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Electrogenic Sulfate/Chloride Exchange in Xenopus Oocytes Mediated by Murine AE1 E699Q

Functional evaluation of chemically modified human erythrocytes has led to the proposal that amino acid residue E681 of the band 3 anion exchanger AE1 lies on the anion translocation pathway and is a proton carrier required for H+/SO4 2− cotransport. We have tested in Xenopus oocytes the functional consequences of mutations in the corresponding residue E699 of mouse AE1. Most mutations tested abolished AE1-mediated Cl− influx and efflux. Only the E699Q mutation increased stilbene disulfonate-sensitive efflux and influx of SO4 2−. E699Q-mediated Cl− influx was activated by elevation of intracellular SO4 2−, but E699Q-mediated Cl− efflux was undetectable. The DNDS (4,4′-dinitrostilbene-2,2′-disulfonic acid) sensitivity of E699Q-mediated SO4 2− efflux was indistinguishable from that of wt AE1-mediated Cl− efflux. The extracellular anion selectivity of E699Q-mediated SO4 2− efflux was similar to that of wt AE1-mediated Cl− efflux. The stoichiometry of E699Q-mediated exchange of extracellular Cl− with intracellular SO4 2− was 1:1. Whereas SO4 2− injection into oocytes expressing wt AE1 produced little change in membrane potential or resistance, injection of SO4 2−, but not of Cl− or gluconate, into oocytes expressing E699Q depolarized the membrane by 17 mV and decreased membrane resistance by 66%. Replacement of bath Cl− with isethionate caused a 28-mV hyperpolarization in SO4 2−-loaded oocytes expressing E699Q, but had no effect on oocytes expressing wt AE1. Extracellular Cl−-dependent depolarization of SO4 2−-preloaded oocytes was blocked by DNDS. AE1 E699Q-mediated inward current measured in the presence of extracellular Cl− was of magnitude sufficient to account for measured 35SO4 2− efflux. Thus, AE1 E699Q-mediated SO4 2−/ Cl− exchange operated largely, if not exclusively, as an electrogenic, asymmetric, 1:1 anion exchange. The data confirm the proposal that E699 resides on or contributes to the integrity of the anion translocation pathway of AE1. A single amino acid change in the sequence of AE1 converted electroneutral to electrogenic anion exchange without alteration of SO4 2−/Cl− exchange stoichiometry.

i n t r o d u c t i o n
The anion exchanger 1 (AE1) 1 (band 3) erythroid anion exchanger is one of the most extensively studied membrane transport proteins (Passow, 1986;Tanner, 1993;Alper, 1994). Erythroid AE1-mediated exchange of Cl Ϫ for HCO 3 Ϫ serves to increase the total CO 2 carrying capacity of blood. Intracellular HCO 3 Ϫ generated by red cell carbonic anhydrase from CO 2 released from respiring tissues is exchanged for extracellular Cl Ϫ in the capillaries of the peripheral circulation. In the pulmonary capillaries the reverse sequence occurs, and ex-tracellular HCO 3 Ϫ enters the cell in exchange for intracellular Cl Ϫ . The newly entered HCO 3 Ϫ is converted to CO 2 by carbonic anhydrase, and the CO 2 can diffuse from the blood cell across endothelium, interstitial space, and alveolar epithelium into the alveolar airspace for expiration.
Despite many years of study, the molecular mechanism by which AE1 mediates anion exchange remains unclear. Only recently has data emerged identifying amino acid residues that may participate directly in binding and translocating anions. Chemical modification studies of red cells have contributed to these identifications. The requirement for liganding anions focused initial attention on cationic amino acid residues. Indeed, chemical modification and pH titration of AE1 in red cells led to the hypothesis that one or more arginine residues play important roles in AE1 transport function (Wieth et al., 1982;Julien and Zaki, 1988). Reductive methylation of AE1 lysines inhibited transport (Jennings, 1982), whereas dansylation of red cells produced reciprocal changes in Cl Ϫ and SO 4 2 Ϫ transport (Lepke and Passow, 1982;Berghout et al., 1988). Mutation of the lysine residues that provide covalent attachment sites to the isothiocyanate moieties of cyanostilbene disulfonates reduced inhibition by these site-directed reagents, but did not change the K 1/2 for extracellular Cl Ϫ of AE1-mediated transport . A different lysine residue has also been identified as exposed to both intracellular and extracellular aqueous media by fluorescence quench of pyridoxal phosphate covalently bound to red cell AE1 ( Bar-Noy and Cabantchik, 1990).
The mildly acidic pK of inhibition by protons of red cell Cl Ϫ /Cl Ϫ exchange led to the implication of histidine residues in AE1 transport function by study of the effect of diethylpyrocarbonate modification of red cells (Izuhara et al., 1989) and by site-directed mutagenesis (Muller-Berger et al., 1995 b ). SO 4 2 Ϫ transport in red cells was found to be stimulated by acidification, with a pK a of 5.5, and was accompanied by proton uptake Gunn, 1982, 1984). The demonstration of H ϩ -SO 4 2 Ϫ cotransport led to the proposal that the residue responsible for proton binding is a residue of acidic pK a . Both titration experiments and chemical modification studies have implicated carboxylate residues in AE1-mediated H ϩ -SO 4 2 Ϫ /Cl Ϫ exchange.
Jennings and colleagues have investigated further the involvement of glutamate residues in human AE1-mediated anion exchange. They reported that reduction of the E681 carboxylate to the corresponding alcohol by treatment with Woodward's reagent K (WRK) followed by reduction with borohydride (BH 4 ) produced a complex pattern of changes in anion transport (Jennings and Anderson, 1987;Jennings and Smith, 1992). Cl Ϫ / Cl Ϫ exchange was inhibited, whereas SO 4 2 Ϫ i /SO 4 2 Ϫ o and SO 4 2 Ϫ i /Cl Ϫ o exchange were stimulated 5-to 10-fold and 80-fold, respectively. Protons were no longer cotransported with SO 4 2 Ϫ , and the activation of SO 4 2 Ϫ transport by acidic pH was abolished. Moreover, in WRK-BH 4 modified cells, trans Cl Ϫ -dependent efflux of 35 SO 4 2 Ϫ was accelerated by cationophores, and ionophore-mediated 86 Rb efflux was activated by extracellular Cl Ϫ . (Jennings and Al-Rhaiyel, 1988;Jennings, 1995). Residue E681 of AE1 was defined as the principal target for WRK-BH 4 modification of intact human erythrocytes (Jennings and Smith, 1992). These workers proposed that E681 is exposed to both intracellular and extracellular aqueous spaces during the anion exchange cycle and is a proton binding site for H ϩ /SO 4 2 Ϫ cotransport. The data further suggested that modification of the AE1 residue E681 to the corresponding alcohol conferred on modified AE1 the ability to mediate electrogenic exchange of intracellular SO 4 2 Ϫ for extracellular Cl Ϫ (Jennings, 1995).
We were prompted by these findings to examine the functional role of the corresponding glutamate residue in mouse AE1, E699, as expressed in the Xenopus oocyte from cRNA. The Xenopus oocyte was the first heterologous system used for functional expression of recombinant AE1 (Bartel et al., 1989;Garcia and Lodish, 1989) and has been developed by subsequent investigators for examination of structure-function relationships in the AE1 polypeptide (Kietz et al., 1991;Groves and Tanner, 1993;Chernova et al., 1995;Muller-Berger, 1995 a , b ). The Xenopus oocyte has been used to confirm the electroneutrality of wild-type (wt) AE1-mediated Cl Ϫ /anion exchange, but also to demonstrate the potential-dependence of transport rates (Grygorczyk et al., 1987).
We have found decreased Cl Ϫ transport in oocytes expressing mouse AE1 mutated at residue E699. Only one among the several amino acid substitutions tested, E699Q, displayed increased SO 4 2 Ϫ transport. Efflux of intracellular SO 4 2 Ϫ was easily detected and required the presence of extracellular Cl Ϫ or other appropriate anions. In contrast efflux of intracellular Cl Ϫ was undetectable regardless of the extracellular anion present. Whereas AE1 E699Q mediated 1:1 electroneutral SO 4 2 Ϫ /SO 4 2 Ϫ exchange, exchange of intracellular SO 4 2 Ϫ for extracellular Cl Ϫ by AE1 E699Q was accompanied by inward currents, consistent with electrogenic outflow of anions. E699Q-mediated anion fluxes and currents were inhibited by the stilbene disulfonate, DNDS (4,4Ј-dinitrostilbene-2,2Ј-disulfonic acid). The stoichiometry of SO 4 2Ϫ efflux to Cl Ϫ influx to inward current was consistent with 1:1 electrogenic anion exchange.
Our results with recombinant mutant AE1 confirm and extend several earlier conclusions from the studies of Jennings and colleagues on red cells: (a) E699 serves as the proton binding site during H ϩ SO 4 2Ϫ /Cl Ϫ exchange by wt AE1; (b) charge neutralization at E699 leads to electrogenic exchange of SO 4 2Ϫ for Cl Ϫ with a 1:1 stoichiometry; and (c) such electrogenic exchange is asymmetric. The results further show that electroneutrality and 1:1 stoichiometry are properties of the AE1 protein that can be uncoupled. Recent results from two additional groups address the functional role of E699. Muller-Berger et al. (1995a) showed that AE1 E699D expressed in Xenopus oocytes exhibits increased pK for Cl Ϫ /Cl Ϫ exchange. Sekler et al. (1995)

Solutions
ND-96 contained (in mM): 96 NaCl, 2 KCl, 1.8 MgCl 2 , 1 CaCl 2 , and 5 HEPES hemisodium, pH 7.40. In some experiments 96 mM NaCl was replaced mole for mole with the sodium salt of either gluconate, isethionate, nitrate, bromide, or iodide. Alternatively, 96 mM NaCl was replaced with 64 mM Na 2 SO 4 or with 70 mM sodium phosphate, pH 7.4. All Cl Ϫ -free solutions included the above mentioned concentrations of K ϩ , Mg 2ϩ , and Ca 2ϩ as the gluconate salts.

Construction of AE1 Mutants
The 563 nt Sma1/Sph1 fragment encoding murine erythroid AE1 nt 2117-2679 (Kopito and Lodish, 1985) was excised from the murine kidney AE1 plasmid pBL (Brosius et al., 1989) and subcloned into M13mp19. Mutations in AE1 E699 were constructed in this phage subclone by the dut Ϫ /ung Ϫ method (Kunkel et al., 1991) using the degenerate oligonucleotide 5Ј-CAT TTTCCTT[C,G,T,A][C,G,T,A]GTCT-3Ј and the Muta-Gene T7 Kit (Bio-Rad Laboratories, Richmond, CA). Mutant phage were detected by sequencing across the mutation site. Doublestranded DNAs from phage in which E699 had been mutated to R, K, G, T, and Q were used to reconstruct full-length mutant AE1 cDNAs. Mutant plasmid cDNAs were again sequenced across the mutation site.

Expression of cRNA in Xenopus Oocytes
Transcription template was made by linearizing plasmids with HindIII. cRNA transcription with T7 RNA polymerase was performed with the Megascript Kit (Ambion Inc., Austin, TX). Manually defolliculated oocytes prepared as previously described (Humphreys et al., 1994) were microinjected with 20 ng cRNA and incubated in ND-96 at 19ЊC for 1-14 d.

Immunoprecipitation of Total and Surface AE Proteins from Xenopus Oocytes
Groups of 10-12 oocytes previously injected with water or with 20 ng cRNA were incubated in ND-96 containing 1-1.5 mCi/ml of 35 S-methionine (20-30 M) for 48-72 h. Metabolically labelled oocytes were washed in modified ND-96, pH 8.0, then incubated for 1 h at 4ЊC in the same medium in the presence or absence of 5 mg/ml chymotrypsin. Oocytes were then washed three times in 10 ml ND-96, pH 7.4 containing 2 mM PMSF and 1 mg/ml BSA Fraction V, and once more in the same medium containing 100 g/ml chymostatin and 1 mg/ml BSA. Groups of washed oocytes were manually homogenized at 4ЊC with a fitted Teflon ® pestle (Kontes, Vineland, NJ) in microfuge tubes with 100 l oocyte immunoprecipitation (IP) buffer containing (in mM) 50 Tris-HCl, 1 EDTA, 1 PMSF, and 0.04 each of leupeptin, pepstatin, and antipain, pH 7.6. The extract was incubated with shaking for 30 min at 4ЊC, then centrifuged 10 min in a microfuge. The resultant supernatants were brought to 500 mM NaCl and precleared with 5% normal rabbit serum. Precleared supernatants were incubated 1 h with rabbit polyclonal antiserum raised against mouse AE1 aa 214-228 , followed by precipitation with protein A-agarose. The protein A-agarose pellets were washed six times in 1 ml IP buffer containing 500 ml NaCl, six more times in 1 ml IP buffer without NaCl, then analyzed by SDS-PAGE fluorography.

Isotopic Flux Studies
Measurement of 35 SO 4 2Ϫ influx was carried out as follows: Oocytes were injected 10 min before initiation of influx measurements with 50 nl of a solution containing (in mM) 130 Na 2 SO 4 , 50 HEPES, pH 7.4. The flux assay was initiated by transfer of groups of 6-10 water-injected or 8-12 cRNA-injected oocytes into microtiter wells containing 149 l of Na isethionate influx medium containing 2 mM Na 2 SO 4 and 1 l (5 Ci) Na 2 35 SO 4 . Influx assays were carried out at room temperature for 15 min, then terminated by rapid transfer of groups of oocytes through three 25ml room temperature washes in isotonic Na gluconate medium. In experiments designed to measure the stoichiometry of SO 4 2Ϫ / SO 4 2Ϫ exchange, influx medium contained 64 mM Na 2 SO 4 instead of 96 mM Na isethionate.
Assay of 35 SO 4 2Ϫ efflux was carried out as follows: Oocytes were injected with 50 nl of a solution containing Na 2 35 SO 4 (0.25-0.5 Ci, 3-6 M) in 50 mM HEPES, pH 7.4, with or without 130 mM Na 2 SO 4 , and maintained for 10 min post-injection in SO 4 -free, Clfree medium containing 96 mM Na isethionate. Efflux was initiated by transfer of individual oocytes into 1 ml of efflux medium. At regular intervals, 950 l of this medium was removed for scintillation counting and replaced with fresh medium. All efflux experiments ended with a final period of efflux into medium containing 100 M DIDS or DNDS before solubilization of the washed oocyte in 100 l 1% SDS. The sum of efflux fractions and residual cpm in the oocyte was Ͼ98% of originally injected cpm. Data were plotted as ln (% cpm remaining) vs. time. Efflux rate constants were measured from linear least squares regressions calculated from the last three time points for each experimental condition, and corrected by subtraction of the efflux rate constant of water-injected oocytes studied in the same experiment. Effluxes were calculated as products of the measured rate constants and intracellular ion concentration (determined as described below). Oocyte water space was assumed to be 450 nl (500 nl acutely following 50 nl microinjection of isotope). As described in results, endogenous oocyte SO 4 2Ϫ concentration was assumed to be 1.1 mM (1.0 mM following microinjection of 50 nl fluid).
Measurement of 36 Cl Ϫ influx was carried out as described by Humphreys et al. (1994) in the presence of 10 M bumetanide. Influx periods were 15-30 min, during which uptake was linear. For some experiments, individual oocytes kept in isethionate medium were injected with 50 nl of 50 mM HEPES, pH 7.40, with or without 130 mM Na 2 SO 4 , then maintained 10 min in isethionate medium before initiation of the influx experiment in ND-96 or other media as indicated.
Measurement of 36 Cl Ϫ efflux was carried out as described previously (Humphreys et al., 1994), modified only in that oocytes were placed into isethionate medium for 10 min after isotope injection. Efflux was initiated by transfer of individual oocytes into medium containing ND-96 or other media as indicated.
Peak anion transport activities were exhibited by wt AE1 at 3-4 d post-injection of cRNA, as noted previously (Brosius et al., 1989;Humphreys et al., 1995). However, anion transport activities of oocytes expressing AE1 E699Q required up to 7 d to reach peak values. Wt AE1-expressing oocytes began to die as soon as 4 d post-injection and were unusable after 10 d. In contrast, AE1 E699Q-expressing oocytes maintained in ND-96 remained healthy and functional for as long as 15 d or more post-injection with cRNA.

Determination of Oocyte (Ovary) Intracellular SO 4 2Ϫ Concentration
Samples for sulfate determination were prepared as follows. Resected fragments of Xenopus ovary were centrifuged in a microcentrifuge at 18,000 rpm for 1 h. 130 l of the supernatant fraction were diluted with water to 500 l and filtered through a Centricon 3 filter (Amicon Corp., Danvers, MA) in a Sorvall RT 6000B at 3,000 rpm for 4 h. This crude cytosol fraction was subjected to sulfate analysis by a barium precipitation method (Greenberg et al., 1980). Costa et al. (1989) have shown that intracellular Na and Cl activities and contents determined in ovary extracts and in pooled, isolated oocytes did not differ.

Measurements of Membrane Potential as a Function of Intracellular Anion
Defolliculated oocytes were incubated for 2-4 d at 19ЊC after injection of cRNA or water. Oocytes were placed in a superfusion chamber, impaled with a 3 M KCl-filled microelectrode, and allowed to recover for several minutes until a stable membrane potential was observed. Pulses of 10 nA were injected at 3-s intervals to monitor input resistance. Monitoring of membrane potential and current injection were performed with a CA 100 voltage clamp amplifier (Biologic, Echirolles, France). A second microelectrode was then introduced into the oocyte, through which the designated K ϩ salts (40-60 nl, titrated to pH 7.4) were injected into the oocytes with 10 s pressure pulses from a pneumatic picopump (WPI, Hertfordshire, UK). Membrane potential and input resistance were monitored for at least 10 min after pressure injection. Data were printed on a thermal array recorder (Graphtec, Japan) or processed through an analog-digital converter at 5 kHz, then transferred to computer for further analysis with custom-written software.

Measurements of Membrane Potential and Current as a Function of Extracellular Anion
After injection of cRNA or water, oocytes were incubated in ND96 for 4-14 d at 19ЊC. Oocytes were then injected with 50 nl of solution containing (in mM) 130 Na 2 SO 4 , 50 HEPES, pH 7.40, and placed in medium in which 96 mM Na isethionate replaced NaCl. Between 1 and 6 h afterward, single oocytes were placed in a 5-ml bath chamber (Model RC-11, Warner Instrument Corp., Hamden, CT) on the stage of a dissecting microscope and impaled with microelectrodes under direct view. Current and potential-sensing electrodes were pulled from borosilicate glass (1.2-mm outer diameter, 0.94-mm inner diameter; Sutter Instrument Co., Novato, CA) on a Flaming/Brown Model P-97 micropipette puller (Sutter Instrument Co.). The electrodes were filled with 3 M KCl and had resistances of 2-3 M⍀.
After impalement, oocyte membrane potential was monitored until it had stabilized (typically 3-7 min). Oocytes were voltage clamped using a Geneclamp 500 amplifier (Axon Instruments, Foster City, CA) interfaced to an 80486 50 MHz IBM-compatible computer (Dell 450/ME, Austin, TX) via a DigiData 1200 AD/D board (Axon Instruments). Electrical connections between the electrodes and the amplifier were made using Ag/AgCl pellets or wires and 3 M KCl/3% agarose bridges. Current was measured as that flowing to ground via the bath reference electrode. Errors induced by voltage drops across the bath ground were eliminated by use of a second reference electrode and a virtual-ground circuit. Current signals were filtered at 200 Hz before digitization. Oocytes were maintained in open-circuit conditions except during measurement of whole oocyte currents, for which membrane potential was clamped at a holding potential of Ϫ50 mV, and test potential steps at 20-mV intervals between Ϫ100 or Ϫ80 mV to ϩ80 mV were imposed for 800-ms time periods. Whole oocyte currents recorded at each test potential were acquired to hard disk for later analysis with pClamp software (Axon Instruments). Oocytes clamped to test potentials of ϩ80 mV or higher displayed activation of endogenous outward currents.

Estimate of Proton Flux Accompanying Sulfate Efflux
Oocytes previously injected with 50 nl of 130 mM Na 2 SO 4 were placed on coverslips in 1 l droplets of modified ND-96 containing 5 M BCECF free acid and 0.5 mM MOPS, pH 7.4, as buffer (Jaisser et al., 1993). The coverslips were mounted in a customized chamber on an inverted microscope stage, and the oocytes in their droplets were alternately irradiated at 440 and 495 nm. BCECF fluorescence excitation ratios were acquired at 530 nm from the extracellular fluid and recorded to optical disk with an Image 1 digital ratio imaging system (Universal Imaging, West Chester, PA) as previously described (Humphreys et al., 1994(Humphreys et al., , 1995. Calibration of the BCECF free acid fluorescence ratio was performed as described (Thomas et al., 1979). J H ϩ from oocyte into the surrounding droplet was calculated by multiplying the measured dpH i /dt of the droplet by the calculated buffer capacity of the extracellular medium. AE1 E699Q-mediated proton efflux was estimated by subtracting outward J H ϩ of water-injected oocytes from that of E699Q-expressing oocytes.
r e s u l t s

Isotopic Chloride Transport by AE1 Mutants
Site-directed mutagenesis was used to modify the wt glutamate residue at position 699 in recombinant murine AE1. Two mutations reversed the wt negative charge with arginine and lysine substitutions. One mutation neutralized the charge with glycine. Two more mutations attenuated the negative charge with the negative dipoles of threonine and glutamine. The threonine hydroxyl resembled the hydroxyl group of the hydroxynorvaline product of WRK-BH 4 modification, whereas the glutamine side chain more closely imitated the steric bulk of the hydroxynorvaline. cRNAs encoding the full-length mutant AE1 polypeptides were expressed in Xenopus oocytes. The ability of the mutant AE1 polypeptides to transport Cl Ϫ was compared with that of wt AE1 48 h after cRNA injection. Fig. 1 A shows a 64-fold activation of unidirectional Cl Ϫ influx by wt AE1, from 0.1 to 6.4 nmol Cl Ϫ per oocyte и 15 min. In contrast, none of the mutants mediated Cl Ϫ uptake under these conditions. 36 Cl Ϫ efflux mediated by these AE polypeptides is shown in Fig. 1 B. Cl Ϫ efflux was evident in wt AE1-expressing oocytes but not in oocytes expressing the AE1 mutants. Exchange of intracellular Cl Ϫ for extracellular sulfate was not observed with either wt AE1 or any of the mutants (Fig. 1 B). This is consistent with a relative rate of red cell Cl Ϫ /sulfate exchange Ͼ10 3 slower than Cl Ϫ / Cl Ϫ exchange, and with a detection threshold of the oocyte 36 Cl Ϫ efflux assay of Ͼ1% of the rate of Cl Ϫ /Cl Ϫ exchange (Humphreys et al., 1994).

Isotopic Sulfate Transport by AE1 Mutants
AE1 E699Q-mediated 35 SO 4 2Ϫ efflux was not reproducibly detectable 48 h after cRNA injection. However, by 5 d after cRNA injection, E699Q-expressing oocytes exhibited sulfate/sulfate exchange (Fig. 2 A). In contrast, neither oocytes expressing wt AE1 nor those expressing E699 mutants in which K, G, R, or T was substituted showed detectable sulfate/sulfate exchange. AE1 E699Q-mediated sulfate/sulfate exchange was DIDSsensitive. Replacement of extracellular 64 mM Na 2 SO 4 with 96 mM NaCl accelerated AE1 E699Q-mediated 35 SO 4 2Ϫ efflux 1.7-fold ( Fig. 2 C) but did not produce detectable wt AE1-mediated 35 SO 4 2Ϫ efflux, unlike the Figure 1. Cl Ϫ transport by mutant AE1 polypeptides. (A) 48 h after injection of cRNA or water, groups of 8-12 oocytes were incubated in 36 Cl Ϫ for 15 min to determine AE1-mediated Cl Ϫ influx. None of the mutants displayed Cl Ϫ -uptake activity. Representative of three similar experiments for all mutants and three additional experiments comparing wt AE1 with AE1 E699Q. (B) 48 h after injection of cRNA or water, oocytes were acutely injected with Na 36 Cl and subjected to isotopic efflux assay as in methods. Extracellular condition noted above x axis. DIDS was added in the continued presence of Cl Ϫ . E699Q displayed no Cl Ϫ efflux activity. One of two similar experiments. Each trace represents a single oocyte. acceleration of wt AE1-mediated 36 Cl Ϫ efflux from oocytes ( Fig. 1 B) or from red cells (Hanke-Baier et al., 1988;Passow et al., 1992) produced by the same extracellular substitution. 35 SO 4 2Ϫ influx into wt AE1-expressing oocytes was undetectable in seven experiments with extracellular [SO 4 2Ϫ ] ranging from 2 to 64 mM, consistent with the low SO 4 2Ϫ /Cl Ϫ exchange rate of unmodified intact red cells. 35 SO 4 2Ϫ influx into AE1 E699Q-expressing oocytes was detectable at low level in about half of experiments performed 48 h after cRNA injection.

Biosynthesis and Surface Expression of AE1 E699Q
Since the only AE1 E699 mutant which transported sulfate in the Xenopus oocyte 48 h after cRNA injection was E699Q, further studies focused on this mutant. Total accumulation of AE1 E699Q polypeptide was at least half that of the wt AE1 polypeptide (Fig. 3, lanes 1 and  3). In Fig. 3, 19% of wt AE1 was cleaved by extracellular chymotrypsin (lane 2), as was 15% of AE1 E699Q (lane 4). These figures represent biochemical estimates of the steady-state proportion of total oocyte AE polypeptide at the oocyte surface. In four similar experiments using oocytes from two frogs, 32 Ϯ 7% of wt AE1 was exposed to chymotrypsin digestion at the oocyte surface. The comparable figure for AE1 E699Q was 29 Ϯ 9%. Therefore, surface accumulation of wt and of E699Q AE1 polypeptides in Xenopus oocytes was of comparable efficiency.

Stilbene Sensitivity of AE1 E699Q
Wt AE1-mediated 36 Cl Ϫ efflux into chloride medium was reversibly inhibited in a dose-dependent manner by the stilbene disulfonate inhibitor, DNDS, with a concentration for half-maximal inhibition of 13 ϩ 8 M (Fig. 4 B). This value is similar to the value of 3-5 M reported by Passow et al. (1992). Half-maximal inhibition by DNDS of AE1 E699Q-mediated 35 SO 4 2Ϫ efflux into chloride medium was 8 Ϯ 2 M (Fig. 4, A and B), a value not significantly different from that of wt AE1 (P Ͼ 0.1). Thus, the apparent affinity of AE1 for an impermeant reversible antagonist applied outside the oocyte was not changed by the single E699Q mutation.   1 and 2), AE1 E699Q (lanes 3 and 4), or no cRNA (lane 5, water-injected) were incubated for 3 h in the absence (lanes 1, 3, and 5) or presence (lanes 2 and 4) of 5 mg/ml chymotrypsin as described in methods. Triton X100 extracts of the oocytes were subjected to immunoprecipitation with anti-AE1 antibody, SDS-PAGE, and autoradiography. Arrows indicate holoprotein and the 60-kD NH 2 -terminal chymotryptic fragment of AE1 (p60). Representative of four similar experiments. 2Ϫ efflux. Each of 5 oocytes was exposed for 9 or 12 min to incrementally increasing DNDS concentrations. SO 4 2Ϫ efflux rates relative to those observed in extracellular Cl Ϫ were 0.97 Ϯ 0.12 (nitrate), 0.67 Ϯ 0.04 (bromide), 0.20 Ϯ 0.03 (gluconate), 0.20 Ϯ 0.04 (isethionate), 0.16 Ϯ 0.02 (iodide), and 0.04 Ϯ 0.01 (phosphate). The values for nitrate, bromide, and iodide were close to those reported previously for wt AE1-mediated Cl Ϫ efflux from Xenopus oocytes and intact mouse red cells . The relative rates of AE1 E699Q-mediated SO 4 2Ϫ exchange with gluconate, isethionate, and phosphate were higher than expected for AE1-mediated Cl Ϫ exchange with the same anions in human red cells, but these rates had not been reported for wt AE1 expressed in Xenopus oocytes. There-fore, relative rate constants were also measured for wt AE1-mediated efflux of 36 Cl Ϫ into extracellular gluconate and isethionate, and were found to be 0.06 Ϯ 0.02 (n ϭ 13 oocytes, 3 frogs) and 0.11 Ϯ 0.03 (n ϭ 17 oocytes, 4 frogs), respectively. Though slightly lower than the corresponding values for AE1 E699Q, these values were consistent with the rates of Cl Ϫ /gluconate exchange exhibited by trout AE1 (Fievet et al., 1995) and by mouse AE2 (Humphreys et al., 1994).

Stoichiometry of AE1 E699Q-mediated Exchange of Intracellular SO 4 2Ϫ for Extracellular Cl Ϫ
To estimate the stoichiometry of AE1 E699Q-mediated SO 4 2Ϫ /Cl Ϫ exchange, it was necessary to measure Cl Ϫ influx. Though AE1 E699Q-mediated 36 Cl Ϫ influx was minimal or absent two days after cRNA injection (Fig. 1  A), 36 Cl Ϫ influx and 35 SO 4 2Ϫ efflux were both detectable 5-7 d after injection of cRNA (Fig. 6). A second requirement was knowledge of the intracellular [SO 4 2Ϫ ]. Cytosolic fractions isolated from ovarian fragments were subjected to sulfate analysis by barium precipitation (Greenberg et al., 1980). This assay indicated an ovarian cytosolic [SO 4 2Ϫ ] of 1.1 Ϯ 0.1 mM (n ϭ 5 frogs). On this basis, oocyte cytosol [SO 4 2Ϫ ] was assumed to be 1 mM after injection of a 50 nl volume into a nominal 450-nl water space.
The stoichiometry of SO 4 2Ϫ /Cl Ϫ exchange by AE1 E699Q was examined Ն5 d after cRNA injection. AE1 E699Q-mediated SO 4 2Ϫ efflux into extracellular Cl Ϫ was 0.31 Ϯ 0.05 pmol cell Ϫ1 s Ϫ1 (n ϭ 59 oocytes, 10 frogs). In 96 oocytes from 10 frogs, AE1 E699Q-mediated Cl Ϫ influx was 0.25 Ϯ 0.06 pmol cell Ϫ1 s Ϫ1 . The ratio of the mean SO 4 2Ϫ efflux over the mean Cl Ϫ influx was 1.24. In two experiments in which both measurements were performed on oocytes from the same frog, the mean of the two flux ratios was 0.94. Thus, exchange of intracellular endogenous SO 4 2Ϫ for extracellular Cl Ϫ by AE1 E699Q was consistent with a stoichiometry of 1:1.
To maximize AE1 E699Q-mediated rates of SO 4 2Ϫ / Cl Ϫ exchange stoichiometry experiments were performed with oocytes acutely injected with 50 nl of solution containing 130 mM Na 2 SO 4 buffered with Na HEPES, pH 7.40, to yield a total estimated intracellular [SO 4 2Ϫ ] of 14 mM. Such intracellular injection of SO 4 2Ϫ produced a 2.2 Ϯ 0.5-fold increase in AE1 E699Q-mediated 36 Cl Ϫ influx (Fig. 6 A, P Ͻ 0.01). This increase presumably reflected increased saturation of the intracellular binding site for sulfate on AE1 E699Q. 36 Cl Ϫ influx assays were performed with AE1 E699Q-expressing oocytes isolated from the same frog and previously injected on the same day 5-15 d earlier with water or with cRNA encoding AE1 E699Q. 10 min before initiation of 36 Cl Ϫ uptake, these oocytes were injected with 50 nl of 130 mM Na 2 SO 4 without tracer, to maximize and efflux. Summary of three similar experiments, with number oocytes evaluated in each condition noted above columns. Relative efflux into iodide medium lacking gluconate salts of Mg 2ϩ , Ca 2ϩ , and K ϩ was further reduced to 0.09 Ϯ 0.03 (n ϭ 5), but omission of these gluconate salts did not change values for other anions (not shown). equalize intra-oocyte sulfate concentrations. For 35 SO 4 2Ϫ efflux experiments, the injected Na 2 SO 4 included carrier-free tracer. The paired influx and efflux experiments carried out with sulfate-loaded oocytes were conducted on the same days (Table I).
In AE1 E699Q-expressing, Na 2 SO 4 -loaded oocytes (n ϭ 7 frogs, 73 oocytes), AE1-mediated 36 Cl Ϫ influx was 0.43 Ϯ 0.11 (SEM) pmol cell Ϫ1 s Ϫ1 . In identically handled AE1 E699Q-expressing, sulfate-loaded oocytes (n ϭ 40) from the same seven frogs, AE1-mediated 35 SO 4 2Ϫ efflux was 0.37 Ϯ 0.04 pmol cell Ϫ1 s Ϫ1 . The ratio of the mean sulfate efflux to the mean chloride influx was 0.86. The mean of the seven individual experimentally determined ratios of sulfate efflux over chloride influx was 1.13 Ϯ 0.23 (Table I). These values were consistent with a 1:1 stoichiometry of SO 4 2Ϫ /Cl Ϫ exchange mediated by AE1 E699Q in Na 2 SO 4 -loaded oocytes. 2 The SO 4 2Ϫ /SO 4 2Ϫ exchange stoichiometry of AE1 E699Q was examined 5 or more days after cRNA injection in the absence of exogenous SO 4 2Ϫ loading. AE1 E699Q-mediated SO 4 2Ϫ efflux into 64 mM extracellular SO 4 2Ϫ was 0.091 Ϯ 0.012 pmol cell Ϫ1 s Ϫ1 (n ϭ 4 frogs, 20 oocytes). 35 SO 4 2Ϫ influx from 64 mM extracellular SO 4 2Ϫ into AE1 E699Q-expressing oocytes was 0.079 Ϯ 0.012 pmol cell Ϫ1 s Ϫ1 (1 frog, 10 oocytes) These values were statistically indistinguishable (P Ͼ 0.5), and yielded a flux ratio of 1.15. Thus, under the nonequilibrium conditions used, AE1 E699Q also mediated SO 4 2Ϫ /SO 4 2Ϫ exchange with a stoichiometry consistent with 1:1 exchange. 2Ϫ -loaded or native oocytes injected 13 d earlier with water or with AE1 E699Q cRNA (*P Ͻ 0.01). Representative of five similar, paired experiments. AE1 E699Q-mediated 36 Cl Ϫ influx was measurable in native oocytes from 8 of 10 frogs tested. Influx was measurable in SO 4 2Ϫloaded oocytes from 9 of 10 frogs tested. (B) Oocytes from the frog of panel A injected 13 d earlier with water (upper trace) or with cRNA (seven lower traces) were acutely injected with 35 SO 4 2Ϫ to a final intracellular [SO 4 2Ϫ ] of 41ف mM, then subjected to the efflux assay. Note that fractional efflux into Cl Ϫ medium was less than observed in Fig. 2  The stoichiometry of Cl Ϫ influx and SO 4 2Ϫ efflux in sulfate-loaded oocytes expressing AE1 E699Q is 1:1. Within each experiment, Cl Ϫ influx and SO 4 2Ϫ efflux were measured on the same day with separate groups of otherwise identically treated oocytes from the same frog. Mean flux ratios are presented as ratios of the mean fluxes and as means Ϯ SEM of the individual experimental flux ratios.
2 Exclusion of the "outlier" experiment 2 from the calculations of the flux ratios resulted only in small changes. Such an edited mean of the individual flux ratios, 1.30 Ϯ 0.19, did not differ statistically from the value in Table I. Such an edited ratio of the individual experimental mean flux values, 1.24 Ϯ 0.05, differed slightly from that in Table I

Effect of AE1 E699Q on Membrane Potential
In WRK-BH 4 -modified, SO 4 2Ϫ -loaded red cells treated with gramicidin, the acceleration of 86 Rb ϩ efflux by addition of 5 mM extracellular Cl Ϫ suggested that the modified AE1 mediated electrogenic anion exchange (Jennings, 1995). Since the 1:1 stoichiometry of AE1 E699Q-mediated SO 4 2Ϫ /Cl Ϫ exchange in oocytes resembled that of WRK-BH 4 -modified red cells, the effects of substrate anions on membrane potential were directly examined in oocytes expressing either wt or mutant AE1 polypeptides. Upon pressure injection of 05ف nl 87 mM K sulfate, E699Q-expressing oocytes underwent a depolarization of 17 Ϯ 3 mV that was maintained during 30 min of observation and was accompanied by a 66% decrease in membrane resistance (Fig. 7). Estimated intracellular [SO 4 2Ϫ ] concentration was 9-10 mM. Injection of equal volumes of KCl or of K gluconate (87 mM) led to transient 3-mV hyperpolarizations accompanied by small increases in membrane resistance. Wt AE1-expressing oocytes, in contrast, displayed minimal change in membrane potential in response to injection of either K sulfate or K gluconate. Similarly, water-injected oocytes showed no voltage response to injection of K sulfate (Fig. 7, Table II). The depolarization and decrease in membrane resistance which characterized SO 4 2Ϫ injection into AE1 E699Q-expressing oocytes suggested outward electrogenic flow of negative charge, and supported the hypothesis of electrogenic SO 4 2Ϫ /Cl Ϫ exchange.
To distinguish between AE1 E699Q-mediated electrodiffusive transport and electrogenic exchange, the extracellular anion selectivity of membrane potential changes was investigated in SO 4 2Ϫ -loaded oocytes (Fig.  8, Table III). Whereas resting potentials in Cl Ϫ medium were Ϫ58 Ϯ 4 mV and Ϫ55 Ϯ 2 mV in water-injected (n ϭ 11) and wt AE1-expressing oocytes (n ϭ 20), respectively, oocytes expressing AE1 E699Q displayed a depolarized resting potential of Ϫ38 Ϯ 2 mV (n ϭ 60). Substitution of extracellular Cl Ϫ by isethionate produced no change in membrane potential in wt AE1-expressing and water-injected oocytes, but caused AE1 E699Qexpressing oocytes to hyperpolarize by 28 Ϯ 2 mV (n ϭ 60). In contrast, replacement of extracellular isethionate by SO 4 2Ϫ , produced little or no change in membrane potential (Table III). Addition of DNDS in the continued presence of extracellular Cl Ϫ hyperpolarized membrane potential in AE1 E699Q-expressing oocytes (Fig. 8 B) by 24 Ϯ 2 mV (n ϭ 25, 4 frogs). Taken together with the isotopic flux data, these results suggest that AE1 E699Q mediated asymmetric, electrogenic 1:1 Figure 7. Intracellular anion dependence of electrogenic anion exchange. (A) Oocytes injected 72 h previously with cRNA encoding AE1 E699Q were acutely injected (arrow) with 50 nl of 87 mM K salts of SO 4 2Ϫ , Cl Ϫ , or gluconate as indicated while membrane potential and resistance were recorded. Regular spaced vertical displacements in potential are in response to 10-nA current pulses. (B) An oocyte injected 72 h previously with cRNA encoding wt AE1 was injected with 50 nl 87 mM K 2 SO 4 . (C) A water-injected oocyte was injected with 50 nl 87 mM K 2 SO 4 .

Injected with
Oocyte sulfate gluconate chloride Changes in Xenopus oocyte membrane potential and transmembrane resistance in response to acute injection of K 2 SO 4 2Ϫ , KCl, and K gluconate. Values are means Ϯ SEM.

Stoichiometry of SO 4 2Ϫ Efflux and Current Mediated by AE1 E699Q
The hypothesis of 1:1 electrogenic SO 4 2Ϫ /Cl Ϫ exchange predicts that AE1 E699Q should mediate outward flow of negative charge from oocytes with properties identical to and of magnitude equal to AE1 E699Q-mediated SO 4 2Ϫ efflux from oocytes. Therefore, SO 4 2Ϫ -loaded oocytes expressing wt and mutant AE1 cRNAs were subjected to two-electrode voltage clamp in order to characterize and to quantitate current flow mediated by the AE1 E699Q polypeptide. Fig. 9 A compares current-voltage relationships of SO 4 2Ϫ -loaded oocytes expressing wt AE1 and AE1 E699Q. The 1ف S conductance measured in oocytes expressing wt mouse AE1 was similar to that of 0.7 S measured by Fievet et al. (1995). The oocytes expressing AE1 E699Q exhibited a small but significant linear inward current not seen in oocytes expressing wt AE1 at membrane potentials more positive than Ϫ80 mV (Fig. 9). The current-voltage relationship remained linear to test potentials as negative as Ϫ150 mV (not shown). The current in wt AE1-expressing oocytes displayed a reversal potential of Ϫ64 mV (Fig. 9 A; 20 oocytes from 6 frogs) similar to the value of Ϫ69 mV measured in water-injected oocytes (not shown; 8 oocytes from 6 frogs). Oocytes expressing AE1 E699Q exhibited substantially increased conductance, with a depolarized reversal potential of Ϫ33 mV (Fig. 9 A; 54 oocytes from 8 frogs). Substitution of extracellular Cl Ϫ by isethionate greatly reduced inward current in Na 2 SO 4loaded oocytes expressing AE1 E699Q, and restored oocyte reversal potential to the more hyperpolarized value of Ϫ74 mV (Fig. 9 B; 44 oocytes from 7 frogs). Substitution of extracellular NaCl by isosmotic Na 2 SO 4 Figure 8. Membrane potential changes in Na 2 SO 4 -loaded oocytes in response to varied extracellular conditions. (A) Membrane potential of a Na 2 SO 4loaded E699Q-expressing oocyte during two sequential transitions from extracellular Cl Ϫ to isethionate. (B) Membrane potential of a Na 2 SO 4 -loaded E699Q-expressing oocyte in extracellular Cl Ϫ medium during introduction and removal of DNDS. (C) Membrane potential of a Na 2 SO 4loaded wt AE1-expressing oocyte during transition from extracellular Cl Ϫ to isethionate. (D) Membrane potential of a Na 2 SO 4loaded oocyte, previously injected with water, during transition from extracellular Cl Ϫ to isethionate. Gaps in the voltage records represent application of stepped voltage protocols (see Fig. 9).

t a b l e i i i
Resting membrane potential (mV) of Na 2 SO 4 -injected oocytes in the presence of different extracellular anions. Values are means Ϯ SEM. AE-expressing oocytes were from 8 frogs; water-injected oocytes were from 2 frogs. ⌬ values (mV) represent means Ϯ SEM of indicated number of individual differences. reduced inward current and hyperpolarized oocyte reversal to the same relative extents (Fig. 9 B; 5 oocytes). Exposure of Na 2 SO 4 -loaded oocytes in extracellular chloride to DNDS (200 M) similarly inhibited AE1 E699Q-associated inward current, and similarly hyperpolarized the reversal potential to Ϫ68 mV (Fig. 9 C; 24 oocytes from 4 frogs). Thus, the conductance of AE1 E699Q-expressing oocytes was restored to that typical of wt AE1-expressing oocytes either by substitution of extracellular chloride by isethionate or by sulfate, or by addition of the anion exchange inhibitor, DNDS. Taken together, these data are consistent with the presence of AE1 E699Q-mediated electrogenic sulfate efflux at inside-negative oocyte resting potential.
To evaluate whether inward current carried by AE1 E699Q sufficed to account for AE1 E699Q-mediated SO 4 2Ϫ efflux, oocyte current at the resting potential in extracellular Cl Ϫ medium was interpolated from the measured I-V relationships. These values were compared to AE1 E699Q-mediated SO 4 2Ϫ efflux into Cl Ϫ medium (Table IV). In 12 experiments examining 104 Na 2 SO 4 -loaded oocytes, AE1 E699Q-mediated SO 4 2Ϫ efflux into extracellular Cl Ϫ was 0.48 ϩ 0.15 pmol cell Ϫ1 s Ϫ1 . SO 4 2Ϫ efflux did not differ significantly (P Ͼ 0.5, two-tailed t test) from AE1 E699Q-mediated inward currents calculated by any of three criteria: 0.58 Ϯ 0.15 pmol charge cell Ϫ1 s Ϫ1 (for current in extracellular Cl Ϫ minus that in extracellular isethionate); 0.37 Ϯ 0.08 pmol charge cell Ϫ1 s Ϫ1 (for current in E699Q-expressing oocytes minus that in wt AE1-expressing oocytes); and 0.43 Ϯ 0.05 pmol charge cell Ϫ1 s Ϫ1 (for current in absence of DNDS minus that in the presence of DNDS). Thus, the outflow of charge mediated by AE1 E699Q in sulfate-loaded oocytes at resting membrane potential sufficed to account for SO 4 2Ϫ efflux from similarly treated oocytes.

Estimate of AE1 E699Q-mediated H ϩ Transport during SO 4 2Ϫ /Cl Ϫ Exchange
WRK-BH 4 -treated red cells mediate SO 4 2Ϫ /Cl Ϫ exchange without evidence for the H ϩ /SO 4 2Ϫ cotransport Figure 9. AE1 E699Q-associated currents. (A) Mean I-V curves and representative current traces from Na 2 SO 4 -loaded oocytes in extracellular Cl Ϫ medium expressing wt AE1 (filled squares, n ϭ 20 oocytes from 6 frogs) and AE1 E699Q (filled circles, n ϭ 54 oocytes from 8 frogs). *P Ͻ 0.04; NS, P Ͼ 0.05. (B) Comparison of E699Qmediated currents in Na 2 SO 4 -loaded oocytes in the presence of extracellular Cl Ϫ (filled circles), extracellular isethionate (open squares, n ϭ 44 oocytes from 7 frogs), and extracellular sulfate (open triangles, n ϭ 5 oocytes from 1 frog). *P Ͻ 0.002. (C) Comparison of E699Q-mediated currents in Na 2 SO 4 -loaded oocytes in extracellular chloride in the absence (filled circles) and presence of 200 M DNDS (open diamonds, n ϭ 24 oocytes from 4 frogs) *P Ͻ 0.03. Voltage was stepped from a holding potential of Ϫ50 mV to test potentials between Ϫ80 and ϩ60 mV in 20-mV increments. At potentials Նϩ80 mV, endogenous outward currents were activated in all groups of oocytes. Data recorded in extracellular chloride from AE1 E699Q-expressing oocytes is replicated in all three panels. (Jennings and Al-Rhaiyel, 1988) that typifies wt AE1 Gunn, 1982, 1984). The electrogenicity of 1:1 SO 4 2Ϫ /Cl Ϫ exchange mediated by AE1 E699Q also suggested the absence of H ϩ cotransport with SO 4 2Ϫ . Therefore, H ϩ efflux from SO 4 2Ϫ -loaded oocytes was estimated from the time-dependent change in BCECF fluorescence ratio of minimally buffered extracellular medium (Jaisser et al., 1993). Water-injected oocytes did not detectably acidify the extracellular medium (n ϭ 3). dpH o /dt in the droplets surrounding AE1 E699Q-expressing oocytes previously injected with Na 2 SO 4 was Ϫ0.011 Ϯ 0.003 min Ϫ1 (n ϭ 5), corresponding to outward J H ϩ of 0.049 pmol cell Ϫ1 s Ϫ1 . With the simplifying assumption of no proton backleak, H ϩ efflux associated with expression of AE1 E699Q was %01ف of the level of AE1 E699Q-mediated SO 4 2Ϫ efflux. However, J H ϩ had the same value in AE1 E699Qexpressing oocytes not previously injected with Na 2 SO 4 (n ϭ 6), suggesting that the proton efflux was not coupled to SO 4 2Ϫ transport. Thus, SO 4 2Ϫ transport by AE1 E699Q was largely unaccompanied by protons.

d i s c u s s i o n
The experiments presented above demonstrate a complex phenotype of anion exchange in Xenopus oocytes expressing mouse AE1 polypeptide with the E699Q missense mutation. The AE1 E699Q polypeptide was processed and delivered to the surface of the oocyte (Fig. 3). There it exhibited both a loss-of-function phenotype with respect to exchange of intracellular Cl Ϫ for extracellular anion (Fig. 1) and a gain-of-function phenotype with respect to exchange of intracellular SO 4 2Ϫ for extracellular anion (Figs. 2, 5, and 6). Thus, SO 4 2Ϫ / Cl Ϫ exchange was asymmetric: whereas exchange of intracellular SO 4 2Ϫ for extracellular Cl Ϫ was increased, exchange of extracellular SO 4 2Ϫ for intracellular Cl Ϫ was undetectable. Though the sensitivity of anion exchange to inhibition by DNDS was unchanged (Fig. 4) and the rank-order of exchange rates with various ex-tracellular anions was little changed from the corresponding wt values (Fig. 5), the mechanism of SO 4 2Ϫ / Cl Ϫ exchange differed dramatically from that of wt AE1. The Xenopus oocyte expression system allowed unambiguous discrimination between two possible mechanisms of SO 4 2Ϫ /Cl Ϫ exchange in the absence of proton cotransport (Jennings, 1995), electroneutral exchange of 2 Cl Ϫ for 1 SO 4 2Ϫ and electrogenic exchange of 1 Cl Ϫ for 1 SO 4 2Ϫ .

Electrogenicity of Anion Exchange by AE1 E699Q
Murine AE1 E699Q-mediated exchange of intracellular SO 4 2Ϫ for extracellular Cl Ϫ with a stoichiometry of 1:1 in Xenopus oocytes (Table I). This exchange was unaccompanied by stoichiometric proton transport and was electrogenic (Tables II and III ,Figs. 7 and 8). In SO 4 2Ϫloaded Xenopus oocytes at inside-negative membrane potentials, AE1 E699Q mediated an inward current that required the presence of intracellular SO 4 2Ϫ and extracellular Cl Ϫ and was inhibited by DNDS (Fig. 9). AE1 E699Q-mediated inward current measured at the resting membrane potential did not differ detectably in magnitude from E699Q-mediated efflux of 35 SO 4 2Ϫ , and so could account entirely for SO 4 2Ϫ efflux (Table IV).
The mutation of glutamate to glutamine at position 699 converted the obligate electroneutrality of wt AE1mediated anion exchange to a more flexible mechanism, in which exchange of intracellular SO 4 2Ϫ for extracellular Cl Ϫ by AE1 E699Q was electrogenic, but AE1 E699Q-mediated SO 4 2Ϫ /SO 4 2Ϫ exchange was electroneutral ( Fig. 9 B). The change in charge of this single amino acid altered the capacity of AE1 to effect transmembrane transport of net charge, without alteration of the wt stoichiometry of 1:1 anion exchange. Electrogenic anion transport by wt mouse AE1 was not observed in Xenopus oocytes ( Fig. 9 A, Tables II and III), in agreement with earlier observations of Grygorczyk et al. (1987) and Fievet et al. (1995). In human red cells in which membrane potential was clamped with gramicidin and imposed K ϩ gradients, Jennings et al. (1990) also found that neither Cl Ϫ /Cl Ϫ exchange nor Cl Ϫ / HCO 3 Ϫ exchange displayed detectable potential dependence.
Wt AE1 in red cells cotransports H ϩ with SO 4 2Ϫ in electroneutral exchange for Cl Ϫ . This cotransport is reflected in stimulation of AE1-mediated SO 4 2Ϫ transport at acid pH Gunn, 1982, 1984). WRK-BH 4 modification of red cells (Jennings, 1995) led to AE1mediated SO 4 2Ϫ transport that was active at neutral pH, not further stimulated at acid pH, and was unaccompanied by H ϩ cotransport. In the present study, the electrogenicity of 1:1 SO 4 2Ϫ /Cl Ϫ exchange by AE1 E699Q suggested the absence of H ϩ cotransport; indeed, AE1 E699Q-mediated SO 4 2Ϫ efflux in Xenopus oocytes was unaccompanied by stoichiometric H ϩ efflux (results). The magnitude of AE1 E699Q-mediated inward current at resting potential sufficed to account for all measured AE1 E699Q-mediated sulfate efflux. n is number of separate experiments, each with 6-12 oocytes. The 12 experiments in which sulfate efflux was measured include the 7 presented in Table I plus 5 additional experiments, not included in Table I because they were not paired with measurements of Cl Ϫ influx.
Moreover, neither SO 4 2Ϫ /Cl Ϫ exchange nor SO 4 2Ϫ / SO 4 2Ϫ exchange were accelerated by intracellular or by extracellular protons (Chernova and Alper, manuscript in preparation).

Asymmetry of Sulfate Transport by AE1 E699Q
SO 4 2Ϫ /Cl Ϫ exchange in WRK-BH 4 -modified human red cells was vectorially asymmetric: exchange of intracellular SO 4 2Ϫ for extracellular Cl Ϫ was 10-fold faster than was exchange of intracellular Cl Ϫ for extracellular SO 4 2Ϫ , though both rates were increased (Jennings, 1995). Xenopus oocytes expressing AE1 E699Q displayed a qualitatively similar asymmetry. Whereas exchange of intracellular SO 4 2Ϫ for extracellular SO 4 2Ϫ or Cl Ϫ was easily detected (Figs. 2, 5, and 6), exchange of intracellular Cl Ϫ for extracellular SO 4 2Ϫ or Cl Ϫ was undetectable not only at 48 h ( Fig. 1 B) but also 15 d after cRNA injection (not shown). These differences in the relative rates of oppositely directed anion hetero-exchange in WRK-BH 4 -modified red cells and in AE1 E699Q-expressing oocytes compared to wt AE1 function may arise from differences in rate constants for both inward and outward translocation. The contribution of the outward translocation step can be more easily estimated, since both wt and mutant proteins transport extracellular Cl Ϫ at operationally maximal rates when measured in the presence of appropriate intracellular anions. In extracellular Cl Ϫ , rate constants for SO 4 2Ϫ efflux reflect the contribution of the E699 negative charge to a local structure in wt AE1 that maintains a low energy barrier for outward translocation of the Cl i Ϫ -carrier complex and a much higher energy barrier for outward translocation of the (H ϩ /SO 4 2Ϫ ) i -carrier complex. Neutralization of the E699 charge by chemical modification or by mutation likely elevates the energy barrier for outward translocation of the Cl i Ϫ -carrier complex. In contrast, this neutralization appears to lower the energy barrier for outward translocation of the SO 4 2Ϫ i -carrier complex, despite the movement of a unit charge across the membrane electric field associated with the mutant transport cycle. The absence of evident H ϩ /SO 4 2Ϫ cotransport by AE1 E699Q is either due to a greatly elevated energy barrier for outward translocation of the (H ϩ /SO 4 2Ϫ ) i -carrier complex, or simply to loss of the H ϩ binding site, with failure to form the (H ϩ /SO 4 2Ϫ ) icarrier complex.
Despite the minimal changes in ID 50 for DNDS (Fig.  4) and in the rank order for most extracellular anions (Fig. 5)  Inward current associated with AE1 E699Q-mediated exchange of intracellular SO 4 2Ϫ for extracellular Cl Ϫ could arise from outward movement of substrate-associated negative charge during the SO 4 2Ϫ efflux step or from inward movement of protein-associated positive charge during the Cl Ϫ influx step. The oocyte experiments did not allow discrimination between these possible mechanisms of charge movement. The experiments of Jennings (1995) with WRK-BH 4 -modified red cells assessed the efflux of SO 4 2Ϫ at varying membrane potentials and varying extracellular Cl Ϫ concentrations. Kinetic analysis with assumption of a ping-pong transport mechanism suggested that the principal chargecarrying limb of the anion transport cycle of WRK-BH 4 -modified AE1 was the influx of Cl Ϫ rather than the efflux of divalent SO 4 2Ϫ . The resulting model of anion translocation by wt AE1 proposed outward transfer through the transbilayer electric field of a neutral complex of two protein-associated positive charges with the two negative charges of transported SO 4 2Ϫ . In contrast, during the inward flux of monovalent Cl Ϫ , one of the protein-associated positive charges (which in wt AE1 may be paired with and neutralized by the negative charge of E699) remains unpaired. Muller-Berger et al. (1995b) have proposed H752 as a candidate residue to ion pair with E699, based on the indirect evidence of similar changes in the apparent pK for AE1-mediated Cl Ϫ efflux produced by the independent mouse AE1 mutations H752S and E699D. Discovery of intragenic second site revertants within AE1 E699 mutants will test more stringently this and other possible charge pairs.

Consequences of the E699Q Mutation to Anion Selectivity and Stilbene Sensitivity
Despite human E681's putative location at the COOH terminus of a stretch of nearly 25 hydrophobic amino acids extending from the exofacial terminus of AE1's putative transmembrane span 8, its WRK-reactivity in red cells suggested its accessibility to the extracellular space (Jennings and Smith, 1992). However, in contrast to the dramatic changes in outward anion translocation rates produced by the E699Q mutation in mouse AE1, changes in inward translocation rates in the presence of elevated intracellular sulfate concentration were minimal for tested anions other than sulfate. The rates of SO 4 2Ϫ efflux in exchange for extracellular gluconate and isethionate were higher than expected from red cell studies, but similar to those measured in oocytes expressing wt AE1 and AE2. It remains unclear why isethionate and gluconate are not impermeant anions with respect to anion exchange in Xenopus oo-cytes. 3 The phenomenon could reflect altered conformation of the anion translocation pathway of either wt or mutant AE1 expressed in oocytes compared to red cells, or activation of endogenous anion transport pathways of the oocyte by expression of heterologous AE polypeptides. The presence of substantial endogenous oocyte permeabilities to extracellular gluconate and isethionate has been suggested by Costa et al. (1989).
The approximately equipotent inhibition by DNDS of AE1 E699Q-mediated SO 4 2Ϫ /Cl Ϫ exchange and of wt AE1-mediated Cl Ϫ /Cl Ϫ exchange suggests preservation in the mutant of those exofacial structures of AE1 which interact with stilbene inhibitors.

Influence of the Host Cell
The origins of the differences between the activities of AE1 E699Q in Xenopus oocytes and of WRK-BH 4 -modified red cell AE1 likely reside in the chemical and steric differences at position 699 between the amide group of glutamine and the hydroxyl group of hydroxynorvaline, in addition to differences in host cell environments. A role for charge-independent steric factors in the interaction between E699 and transported anions was suggested by the loss of SO 4 2Ϫ /SO 4 2Ϫ exchange in microsomes from 293 cells expressing AE1 E699D, in contrast to the increased activity observed in microsomes from cells expressing either E699Q or E699K (Sekler et al., 1995). However, expression of these mutant AE1 polypeptides in Xenopus oocytes had different consequences. AE1 E699K expressed in Xenopus oocytes did not confer measurable SO 4 2Ϫ /SO 4 2Ϫ exchange either 2 d (Fig. 2) or 2 wk after cRNA injection (data not shown). In addition, AE1 E699D expressed in Xenopus oocytes retained %04ف of wt levels of 36 Cl Ϫ influx activity (Muller-Berger et al., 1995a). Thus, the consequences of selected mutations in AE1 E699 differed in different host cell expression systems. Since the WRK-BH 4 protocols optimized for the human red cell (Jennings, 1995) did not inhibit wt AE1-mediated Cl Ϫ /Cl Ϫ exchange in Xenopus oocytes (Chernova and Alper, unpublished observations), direct comparison of WRK-BH 4 -modified AE1 in red cells and in oocytes was not possible.

Relationship to Chloride Conductance Attributed to Wt AE1
The DIDS-inhibitable portion of human red cell Cl Ϫ conductance has been attributed to conductive anion "tunneling" through the AE1 protein (Frohlich, 1988). Jennings (1995) noted that WRK-BH 4 -modification of human red cells, in addition to its effects on anion exchange, increased Cl Ϫ conductance 8-10-fold. In addition to the absence of trans-anion dependence, a property distinguishing red cell Cl Ϫ conductance from anion exchange is the insensitivity of Cl Ϫ conductance to concentrations of phloretin that inhibit anion exchange (Frohlich, 1988). However, 200 M phloretin inhibits both SO 4 2Ϫ /Cl Ϫ exchange (n ϭ 6) and inward current (n ϭ 4, not shown) mediated by AE1 E699Q. Together with the demonstrated trans-anion dependence, this result suggests that anion tunneling contributed minimally to AE1 E699Q-mediated electrogenic anion transport in oocytes.
Expression in oocytes of wt trout AE1 cRNA was reported to confer on oocytes a Cl Ϫ conductance (Fievet et al., 1995). This conductance differed in at least three ways from mouse AE1 E699Q-mediated electrogenic exchange of intracellular SO 4 2Ϫ for extracellular Cl Ϫ . First, trout AE1-mediated Cl Ϫ conductance took place in the presence of the conserved glutamate at the position corresponding to mouse E699. Second, Cl Ϫ conductance and Cl Ϫ exchange by trout AE1 displayed very different stilbene sensitivities, whereas electrogenic and electroneutral variants of anion exchange by mouse AE1 E699Q exhibited similar stilbene sensitivities. Third, trout AE1 expressed in Xenopus oocytes exhibited maximal anion exchange activity comparable in magnitude to that of mouse AE1 but exhibited a Cl Ϫ conductance -05فfold larger than the AE1 E699Q-mediated currents in the present study. Interestingly, however, trout AE1 expression at levels lower than 15% of maximal anion exchange rates was unassociated with Cl Ϫ current, whereas large currents accompanied higher levels of AE1 expression (Fievet et al., 1995). Though the mechanistic relationship between trout AE1-associated Cl Ϫ conductance and AE1 E699Q-mediated electrogenic anion exchange is unclear, each may require for expression of electrogenic transport some minimal level of electroneutral exchange activity. 3 Though AE1 E699Q-mediated inward current at clamped resting potential was of sufficient magnitude to conclude that all anion exchange flux measured under open circuit conditions was electrogenic (Table IV), extracellular isethionate supported a rate of AE1 E699Q-mediated 35 SO 4 2Ϫ efflux of 0.2 relative to extracellular Cl Ϫ (Fig. 5) but supported no greater inward current than did extracellular sulfate. Several factors might account for this discrepancy without invoking an alternate transport mechanism. First, current values of 0.2 relative to those recorded in extracellular Cl Ϫ are near or below the threshold of detection against the variable background current of individual oocytes. Second, the two assays were performed under different experimental conditions. Sulfate efflux into extracellular isethionate (Fig. 5) was measured 10 min after injection of oocytes with 50 nl carrier-free Na 2 35SO 4 in 50 mM Na HEPES, pH 7.4, whereas membrane potential change in response to extracellular anion substitution (Fig. 8, Table III) was measured in oocytes maintained in isethionate medium for 1-6 h after injection with 50 nl of 130 mM Na 2 SO 4 , 50 mM Na HEPES, pH 7.4. Lastly, evaluation of the effect of extracellular isethionate on the membrane potential of AE1 E699Q-expressing oocytes requires comparison with the effect of an extracellular monovalent anion to which the oocyte is "absolutely" or maximally impermeable (at least as judged by 35 SO 4 2Ϫ efflux). Experiments to address this issue are underway.


extfieldsofstudy: Biology Medicine

s2fieldsofstudy: Biology Chemistry

text:
Investigation of the Pyrimidine Preference by the c-Myb DNA-binding Domain at the Initial Base of the Consensus Sequence*

The principal determinant of the pyrimidine preference by the c-Myb DNA-binding domain at the initial base of the consensus sequence was investigated by mutation of both the protein and the DNA base pairs, with analysis by a filter binding assay. Amino acid residue 187 was revealed to interact with the pyrimidine base position, as estimated from our previous complex structure. Unexpectedly, since the pyrimidine preference is retained even in the Gly187 mutant, the principal origin of the base specificity should not occur via the direct-readout mechanism, but by an indirect-readout mechanism, namely in the intrinsic “bendability” of the pyrimidine-purine step of the DNA duplex. A significant but rather small positive base pair roll is detectable in the conformation of DNA in complex with the c-Myb DNA-binding domain. Following the conventional chemical rules of the direct-readout mechanism, amino acid mutagenesis at position 187 yielded several new base preferences for the protein.

Specific interactions between proteins and DNA are critical to gene expression and regulation, so a general readout mechanism of the information encoded in DNA has been sought (1)(2)(3). However, a number of complex structures of DNA duplexes and proteins determined at atomic resolution have revealed that nature uses a great variety of readout mechanisms (4 -11).
In most complex structures, the direct-readout mechanism is mediated by intermolecular hydrogen bond networks and hydrophobic interactions between DNA duplexes and proteins. The interaction modes have been classified into: (i) the intrinsic chemical features of bases and amino acids (1,3,12), and (ii) the stereochemical relations between the amino acids and the bases inside the DNA major grooves (12,13). In contrast, the indirect-readout mechanism works in several systems, such as the trp repressor/operator (4,5), where the DNA bases are specifically recognized by proteins without the use of particular hydrogen bonds or non-polar contacts. Instead, each sequencedependent deformation of the DNA conformation stabilizes the characteristic geometry of the phosphate backbone, which directly interacts with the protein through polar contacts (6). Water molecules are often observed to mediate the specific interaction through additional hydrogen bonds (4,5). One common type of DNA deformation is a steep kink of the duplex (7)(8)(9), which substantially contributes to readout of the minor groove (8,9).
In general, a combination of the direct-and indirect-readout mechanisms results in specific base pair recognition. In other words, both the specific binding affinity and the DNA bending contribute to the free energy of complex formation (6,14). This situation has made it difficult to determine how each consensus base sequence is recognized by the corresponding protein, even when the precise complex structure is known.
The c-myb gene product (c-Myb) is a transcriptional activator that specifically binds to DNA fragments containing the consensus sequence PyAAC(G/T)G, where Py indicates a pyrimidine (15)(16)(17). The DNA-binding domain (DBD) 1 of c-Myb consists of three imperfect 51-or 52-residue repeats (designated R1, R2, and R3 from the N terminus) (18 -20). The last two repeats, R2 and R3, are sufficient for the recognition of the specific DNA sequences (20,21). NMR analysis revealed that both R2 and R3 contain three helices, and the third helix in each is a recognition helix (22)(23)(24). R2 and R3 are closely packed in the major groove, so that the two recognition helices directly contact each other to bind cooperatively to the specific base sequence.
In the complex of c-Myb R2R3 with the Myb-binding DNA sequence (MBS-I), the consensus A4, the counterpart guanine of C6, and the last G8 directly interact with Asn 183 in R3, Lys 182 in R3, and Lys 128 in R2, respectively ( Fig. 1) (23). The strong cooperativity between R2 and R3 originates from the putative polar interactions between the side chains of Glu 132 and Asn 179 , and between those of Arg 131 and Asp 178 . However, it is not clear why the initial Py corresponding to the third base position in the MBS-I fragment is preferred by c-Myb R2R3, although this Py3 is less specific than the other A4, A5, C6, and G8 sites in the consensus DNA sequence (17). In our NMR structure shown in Fig. 1, Ser 187 is the only candidate that interacts with the T3 base, and this ability was suggested in our previous paper (23). The hydroxyl group in the Ser side chain could form a hydrogen bond with the O 4 oxygen of the T3 base, either directly or through water molecules.
Thus far, the Myb-homologous DBD has been found in over 30 proteins from many species. An alignment of the DBDs shows that the Ser at position 187 is highly conserved in the animal sequences, whereas it is variable in the plant sequences (23,25).
Here, to investigate the role of Ser 187 and the origin of this * 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.
‡ ‡ To whom all correspondence should be addressed. Tel.: 81-6-872-8212; Fax: 81-6-872-8219. pyrimidine preference at the third base position, both Ser 187 in the c-Myb R2R3 and the third T-A base pair in the 22-mer MBS-I fragment containing the Myb-binding site were substituted by other amino acids and other base pairs, respectively. The interactions between them were examined using a filter binding assay, whose efficiency has already been shown (17,26,27). The recognition mechanism will be discussed.

EXPERIMENTAL PROCEDURES
Plasmids and Site-directed Mutagenesis-A DNA fragment encompassing R2R3 (Leu 90 -Val 193 ) in the DNA-binding domain of c-Myb was amplified by polymerase chain reaction, using pact-c-myb (28) as the template and two synthetic primers, to generate an NcoI site and a BamHI site at the 5Ј-and the 3Ј-end of the amplified fragment, respectively. After digestion with NcoI and BamHI, the DNA fragment was cloned into pAR2156NcoI (17) to yield the expression plasmid, pRP23. An additional Met-Glu-sequence was introduced at the N terminus of R2R3. Site-directed mutagenesis was performed by two-step polymerase chain reaction, as described by Higuchi (29). Here the name of each mutant protein is indicated as, for example, C130I/S187G for the simultaneous mutations that replace Cys 130 with Ile and Ser 187 with Gly.
Protein Expression and Purification-Escherichia coli BL21(DE3) was transformed with the wild type and mutant plasmids (30). Freshly precultivated cells were inoculated into growth medium containing 100 g/ml ampicillin and were grown at 37°C. When the culture reached an A 600 of about 0.4, isopropyl-1-thio-␤-D-galactopyranoside was added to a final concentration of 0.5 mM. The cells were cultured at 22°C for another 12 h. The harvested cells were suspended in 50 mM Tris-HCl buffer (pH 7.8) containing 5 mM MgCl 2 , and were lysed by sonication at 4°C. After the cell debris was removed by centrifugation, ammonium sulfate was added to the supernatant to 50% saturation. After an incubation at 4°C for 1 h, the supernatant was dialyzed against 50 mM potassium phosphate buffer (pH 7.5) containing 200 mM NaCl, and was then applied to a phosphocellulose column (Whatman, P11). The purified fractions were pooled, and the buffer was exchanged to 100 mM potassium phosphate buffer (pH 7.5) containing 20 mM KCl. The protein concentrations were determined from UV absorption at 280 nm and were calculated by using the molar absorption coefficient of 3.7 ϫ 10 4 M Ϫ1 cm Ϫ1 (17). CD Measurements-Circular dichroism (CD) spectra were measured at 20°C on a Jasco J-600 spectropolarimeter equipped with a watercirculating cell holder. The spectra were obtained in 100 mM potassium phosphate buffer (pH 7.5) containing 20 mM KCl, using a 0.2-cm optical path length cell. The protein concentration was 0.1 mg/ml. CD spectra between 200 and 250 nm were obtained using a scanning speed of 20 nm/min, a time response of 1 s, a bandwidth of 1 nm, and an average over 8 scans.
Preparation of Oligonucleotides-The 22-mer oligonucleotide CAC-CCTAACTGACACACATTCT, containing the Myb-binding site in the simian virus 40 enhancer sequence (MBS-I) (31), and the third base substituted variants were synthesized and purified by high performance liquid chromatography with a C 18 reverse-phase column (Fig. 2). The purified DNA was suspended in STE (10 mM Tris-HCl (pH 8.0), 100 mM NaCl, 1 mM EDTA), and complementary strands were annealed and end-labeled with [␥-32 P]ATP (Amersham) using T4 polynucleotide kinase (Toyobo, Osaka, Japan). The labeled DNAs were purified by passage through spin columns (Pharmacia Biotech Inc., HR-300). Here the name of each variant DNA is indicated as, for example, [C3]MBS-I, for the substitution of the T-A base pair at the third position by a C-G base pair.
Filter Binding Assay-All filter binding assays for the protein-DNA binding were carried out essentially as described (32)(33)(34). [ 32 P]DNA and various amounts of the c-Myb R2R3 mutant proteins were incubated in 100 l of binding buffer (100 mM potassium phosphate buffer (pH 7.5), 20 mM KCl, 0.1 mM EDTA, 500 g/ml bovine serum albumin, and 5% (v/v) glycerol) on ice for 30 min. The final concentration of the [ 32 P]DNA in binding buffer was 0.4 nM, which was always a lower concentration than the K d value. The incubated samples were filtered through a nitrocellulose membrane (Schleicher & Schuell, BA-85, 0.45 m) in approximately 10 s with suction. The filters were dried and counted by a liquid scintillation counter. The equilibrium dissociation constants K d were obtained from the binding titration curve, based on the least square fitting to the normalized bound DNA (y) with the protein concentration (x) using the formula, y ϭ x/(x ϩ K d ).

RESULTS
Prior to the mutational analyses of Ser 187 , the Cys 130 in R2, which is the only cysteine residue in the c-Myb R2R3 and is located at a position equivalent to an isoleucine in R3, was replaced with Ile, to facilitate the protein purification and the DNA-binding assay (35). It was reported that this mutation has little effect on DNA binding (36). The affinity of the C130I mutant was also measured in our own assay system, and it was shown to be almost equal to that of the wild type, and to maintain the pyrimidine preference at the third base position (Table I).
A series of 10 amino acids, Gly, Ala, Thr, Asn, Gln, Val, Leu, Lys Arg, and Asp, were introduced into position 187 of the c-Myb R2R3, which is a Ser residue in the wild type. The purity of each mutant protein was about 95%, as monitored by SDS- polyacrylamide gel electrophoresis. All of the mutant proteins have secondary structure contents similar to the wild type, as confirmed by the CD spectra at the far UV region (Fig. 3). The perfect coincidence of all the spectra suggests that the global tertiary structures of the mutant proteins were not deformed.
The binding affinities of the mutants to the cognate 22-mer MBS-I fragments and the third base pair substituted variants were analyzed using the filter binding assay, and the results are summarized in Table I. All measurements were repeated at least twice, and typical experimental errors for the K d value were less than 10%, although the retention efficiency was 20 Ϯ 10% depending on the experimental conditions. From the methylation interference experiments (17) and the NMR analyses (23), the number of bound DNA duplexes per the c-Myb is considered to be one within the concentration used in this assay. As already indicated in the previous experiments (17,26,27), the filter binding assay was validated for this investigation.
The C130I/S187G mutant protein binds about one-third less strongly to the cognate MBS-I than the standard C130I mutant. The relative binding free energy change for the replacement of Ser with Gly, calculated from the K d values, is 0.65 kcal/mol. It should correspond to the free energy derived from the interaction between the Ser side chain and the T3 base. This Gly mutant preferentially binds to both the cognate [T3]MBS-I and the substituted [C3]MBS-I. That is, even when residue 187 has no side chain, the mutant protein prefers the third pyrimidine as well as the wild type and the C130I mutant proteins.
The substitutions of Ser 187 by Ala (C130I/S187A), Thr (C130I/S187T), or Val (C130I/S187V) reveal slightly reduced binding affinities, although the sequence specificities are retained like the standard C130I. In contrast, the C130I/S187N mutant preferentially binds to the [A3]MBS-I. The affinity for the A3 base is similar to that of the wild type, although those for the other three bases (T, C, and G) are greatly reduced, by approximately one-half to one-sixth. The specific interaction between the Asn residue and the A3 base closely follows the intrinsic chemical features. Interestingly, for the substitution by Gln, which is one methylene group longer than Asn, the C130I/S187Q mutant loses the preference for the A3 base. Also, in the case of the C130I/S187L mutant, in which Leu is one methylene group longer than Val, the binding affinity is greatly reduced.
The mutant proteins C130I/S187K and C130I/S187R, which introduced basic amino acids into position 187, specifically prefer to bind to the [G3]MBS-I and [C3]MBS-I variants. In contrast, for the substitution of Ser 187 by acidic Asp (C130I/ S187D), the binding affinity is completely reduced and is no longer sequence-specific. DISCUSSION Thus far, many amino acid replacements in the c-Myb R2R3 have been created and assayed by specific DNA binding (37)(38)(39), and almost all of their effects have been explained by the specific polar contacts between the R2R3 and the DNA in the three-dimensional structure of the R2R3-DNA complex (23). The current mutational study clearly indicates that residue 187 in R2R3 is also able to interact with the T3 base, as estimated from the geometry of Ser 187 in the NMR complex structure (23). This specific DNA-binding mode is very different from the telomeric DNA recognition by the yeast RAP1-DBD (40), whose amino acid sequence is weakly homologous to that of the c-Myb R2R3.
However, the substitution of Ser 187 with Gly, Ala, or Val unexpectedly resulted in only about a 3-fold decrease in the binding affinity toward any base, which would be a consequence of a direct-readout mechanism, while the pyrimidine base preference at the third position in the MBS-I fragment was retained. Ser is thought to have weak specificity, because its side chain can act as either a hydrogen bond donor or an acceptor, and thus can bind to any base. Nevertheless, Ser 187 of the R2R3 preferentially binds to the pyrimidine bases. If this interaction were attributable only to the direct-readout mechanism, then the substitution of Ser 187 should have resulted in an over 100-fold reduction of the binding affinity and a loss of the sequence specificity, like the substitution of Lys 128 by Ala a An additional Met-Ala-sequence was introduced at the N terminus of R2R3, which was used in the NMR experiment (23). (23), and those of Asn 136 and Asn 186 by Ala (38). These results suggest that the preference of the pyrimidine bases at the third position of MBS-I should occur primarily by an indirect-readout mechanism.
In our previous structural study, no distinct deformation of the global DNA conformation was observed (23). However, when the local bending of the DNA duplex was carefully analyzed in 25 NMR complex structures and the refined average structure (Protein Data Bank codes 1MSF and 1MSE, respectively), significantly positive roll angles were always observed between the third pyrimidine and the fourth purine, as indicated by an arrow in Fig. 4. Characteristic negative slides (Ϫ1.1 Ϯ 0.3 Å) were also observed at the same pyrimidinepurine step, corresponding to positive rolling, while the twist angles at this step were 34.1 Ϯ 2.3°, nearly equal to the twist angle in standard B-form DNA. Similar significant, positive rolls at pyrimidine-purine steps are general phenomena (42), observed in many complex crystal structures of repressors and homeodomains with the helixturn-helix motif, as summarized in Table II. In every case, as a part of the consensus base sequence, the base pair roll bends the DNA so that the recognition helix is wrapped by the DNA duplex in the major groove (3,42). Consequently, a large contact area is created between the recognition helix and the DNA major groove, facilitating the preferable polar contacts between the protein side chains and the DNA phosphate backbone. The local roll in the MBS-I fragment may be associated with the small magnitude of observed bending in long DNA duplexes bound with the c-Myb R2R3 (52). This bending may be enhanced by other regions in the protein, like the transactivation domain.
Due to the intrinsic propeller-twist of the DNA base pairs, the pyrimidine-purine step has two stable conformations, with rolling of 0°and around 10° (53,54), from the physical requirements of the base stacking (55). There is negligible additional free energy cost required for the 10°rolling at the pyrimidinepurine step, even for a free DNA duplex without a protein. This is the physical origin of the so-called "bendability" of kinked DNA duplexes, commonly observed in the minor groove readout mechanism (8,9). At the other pyrimidine-pyrimidine, purine-purine, and purine-pyrimidine steps, no such tendency toward a strongly bistable step is observed (53). In fact, the binding free energy differences between the pyrimidine bases and the purine bases at the third base position for the current Gly 187 , Ala 187 , and Val 187 mutants are 0.4 Ϯ 0.1 kcal/mol, as calculated from the dissociation constants in Table I. Fig. 5 shows the results of the relative binding free energy changes ⌬⌬G toward the C130I/S187G mutant: ⌬⌬G ϭ ⌬G bind (mutant against the third N base) Ϫ ⌬G bind (C130I/S187G against the same third N base), where ⌬G bind ϭ RT ln K d . Here, the difference was calculated while keeping the same third position base pair. We can now separate the bendability effect from the total binding free energies between the c-Myb R2R3 mutants and the variety of DNA sequences, unless the binding modes vary from the wild type. Each positive and negative free energy corresponds to a decrease and an increase of the binding affinity, depending upon the intrinsic chemical features of the amino acids and the bases, and subtracting the DNA bending effect.
For the Ala substitution, the binding affinity is increased as compared with Gly 187 , independent of the bases at the third position, probably due to the hydrophobic contacts. When the side chain volume is larger in the Val substitution, a similar binding affinity to the pyrimidines remains, but the affinity becomes neutral to the purines. Therefore, the volume of space created between residue 187 and the third base may allow at most the Val-pyrimidine pair, but the Val-purine pair would be slightly too large for the space. In fact, other amino acids, such as Leu and Gln, with larger side chain volumes than Val, significantly lack binding affinity, as indicated in Fig. 5. Moreover, the Val-, Leu-, and Gln-substituted mutants always have lower affinities for adenine than for guanine. This is also supported by the fact that the amino N 6 of adenine occupies a larger volume than the oxygen O 6 of guanine, which should be located at the position nearest to the side chain of residue 187.
From this consideration of the space volume around residue 187 and the third base, the native and the optimum interaction between Ser 187 and T3 should be mediated by water molecules, as long as the binding mode is assumed to be the same in all of the mutant proteins and DNAs. In the Thr mutant, the disposition of the water molecules could be different from that in the wild type, thus yielding a slight decrease in the binding affinity. Since there is no possible conformation on the helix in which the methyl group of the Thr side chain would be able to access the methyl group in T3, as shown in a modeling study, a specific non-polar contact between the Thr mutant and T3 is not expected.
Following the conventional chemical rules for specific binding between amino acids and bases (1,3,12), the current Asn mutant specifically binds to the A3 base relative to the other bases, as indicated in Fig. 5. The Asn side chain size is less than that of Val, and there should be enough space for the Asnadenine pair, resulting in the formation of direct hydrogen bonds with a free energy gain of about 0.5 kcal/mol. In addition, the Lys and Arg mutant proteins prefer to bind to the G3 base. From their intrinsic chemical nature, both basic amino acids can bind to the guanine base almost exclusively by electrostatic interaction. In contrast, these mutant proteins bind to the [A3]MBS-I and [T3]MBS-I bases with only weak affinity, probably because of the bulky side chains of the amino acids, like the Leu mutant. It is interesting that their long side chains seem to interact with the guanine base on the opposite side of C3. The acidic Asp substitution results in a severe reduction of its DNA binding, which is much lower than the Gly substitution, suggesting that the Asp side chain cannot interact with any base, including cytosine, in this geometry. Rather, the negative ionic charge may disturb other specific hydrogen bonds between the protein and the DNA.
The wild type protein and the C130I mutant with Ser 187 bind to the cognate DNA most tightly among the mutant proteins, and their K d values are in the nanomolar order. Generally, transcriptional regulator proteins bind to their target genes with greater affinity (57). These results are consistent with the conservation of Ser in position 187 of c-Myb among animal species (23). In contrast, among plant species, the amino acid in this position varies (25). This suggests that the recognition mode in the plant Myb homologues may be different from that of the c-Myb DBD from animal species. In fact, in the case of the yeast RAP1 domain 1, the corresponding Val 409 residue does not interact with the DNA in the complex structure (40), although the free domain structure is similar to that of the c-Myb R3.
In conclusion, the current mutational analysis revealed that the pyrimidine preference of the native c-Myb DBD for the initial base of the consensus sequence originates principally in the intrinsic positive roll at the pyrimidine-purine step of the DNA duplex. For the purine-purine step, as much as 0.4 kcal/ mol of additional free energy would be necessary, corresponding to the bendability. When these bending energies are separated, the conventional chemical rules between the amino acids and the bases are distinctively observed in the c-Myb R2R3 mutants.
It is still difficult to extract a definite "recognition code" from the variety of DNA information readout mechanisms. The situation becomes much more complicated when the DNA flexibility is considered. Only a screening technology, such as a phage display library (58 -60), would be expected to reveal a novel, specific form of DNA recognition, instead of an artificial  5. Relative binding free energy changes (⌬⌬G) toward the C130I/S187G mutant protein binding of the Ser 187 -substituted R2R3 mutant proteins to the cognate MBS-I and its variants. Each ⌬⌬G for the particular base pair was calculated from the K d values, as described under ''Discussion.'' The side chain volumes (Å 3 ) of the substituted amino acids (56) are indicated in the parentheses below the individual residues. molecular design. However, based upon the complex structure and the mutational analysis, one may be able to dissect the sequence specific affinity into the DNA bendability and the specific interaction between the amino acids and the bases. Without this kind of precise analysis, we may never reach a complete understanding of the readout mechanism, nor produce any novel devices for molecular readout.