdehyde (Fig. 5E). In contrast to the small amount of monomeric prestin visible in the BN-PAGE gel (Fig. 5F, lane 1), no such monomer could be detected in the cross-linked sample (Fig. 5F, lane 2). We conclude that zebrafish prestin exists as a homogenous population of dimers when expressed in Xenopus oocytes.

A prokaryotic SLC26 is also assembled as dimer (Fig. 5G). After expression in Xenopus oocytes, PASulP migrated as one distinct band of ~160 kDa in its non-denatured form (Fig. 5G, lane 1). Incubation for 1 h at 37 °C in the presence of Coomassie dye led to the additional appearance of a ~82-kDa band (Fig. 5G, lane 2). Denaturing with SDS resulted in one distinct band of ~62 kDa, close to the calculated molecular mass of 64 kDa of the PASulP monomer. We conclude that the 160- and 82-kDa bands represent the PASulP dimer and monomer, respectively, and that SDS increases the mobility of the PASulP monomer in the BN-PAGE gel.


Figure 6
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  		FIGURE 6.
Oligomeric state of human SLC26A3 and rat and zebrafish prestin in mammalian cells as determined by BN-PAGE. BN-PAGE analysis of fluorophore-tagged proteins heterologously expressed in tsA201 cells or FRT/TO HEK293 cells. If indicated, samples were treated with SDS for 1 h at 37 °C to induce dissociation into lower order intermediates before gel loading. YFP-tagged proteins were visualized by fluorescence scanning the wet BN-PAGE gel. Numbers specify the oligomeric state of P2X1 protein.

 


Figure 7
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  		FIGURE 7.
PFO and PFO-PAGE exert protein-denaturing functions. A, autoradiography of a PFO-PAGE gel (8% acrylamide) of rat prestins isolated from 1% digitonin extracts of X. laevis oocytes after a 24-h chase following overnight [35S]methionine labeling. Digitonin (0.5%) and PFO (1%) indicate the detergent used for elution from the Ni2+-NTA-agarose. Where indicated, samples were partially denatured with 0.1% SDS (1 h at 37 °C). GA indicates that digitonin-solubilized prestin was cross-linked by 10 mM glutardialdehyde before elution from the beads. B, PFO-PAGE gel (6% acrylamide) of YFP-rprestin extracted with 1% digitonin or 1% PFO from Flp-In-T-Rex cells stably expressing YFP-rprestin cells induced by 1 µg/ml of tetracycline for 24 h. Where indicated, samples were incubated with 0.1% SDS or 0.1 M DTT for 1 h at 37 °C before being loaded onto the gel. C, YFP-rprestin samples from the same experiment as shown in B were resolved by non-reducing SDS-PAGE. IAA indicates whether or not 10 mM iodoacetamide was present in the extraction buffer. D, the same YFP-prestin sample as in lane 4 of C was deglycosylated with EndoH or PNGase F as indicated and resolved by reducing SDS-PAGE. Masses of the All Blue Precision Plus Protein standard (Bio-Rad) are given on the left.

 
Subunit Stoichiometry of SLC26 Is Not Affected by the Expression System—To test whether oligomerization of eukaryotic SLC26s might be affected by the cellular environment, we resolved rat prestin, zebrafish prestin, and SLC26A3 by BN-PAGE after heterologous expression as YFP fusion protein in mammalian cells. The expressed proteins were extracted with 1% (w/v) digitonin and subjected to BN-PAGE either by application of the full lysate (rat and zebrafish prestin) or after purification by metal affinity chromatography (His-SLC26A3) (Fig. 6). In all cases, YFP-tagged SLC26 transporters migrated predominantly as bands of ~260 kDa (Fig. 6, lanes 3, 6, and 9) in BN-PAGE gels. The carboxyl-terminally GFP-tagged rP2X1 receptor served as a mass marker (Fig. 6, lanes 1 and 2). Incubation in SDS (Fig. 6, lanes 4 and 5, 7 and 8, and 10 and 11) led to a disappearance of the ~260-kDa SLC26 proteins and the appearance of one band with about half of the molecular mass, in agreement with a dimeric structure also in mammalian cells.

PFO-PAGE Does Not Display a Multimeric Structure of Rat Prestin—Previous experiments on the subunit stoichiometry of prestin used a different detergent and PAGE systems. Whereas we employed digitonin as detergent and BN-PAGE, Zheng et al. used PFO and PFO-PAGE (34). To assess the consequences of this difference, we purified WT and glycosylation-deficient mutant rat prestin from oocytes using digitonin and PFO as detergents. When resolved by PFO-PAGE, WT, and glycosylation-deficient rat prestin migrated entirely as monomers, irrespective of the detergent used for purification (Fig. 7A, lanes 2 and 3, and 8 and 9). On PFO-PAGE gels, prestin dimers were only observed when digitonin-purified prestin was cross-linked with glutardialdehyde before application to the gel (Fig. 7A, lanes 6 and 7). Hence, the dimeric state of prestin is preserved during purification with digitonin, but lost during PFO-PAGE unless stabilized by cross-linking. PFO not only dissociates prestin oligomers, but also GlyR pentamers (Fig. 7A, lane 1). The appearance of GlyR monomers in the non-denatured GlyR sample that was co-analyzed as a mass marker also indicates a significantly stronger denaturing effect of PFO than of digitonin. On BN-PAGE gels, GlyR behaves as a very stably assembled homopentamer; monomers are only observed after denaturation with SDS (22).

We next extracted a YFP fusion protein of rat prestin from stably transfected mammalian cells using digitonin or PFO. Digitonin-extracted YFP-rprestin migrated in the PFO-PAGE gel exclusively in the monomeric state (Fig. 7B, lane 2). Two proteins bands were resolved by PFO-PAGE and could be identified by deglycosylation analysis as the predominant complex-glycosylated (Endo H-resistant) form of prestin and a less abundant (Endo H-sensitive) core-glycosylated form (Fig. 7D). The dimeric state that is predominant on BN-PAGE gels (Fig. 6) is not present under these conditions. PFO-extracted YFP-prestin migrated neither in BN-PAGE gels (data not shown) nor in PFO-PAGE gels (Fig. 7B, lane 1) as oligomer. Neither the addition of DTT nor of iodoacetamide resulted in the appearance of protein bands with lower molecular weight, verifying the absence of disulfide-bonded YFP-rprestin oligomers (Fig. 7C). We conclude that PFO does not conserve the oligomeric state of rat prestin.


Figure 8
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  		FIGURE 8.
Non-linear capacitance in tsA201 cells co-expressing mutant rat prestin. A, voltage dependence of the non-linear capacitance of tsA201 cells expressing either D154N or D342Q prestin. B, simulated voltage dependence of the non-linear capacitance of cells co-expressing D154N and D342Q prestin without functional interaction between the subunits. C-E, voltage dependence of the non-linear capacitance measured on three different cells co-transfected with D154N and D342Q prestin. The solid black line represents a fit of a sum of three first derivatives of a Boltzmann term to the measured values. Colored lines show the voltage dependence of the three components: red and blue correspond to the capacitance conferred by the homodimeric motor proteins, green corresponds to the hetero-oligomers.

 
Functional Interaction between Prestin Subunits—In a dimeric assembly, each subunit can be functionally independent, or alternatively, the two neighboring subunits can cooperate in performing a certain protein function. To study whether the two subunits of the motor protein prestin interact functionally, we co-expressed two mutant proteins - D154N and D342Q (29). In agreement with an earlier report (29), D154N shifts the voltage dependence of the non-linear capacitance to more negative voltages and D342Q shifts the voltage dependence to more positive ones (Fig. 8A). The voltage dependence of the non-linear capacitance of prestin can be fit with a derivative of a Boltzmann equation, providing values for the potential at which the half-maximal charge transfer has occurred (V1/2) and the number of elementary charges displaced across a fraction of the membrane during the voltage-dependent conformational change (Table 1).


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  		TABLE 1
Fit parameters of the voltage dependences of non-linear charge movement of homodimeric mutant and heterodimeric rat prestin

 
In case of a parallel action of the two subunits without functional interactions, the voltage dependence of the non-linear capacitance of heterodimeric prestin will be the addition of the respective voltage dependences of each homo-dimeric transporter. In contrast, any cooperative interaction during the voltage-dependent conformational change will cause a deviation of the voltage dependence of the heterodimeric motor protein from this superposition. Fig. 8B shows the simulated voltage dependences of the non-linear capacitance (Cnonlin-V) curves, assuming no interaction between D154N and D342Q prestin in a heterodimeric motor protein. All the simulated Cnonlin-V curves showed two clearly separated maxima with amplitudes that correspond to the relative amplitude of the individual components.

Non-linear capacitances from co-transfected cells are different from the simple sum of the capacitances generated by the two mutant motor proteins (Fig. 8, C-E). The Cnonlin-V curves exhibit a single peak that is significantly broader than the ones observed in cells expressing only a single type of prestin. Heterodimeric motor proteins thus undergo conformational changes whose voltage dependence is jointly determined by both subunits. Such a behavior can only be found in case of a functional interaction of the two subunits. Conformational changes of one prestin molecule are modified by the contralateral subunit in the dimeric motor protein.

This functional assay did not only provide evidence for a functional interaction between subunits but also further support for a dimeric structure of prestin. For a dimeric protein, cells co-transfected with two plasmids are expected to express three populations, homodimeric D154N, homodimeric D342Q, and heterodimeric motor proteins consisting of one D154N and one D342Q subunit. We thus fitted the experimentally observed voltage dependences of non-linear capacitances with the sum of three first derivatives of a two-state Boltzmann function. For the two of the three that corresponded to the homodimeric populations, Vo and z{delta} were fixed to the values obtained from cells only transfected with one mutant, and only the relative amplitude Qmax was adjusted as a fit parameter. For the third term, Vo, z{delta}, and Qmax were optimized as fit parameters. For all tested cells, the values for Vo and z{delta} of the third fitted component were very similar (Table 1). Such behavior is in full agreement with a dimeric assembly. It is inconsistent with a tetramer, for which the third capacitance component would represent three hetero-multimeric components, whose relative contribution would depend on the relative number of the two mutant subunits in the oligomer.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
SLC26 is a gene family encoding anion transporters, anion channels, and membrane-bound motor proteins. The first evidence for SLC26 multimerization came from fluorescence resonance energy transfer experiments that demonstrated a close proximity between fluorescently tagged carboxyl termini of two prestin subunits (42-44). The number of subunits necessary to form a functional motor protein was first addressed by Zheng et al. (34). In that study, native and recombinant prestin were shown to form stable oligomers that resisted dissociation into monomers by SDS. Chemical cross-linking and PFO-PAGE combined with immunoblotting and affinity purification suggested a tetrameric subunit stoichiometry of prestin. SDS dissociated the tetramer into dimers that could be converted to monomers by hydrophobic reducing agents but not by the hydrophilic ones. The authors concluded that prestin monomers are covalently linked to dimers by disulfide bonds located in the hydrophobic membrane core and that these covalently linked dimers associate to form a tetramer.

Our results are in contrast to those published findings. BN-PAGE and chemical cross-linking demonstrated that neither prestin nor any other tested SLC26 isoform was linked by disulfide bonds, and all SLC26s formed dimers as predominant oligomeric state. Oligomers dissociated entirely into monomers under non-reducing conditions in the presence of low concentrations of SDS. The use of a tandem concatamer as a mass marker corroborated the correct assignment of BN-PAGE bands to certain oligomeric states of rat prestin. Not only all tested eukaryotic, but also a prokaryotic SLC26 exhibits a dimeric subunit stoichiometry. After expression in Xenopus oocytes, a paralog from P. aeruginosa migrates exclusively as dimer in BN-PAGE.

Because our experiments were performed with rat and zebrafish prestin and those of the other study with gerbil SLC26A5/prestin (34), these differences might be due to isoform-specific locations of cysteine residues. This is not the case: all cysteines are fully conserved in human, rat, mouse, and gerbil prestin, but not in the zebrafish isoform (supplemental Fig. S2). It appears furthermore possible that SLC26s exhibit both oligomerization states, dimers as well as tetramers, as reported for a member of another anion transporter family (SLC4) (45). However, the complete absence of tetramers in all our assays argues against this possibility.

We can thus only speculate about the reasons for the different results of our group and those by Zheng et al. (34). We solubilized prestin with digitonin, whereas Zheng et al. used PFO (34). Digitonin has a strong record as an efficient and gentle detergent for the extraction of membrane protein complexes prone to dissociation by treatment with other detergents (46, 47). The combination of digitonin solubilization and BN-PAGE has been demonstrated to faithfully display the subunit stoichiometry of various membrane proteins, such as the mitochondrial protein import machinery, oxidative phosphorylation supercomplexes (47), various ligand-gated ion channels (18, 32, 35), transporters (32, 48), and membrane-bound enzymes (49). In contrast, PFO-PAGE dissociates membrane-bound enzymes (50), ion channels (ionotropic {gamma}-aminobutyric acid (24) and glycine receptors (Fig. 7)), as well as transporters (a bacterial glutamate transporter6). PFO also dissociates prestin (Fig. 7, A and B). To explain the occurrence of oligomers resistant to both SDS and DTT (see Zheng et al. Fig. 6, A and B), we suggest that prestin may have aggregated during or after protein solubilization to form random oligomers. Alternatively, intersubunit disulfide bond formation could have occurred during purification in those experiments in which iodoacetamide was not added. Even small amounts of such non-physiological higher order oligomers can be detected by the highly sensitive Western blot technique (34). In contrast, we used metabolic labeling or labeling with fluorescent proteins, both permitting a quantification of the relative amounts of several protein fractions.

The difference in detection might also explain the dissimilar outcome of the chemical cross-linking. In the previous study, dimers were the predominant state, although trimers and tetramers were observed (34). Cross-linking combined with Western blotting provides semi-quantitative data about the abundance of the distinct oligomeric states. Moreover, at high membrane densities, which are likely to occur in the mammalian cochlea, unspecific cross-linking can occur between neighboring protein complexes.

A limitation of BN-PAGE analysis in determining the native oligomeric state of proteins is that solubilization of the protein might dissociate weak protein-protein interactions. It thus appears possible that SLC26s form stable dimers that associate to higher order oligomers in the native membrane, i.e. tetramers that are dissociated during solubilization or gel electrophoresis. The results of our cross-linking experiments (Figs. 4, 5E, and 7A) and the functional analysis of cells co-transfected with two mutant prestin proteins with different voltage dependences of the non-linear capacitance (Fig. 8) argue against this possibility. Co-transfection resulted in the formation of hetero-oligomers with distinct voltage dependences. The voltage dependence of the non-linear capacitance of co-transfected cells could be well fit assuming only a single population of hetero-oligomers without evidence for a cooperative interaction of more than two subunits. These data demonstrate that a dimeric assembly is the functional unit of prestin.

Heterodimeric D154N-D342Q prestin exhibits voltage-dependent capacitances that are different from D154N and D342Q homodimeric proteins. The two mutations thus do not exert an additive effect, as expected in case of two independently functioning subunits. The two subunits rather jointly determine the voltage dependence of conformational changes of the dimeric protein. This inter-subunit interaction explains the recent finding of a missense mutation in the pres gene found in a heterozygous hearing-impaired patient (51). The mutation results in a shift of the voltage dependence of non-linear capacitance. It can result in a dominant inheritance mode, because heterodimeric prestin consisting of WT and mutant prestin exhibits an intermediate phenotype. At present, it is unknown if inter-subunit interactions occur in SLC26 transporters and channels. For all inherited diseases linked to these SLC26 genes, the inheritance mode is recessive (3).

The interaction between prestin subunits is weak. BN-PAGE of rat prestin reveals a significant percentage of monomeric proteins (Fig. 3), and cross-linking increases the percentage of dimers to only ~80% (Fig. 4). Hence, a certain fraction of monomers exists in intracellular compartments or in the surface membrane.

Many known transporter families have been shown to form oligomeric proteins. Members of the SLC1 family are assembled as trimers (32, 38), whereas SLC4, SLC6, and ClC transporters form dimers (40, 52, 53). In oligomeric transporters two different forms of intersubunit interactions are possible. The subunits could jointly contribute to a central carrier or motor protein domain, or each subunit might be capable of functioning by itself. SLC1 (38), SLC4 (52), and ClC (40) transporters exhibit multibarreled structures with two or three carrier domains each formed by a single subunit. The ClC family contains channels and transporters, and for ClC channels cooperative gating steps have been reported (54). In SLC1 transporters, each subunit appears to function independently of each other (55). Because prestin dimers are not very stable (Fig. 3) and because the inheritance mode of SLC26-linked diseases is in general recessive, the formation of a common carrier or motor protein domain appears unlikely. A likely explanation for all our results is that two SLC26 subunits work in parallel but interact allosterically.

Two different transmembrane topology models of prestin are currently considered. Both place the amino and the carboxyl termini at the intracellular membrane side, but differ in the number of transmembrane helices. Although Zheng and colleagues postulated 12 transmembrane helices (56), a revised topology model contains only 10 transmembrane domains (42). We here confirm that rat prestin is a glycoprotein with two N-glycans at Asn163 and Asn166 (26) and demonstrate that zebrafish prestin and human SLC26A3 carry two N-glycans at corresponding localizations. SLC26A3 harbors one additional N-glycan in position Asn153, which is also located in the predicted second extracellular loop. Three N-glycosylation sequons in the rat prestin sequence remain unused: 589NATV, 603NATK, and 736NATP. The non-usage of 736NATP does not provide topological information, because a proline in the +4 position is known to prevent N-glycosylation. In contrast, the lack of glycosylation of 589NATV and 603NATK supports the cytosolic localization of these residues. The glycosylation states of various SLC26s strongly support the topology model with 12 transmembrane domains.

We conclude that a dimeric subunit stoichiometry is general to prokaryotic and eukaryotic SLC26 isoforms. Three different approaches demonstrated a dimeric subunit stoichiometry for one mammalian anion exchanger, one mammalian motor protein, one zebrafish prestin homologue, and one prokaryotic paralog. Isoform-specific functional differences therefore do not originate from differences in the quaternary structure.


   FOOTNOTES
 
* This work was supported by the Deutsche Forschungsgemeinschaft (Grant FOR450 to P. H., G. S., and Ch. F.). 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

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1 and S2. Back

1 Both authors contributed equally to this work as first authors. Back

4 Both authors contributed equally to this work as senior authors. Back

2 To whom correspondence may be addressed. Tel.: 49-241-808-9130; Fax: 49-241-808-2433; E-mail: gschmalzing{at}ukaachen.de. 3 To whom correspondence may be addressed. Tel.: 49-511-532-2777; Fax: 49-511-532-2776; E-mail: fahlke.christoph{at}mh-hannover.de.

5 The abbreviations used are: SLC26, solute carrier 26; BN, blue native; EndoH, endoglycosidase H; HEK293, human embryonic kidney 293; NTA, nitrilotriacetic acid; PFO, perfluoro-octanoate; PNGase F, peptide:N-glycosidase F; YFP, yellow fluorescent protein; DTT, dithiothreitol; WT, wild type; zf, zebrafish; zfprestin, zebrafish prestin; rprestin, rat prestin; PASulP, bacterial isoform from P. aeruginosa; DIDS, 4,4'-diisothiocyanostilbene disulfonic acid; DMA, dimethyl adipimidate; GA, glutardialdehyde; GlyR: glycine receptor. Back

6 S. Detro-Dassen and G. Schmalzing, our unpublished observations. Back


   ACKNOWLEDGMENTS
 
We thank Drs. B. Fakler and D. B. Mount for providing expression constructs for rat prestin, zebrafish prestin, and human SLC26A3.



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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