©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Role of Cysteine Residues in the Erythrocyte Plasma Membrane Anion Exchange Protein, AE1 (*)

Joseph R. Casey (§) , Yue Ding (¶) , Ron R. Kopito (**)

From the (1) Department of Biological Sciences, Stanford University, Stanford, California 94305-5020

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

AE1 (Band 3), a ≅110-kDa integral plasma membrane protein, facilitates the electroneutral movement of Cland HCO3- across the erythrocyte membrane and serves as the primary attachment site for the erythrocyte spectrin-actin cytoskeleton. In this investigation, we have characterized the role of native cysteines in the function of AE1. We have constructed a mutant version of human AE1 (AE1C) in which all five cysteines of AE1 were replaced with serines. Wild-type and AE1CcDNAs were expressed by transient transfection of human embryonic kidney cells. Two of the mutated cysteines in AE1Care in a region involved in ankyrin binding, and ankyrin binding has previously been shown to be sensitive to the oxidation state of these cysteines. However, the Kvalues for ankyrin binding by AE1 and AE1Cwere indistinguishable, suggesting that AE1 cysteines are not essential components of the ankyrin-binding site. Using size exclusion chromatography, both AE1 and AE1Cwere found to associate as a mixture of dimers and high molecular mass complexes. The rate of anion exchange by AE1C, as measured in a reconstituted microsome sulfate transport assay, was indistinguishable from that by AE1 and was inhibited by 4,4`-diisothiocyanodihydrostilbene-2,2`-disulfonate. We conclude that the cysteines of AE1 are not required for the anion exchange or cytoskeletal binding roles of the protein.


INTRODUCTION

AE1 is the best studied member of a family of plasma membrane anion exchange proteins that are widely distributed across mammalian tissues. These proteins include AE1 (Band 3) expressed in erythrocytes and kidney; AE2 found in kidney, lymphocytes, and stomach; and AE3 found in brain, heart, and retina (1) . All three anion exchangers function to exchange the physiological substrates Cland HCO3- across the plasma membrane. This anion exchange process contributes to the regulation of intracellular pH, [Cl], and volume and is involved in proton, bicarbonate, and chloride secretion in some epithelial tissues.

Anion exchangers are bifunctional proteins that have two clearly separated domains: a highly conserved 55-kDa membrane domain that is sufficient to mediate anion exchange and a more divergent 45-110-kDa cytoplasmic domain (2) . In erythrocytes, the cytoplasmic domain of AE1 is required to anchor the plasma membrane to the spectrin-actin cytoskeleton via interaction with ankyrin (3) . AE1 transports a wide range of small anions including Cl, HCO3-, HPO4-, and SO42- (4) . Sulfate is frequently used to measure anion exchange activity because it is transported 10times more slowly than Cl, allowing measurement of sulfate transport on a longer time scale (4) .

An important aspect of AE1 structure is its oligomeric state. Early studies indicated that AE1 could be cross-linked covalently to dimers by oxidation of its cytoplasmic cysteine residues (5) . A wide range of techniques including electron microscopy, radiation inactivation, spectroscopy, cross-linking, and hydrodynamics have been used to examine the oligomeric structure of AE1, with the conclusion that AE1 is a mixture of homodimers and homotetramers in the erythrocyte membrane and in detergent solution (6) . Monomeric AE1 can be observed only after protein denaturation with dimethylmaleic anhydride or SDS (7) . Furthermore, the electron diffraction structure of AE1 has 2-fold symmetry (8) , suggesting, together with the above data, that the oligomeric unit of native AE1 in the membrane is minimally a homodimer.

The affinity of ankyrin binding to AE1 is dependent upon AE1 oligomeric structure. The population of AE1 released from erythrocyte membranes by CE() detergent is purely homodimeric, suggesting that the tetrameric fraction remains bound to the cytoskeleton via ankyrin binding (9) . Furthermore, using time-resolved phosphorescence anisotropy, two populations of AE1 were resolved, one smaller and more mobile and the other larger and immobilized (10) , which was interpreted to mean that AE1 tetramers bind to ankyrin. Taken together, it is likely that AE1 tetramers form the high affinity ankyrin-binding site.

Human AE1 protein contains five cysteine residues at amino acids 201, 317, 479, 843, and 885. As seen schematically in Fig. 1 , two of these cysteines are found in the cytoplasmic domain of AE1, and three are in the membrane domain. We have mutated each of these cysteines to serine to examine their role in the function of AE1.


Figure 1: Folding model of human AE1 protein showing positions of cysteine residues. The dashed lines represent the lipid bilayer. The stars represent the five cysteine residues at amino acids 201, 317, 479, 843, and 885. The Y on the protein represents the single chain of N-linked carbohydrate on the protein. The site that was shown to be palmitylated in erythrocyte Band 3 protein (15) is marked by the unfilled star.



The regions of the cytoplasmic domain that are involved in ankyrin binding have been mapped to amino acids 190-203 and 317-359 because antibodies directed against these sequences block ankyrin binding (11, 12, 13) . Chemical modification or oxidative cross-linking of Cys-201 and Cys-317 has been shown to impair ankyrin binding (11) , suggesting that they either are directly essential for ankyrin binding or are in a region that is essential for ankyrin binding. The ability to cross-link Cys-201 and Cys-317 suggests that they are close to each other in the protein's folded structure (11) . Since they can also be cross-linked intermolecularly between subunits of an AE1 dimer (5) , Cys-201 and Cys-317 may be oriented along the dimeric interface. Kidney AE1, which lacks the amino-terminal 79 amino acids of AE1, was recently shown not to bind ankyrin (14) . Furthermore, the presence of ankyrin impaired the ability to phosphorylate two tyrosine residues within this amino-terminal region (12) . Together, these data suggest that the NH-terminal region is essential for ankyrin binding, although the region containing the two cytoplasmic cysteine residues may also play a role.

One cysteine residue of human AE1, Cys-843, has been shown to be post-translationally modified by palmitylation in human erythrocytes (15) . It has been suggested that palmitylation could serve to localize a residue to the inner leaflet of the plasma membrane and thereby stabilize a transmembrane helix. However, palmitylation of the residue corresponding to Cys-843 in mouse AE1 was recently shown not to be required for anion exchange activity when expressed in Xenopus laevis oocytes (16) .

In this study, we have examined the role of native sulfhydryls in AE1 function and oligomeric structure. Our data demonstrate that the sulfhydryls of AE1 have no role that cannot be replaced by serine. The availability of such a mutant will facilitate future structure-function studies of AE1 that employ chemistry of engineered -SH groups.


EXPERIMENTAL PROCEDURES

Human AE1 Expression Construct

A human AE1 cDNA (pHB3) (17) on an AccI- HindIII fragment was cloned into the HindIII and EcoRI sites of expression vector pRBG4 (18) using an AccI- EcoRI linker. The resulting human AE1 expression construct was called pJRC9 and contains 27 bases of 5`-untranslated sequence and the full coding sequence. Expression vector pRBG4 contains the cytotomegalovirus immediate early gene promoter (18) .

Site-directed Mutagenesis

Mutagenesis was performed using a polymerase chain reaction megaprimer mutagenesis strategy as described (19, 20) . Polymerase chain reaction primers were designed using the Primers program (Whitehead Institute for Medical Research). Polymerase chain reaction was performed using an ERICOMP thermal cycler and Vent DNA polymerase (New England Biolabs Inc.). Sequences generated by polymerase chain reaction were sequenced in their entirety to ensure that no polymerase errors were introduced. The human cysteineless AE1 mutant was constructed by cloning the mutant regions of each individual Cys-Ser mutant consecutively into pJRC9. The construct coding for human cysteineless AE1 cDNA in the pRBG4 expression vector was called pJRC26. The cysteineless protein encoded by pJRC26 was called AE1C.

Protein Expression and Membrane Isolation

Anion exchangers were expressed by transient transfection of human embryonic kidney (HEK) 293 cells (21) as described previously (22) , except that calcium phosphate-precipitated plasmid was added at 12 µg of plasmid/150-mm tissue culture plate. Eight 150-mm dishes of cells were harvested by scraping from the plate, and cells were washed twice with 140 m M NaCl, 10 m M Tris/HCl, pH 7.5. Cells were swollen in 10 ml of lysis buffer (1 m M phenylmethylsulfonyl fluoride, 10 m M Tris/HCl, pH 7.5) for 20 min at 4 °C and then lysed by 50 strokes in a Dounce homogenizer on ice. The lysate was diluted to 20 ml with lysis buffer and made to 10% (w/v) sucrose by the addition of 50% (w/v) sucrose. Nuclei were sedimented by centrifugation at 500 g for 5 min. The resulting supernatant was centrifuged at 4000 g for 5 min. The two pellets were pooled, extracted with 25 ml of lysis buffer containing 10% (w/v) sucrose, and centrifuged at 4000 g for 5 min. This supernatant was pooled with the previous supernatant and made to 25 m M KCl and 10 m M Hepes/NaOH, pH 7.4. The membrane extract was centrifuged at 35,000 rpm in a Beckman Ti-45 rotor for 50 min. This membrane pellet was resuspended in 0.5 ml of 7% (w/v) sucrose, 25 m M KCl, 1 m M MgCl, 15 m M Hepes/NaOH, pH 7.4, by 10 gentle strokes in a Dounce homogenizer. Microsomal membranes were used either fresh or frozen as aliquots in liquid nitrogen. Erythrocyte ghost membranes and purified Band 3 protein were isolated as described (23) .

Size Exclusion HPLC

Membranes (1.4 mg of protein) were suspended in 1 m M dithiothreitol, 5 m M sodium phosphate, pH 8, and centrifuged for 30 min at 35,000 rpm in a Beckman Ti-50 rotor. Membranes were solubilized by resuspension in 200 µl of 2% (v/v) CE, 0.1% (v/v) 1 m M dithiothreitol, 5 m M sodium phosphate, pH 8.0. After 20 min of incubation on ice, samples were centrifuged for 5 min at 45,000 rpm in a Beckman TLA-100 rotor. Supernatants were subjected to size exclusion HPLC on a 0.75 30-cm TSK 4000SW column (Beckman Instruments) eluted with 0.1 M sodium chloride, 0.1% (v/v) CE, 5 m M sodium phosphate, pH 7.0, as described previously (9) . Flow rate was 0.5 ml/min using a Beckman 114M pump. The column was calibrated with protein standards (Pharmacia Biotech Inc.) that were shown not to bind detergent (24) . To assay the elution position of the anion exchange protein, 30-s fractions of column eluate were collected, made to 1% (w/v) SDS, and dot-blotted onto nitrocellulose membrane. Dot blots were processed as immunoblots as described below and quantified using a Scanjet Plus scanner (Hewlett-Packard Co.) and Image 1.44 software (National Institutes of Health, Bethesda, MD).

Ankyrin Binding Assay

A truncated 43-kDa fragment of ankyrin was expressed in Escherichia coli, purified (25) , and I-labeled as described (26) . Radioiodinated ankyrin was diluted with unlabeled ankyrin to a specific activity of 2 10cpm/µg. Ankyrin binding assays were performed as described (14) . Briefly, frozen HEK cell membranes were thawed, and aliquots (30 µg of protein) were added to I-labeled ankyrin in 150 µl of binding buffer (1 mg/ml bovine serum albumin, 90 m M NaCl, 1 m M EDTA, 0.5 m M dithiothreitol, 0.02% (v/v) Tween 20, 10 m M sodium phosphate, pH 7.5) and incubated for 2 h at room temperature. The suspension was then loaded onto 0.25 ml of binding buffer containing 20% (w/v) sucrose in a 0.4-ml microtest tube (4 43 mm; Eppendorf North America, Inc.) and centrifuged at 16,000 rpm in an SS34 rotor for 20 min. Tubes were then frozen in dry ice. The bottom of the tube was cut off with a razor blade, and the pelleted membranes were subjected to -counting in a Beckman -5500B counter.

Sulfate Transport Assay

The reconstitution method was as described.() In a typical time course, 150 µg (20 µl) of membrane protein was added to 500 µl of 2 reaction buffer (40 m M sodium sulfate, 4 m M MgCl, 20 m M Mes/KOH, pH 6.0), 40 µCi of [S]sulfuric acid (DuPont NEN), and 500 µl of sonicated soybean phosphatidylcholine (Sigma) in water (20 mg/ml). To fuse the lipid vesicles with the microsomes (forming larger, less leaky vesicles), the mixture was frozen in liquid nitrogen, thawed at room temperature, and sonicated twice for 1 s each time in a G112SP1G bath sonicator (Laboratory Supplies Co., Hickesville, NY). Extravesicular [S]SO42- was removed on a 10-ml spin column of Sephadex G-50 (fine) equilibrated with 1 reaction buffer. The eluate from the column was placed into an ice-cold glass tube. HDIDS-treated samples also contained 160 µ M HDIDS (Molecular Probes, Inc.) in the reconstitution mixture and in 1 reaction buffer. Transport was initiated by placing the glass tube in a water bath at 25 °C. For each time point, samples in triplicate were removed from the tube and pipetted onto an ice-cold 1.5-ml column of Dowex 1 resin equilibrated with 0.1 M sucrose. The columns were rapidly washed with 2 0.75 ml of ice-cold 0.1 M sucrose. The radioactivity associated with each eluate was then measured by scintillation counting.

Electrophoresis and Immunoblotting

SDS-polyacrylamide gel electrophoresis was performed (28) , and proteins were transferred to nitrocellulose as described (29) . Immunoblots were blocked by incubation for 30 min in antibody buffer (5% (w/v) nonfat dry milk, 137 m M NaCl, 20 m M Tris/HCl, pH 7.6). Blots were incubated with anti-AE1 antibody 5-297, an antipeptide antibody raised against a synthetic peptide corresponding to the COOH-terminal 12 amino acids of mouse Band 3 protein (30) . Conditions were 10 ml/blot 1:2500 diluted antibody in antibody buffer for 2 h at room temperature, followed by 10 ml/blot 1:2500 diluted donkey anti-rabbit IgG conjugated to horseradish peroxidase (Amersham Corp.) incubated for 1 h at room temperature. Blots were visualized using Renaissance chemiluminescent reagent (DuPont NEN) and Hyperfilm (Amersham Corp.).

Molecular Biological Methods

Plasmids for transfections were prepared using QIAGEN columns (QIAGEN Inc.). DNA sequencing of plasmids was performed with Sequenase 2.0 (U. S. Biochemical Corp.) following the manufacturer's instructions. All other procedures followed standard protocols (31) .

Analytical Methods

Protein concentrations were determined with bicinchoninic acid reagent (Sigma) in the presence of SDS. Amino acid sequences were aligned with Intelligenetics software.


RESULTS

Expression of AE1 and AE1C-Fig. 2 shows an immunoblot of wild-type AE1 and AE1C. In the experiment shown, equal amounts of transfected HEK cell membrane protein were loaded, yet slightly more AE1 is observed in the lane containing the cysteineless mutant. This higher expression level was not observed consistently, so the mutations in AE1Cdo not influence biosynthetic levels. The recombinant proteins have a faster electrophoretic mobility than erythrocyte Band 3 because erythrocyte AE1 is glycosylated with up to 10 kDa of heterogeneous carbohydrate (32) . Since AE1 expressed in HEK cells is retained in the endoplasmic reticulum, it receives only core carbohydrate (22) .

Oligomeric Structure of AE1 Proteins

One measure of AE1 native structure is its oligomeric state. Several lines of evidence suggest that in the native membrane and in detergent solution, the minimal oligomeric unit of AE1 is the homodimer (6) . Size exclusion HPLC has been shown to resolve Band 3 oligomers and has revealed that the protein is a mixture of dimers and tetramers in CEsolution (9) . In size exclusion HPLC experiments, the elution position of protein is monitored, usually spectrophotometrically, and the Stokes radius of the eluted molecule can be determined after appropriate calibration of the column. Since AE1 in HEK cell membranes is expressed at 1% of the level of Band 3 in the erythrocyte, isolation of sufficient pure recombinant AE1 to allow spectrophotometric detection of protein elution is not possible. Therefore, to determine the oligomeric state of AE1 protein expressed in HEK cells, we combined size exclusion HPLC with detection of eluted protein by immunoblots. In the experiment shown in Fig. 3, CE-solubilized HEK cell membranes were applied to the size exclusion HPLC column. Fractions of the eluate were applied to nitrocellulose by ``dot blotting,'' and the nitrocellulose was processed as a conventional immunoblot. The amount of AE1 eluting in each fraction was then quantitated relative to standard amounts of AE1.

As seen in Fig. 3( upper panel), the elution profile of purified erythrocyte Band 3 protein has four peaks. These peaks were previously identified as highly associated AE1 protein, which elutes at the void volume (5.03 ml); tetrameric AE1 (6.75 ml); dimeric AE1 (7.85 ml); and detergent micelles (10.35 ml) (9) . Fig. 3( lower panel) shows that both wild-type AE1 and AE1Cfrom HEK cell membranes elute as two peaks; the first is at the void volume of the column and therefore represents a large protein complex, while the second elutes close to the position of dimeric erythrocyte AE1 protein. Although the peak positions of AE1 and AE1Cdo not coincide, the elution position of AE1Cis not consistent with that of the tetramer. In some preparations, wild-type AE1 eluted as a single major peak at a position corresponding to dimeric protein; however, in no preparation of AE1Cwas the protein peak at the void volume absent. The oligomeric state of AE1 expressed in HEK cells is predominantly dimeric, but the protein also associates into very large complexes. Oligomeric structure is not grossly altered in AE1C.


Figure 3: Resolution of AE1 oligomers by size exclusion HPLC. Upper panel, shown is the elution profile monitored at 215 nm of purified erythrocyte AE1 protein (6 µg) applied to a TSK 4000SW column eluted with 0.1 M sodium chloride, 0.1% (v/v) CE, 5 m M sodium phosphate, pH 7.0. Lower panel, the elution positions of AE1 () and AE1C() from CE-solubilized HEK cell membranes (20 µl) were determined by immunoblotting of the eluted protein. Shown at the bottom are the elution positions of the following standard proteins: thyroglobulin ( T; Stokes radius = 86 Å), ferritin ( F; 63 Å), catalase ( C; 52 Å), and aldolase ( A; 46 Å). The void volume ( V) was determined from the elution position of blue dextran 2000 (average M= 2 10), and the total volume ( V) was determined from the elution position of 2-mercaptoethanol.



Ankyrin Binding to AE1 Proteins

Erythrocyte Band 3 protein (AE1) anchors the cytoskeleton to the membrane via interactions between the cytoplasmic domain of Band 3 and ankyrin (3) . Since cysteines 201 and 317 reside in the predicted ankyrin-binding region of AE1, one possible consequence of the mutation of AE1 cysteines to serines is to alter the binding of ankyrin. To examine the role of these cysteine residues in ankyrin binding, AE1 proteins were expressed in HEK cells, and the binding of a radioiodinated truncated form of ankyrin was measured. In this system, AE1 has an inside-out orientation (22) such that ankyrin may interact with the cytoplasmic surface of AE1 in the binding assay. Full-length ankyrin has a molecular mass of 206 kDa and contains 24 repeats of a 33-amino acid sequence that has affinity for the cytoplasmic domain of AE1 (33) . In the ankyrin binding assay used here, a 43-kDa truncated ankyrin molecule, with 12 33-amino acid AE1-binding repeats, was used because it is more readily overexpressed and purified, yet is able to displace bound full-length ankyrin from erythrocyte membranes with similar affinity (25) . We have recently demonstrated that AE1 expressed in HEK cells retains the ability to bind the 43-kDa fragment of ankyrin (14) .

Fig. 4 shows the binding of the 43-kDa ankyrin fragment to AE1 and AE1C. The observed binding is saturable (Fig. 4 A) and from Scatchard analysis (Fig. 4 B) resolves into high and low affinity components. In Fig. 4 A, a single line can be drawn through the binding data from the two protein samples. The Scatchard plot (34) of the same data shows that the data cluster around a common line, indicating that the binding affinity for ankyrin (curve slope = -1/ K) and the binding capacity of the membranes (from the x intercept) are not altered by mutating the cysteines to serines. The high affinity component has a Kof 14 n M and represents 76 pmol of binding sites/mg of membrane protein, while the low affinity component has a Kof 106 n M and represents 155 pmol of binding sites/mg of membrane protein. The binding affinity observed in this assay is consistent with the Kof 5-10 n M found for the interaction of native ankyrin with AE1 in erythrocyte membranes (35, 36) . However, the density of AE1 protein in the microsomal membranes is much lower than in the erythrocyte membrane, which has 49 nmol of binding sites/mg of membrane protein (37) or 644 times the density of binding sites. Together, these data suggest that the cysteine residues of AE1 can be replaced by serine without altering the affinity of AE1 for ankyrin. Anion Exchange Activity of AE1 and AE1C-The role of the membrane domain of AE1 is to transport anions across the plasma membrane. Thus, the best criterion of whether mutation of AE1 cysteines to serines interferes with the protein's native state is the effect upon anion exchange activity. Anion exchange proteins will transport a range of anions (4) . The assay for anion exchange activity makes use of the ability of AE1 to transport SO42-.In this assay, microsomal membranes were reconstituted with exogenous soybean phosphatidylcholine and loaded with [S]SO42-. Exchange for extravesicular nonradioactive SO42- resulted in a decrease in vesicular [S]SO42- as a function of time.


Figure 4: Ankyrin binding by HEK cell membranes expressing AE1 and AE1C proteins. A, net ankyrin binding by membranes (30 µg of membrane protein/point) containing wild-type AE1 protein () or AE1Cprotein (). The binding by an equivalent amount of membranes from vector alone (pRBG4)-transfected HEK cells was subtracted from each value. B, Scatchard plot (34) of the binding of I-labeled ankyrin to membranes isolated from HEK cells transfected with wild-type AE1 (), AE1C(), and the pRBG4 vector alone (). The units of bound and free ankyrin are picomoles of ankyrin/milligram of membrane protein and picomoles/milliliter, respectively. Error bars that cannot be seen are smaller than the data point.



Fig. 5 shows [S]SO42-/SO42- exchange mediated by vesicles prepared from HEK cells transfected with AE1 or AE1CcDNA. From Fig. 5, the half-times of [S]SO42- efflux for AE1 and AE1Cwere both 2.5 min. Transport in AE1 and AE1Cvesicles could be inhibited by HDIDS to a similar extent. To test whether the anion exchange assay was sensitive to the number of functional AE1 molecules, microsomes were prepared from HEK cells transfected with either pJRC9 (AE1 cDNA) or pRBG4 (vector alone). Microsomes were mixed at a range of ratios, and the initial rate of anion exchange was proportional to the amount of AE1 protein present over the protein range in which transport experiments were performed here.() In Fig. 5, the two initial slopes (over the first 5 min) are not different from one another within the error of the experiment (AE1, 3.1% min; AE1C, 2.8% min), suggesting that the anion exchange activity of AE1Cis not compromised by the cysteine mutations. In control experiments, vesicles prepared from cells transfected with the pRBG4 vector alone had anion exchange activity similar to that seen in Fig. 5 for AE1 vesicles assayed in the presence of HDIDS.Human embryonic kidney cells were originally chosen for expression of anion exchange proteins because of their very low level of background anion exchange activity (18) ; therefore, the activity seen in Fig. 5is not due to an endogenous anion exchanger. The slight increase in intravesicular [S]SO42- as a function of time was observed reproducibly in HDIDS-treated samples, although the explanation for the observation is unclear. We conclude that the anion exchange function of the AE1 membrane domain is not influenced by mutation of the protein's cysteine residues to serines.


Figure 5: [S]SO42-/SO42- anion exchange assay of AE1 proteins. Microsomal membranes from transfected HEK cells were reconstituted with exogenous phosphatidylcholine in the presence of [S]SO42-. At each time point, triplicate samples were removed and pipetted onto a Dowex 1 anion exchange column to remove effluxed [S]SO42-. Radioactivity remaining associated with the vesicles eluted from the Dowex 1 columns was measured by scintillation counting. Shown is the efflux of intravesicular [S]SO42- mediated by HEK cell membranes expressing wild-type AE1 (200 µg of protein/time course) ( A) and AE1C(120 µg of protein/time course) ( B) in the absence () and presence () of 160 µ M HDIDS. The amount of protein in each assay was normalized to contain the same amount of anion exchange protein based on the intensity of staining on an immunoblot probed with anti-AE1 antibody 5-297. Error bars that cannot be seen are smaller than the data point. Initial time points represent 3-4 10cpm.




DISCUSSION

We have constructed and expressed a version of the plasma membrane anion exchange protein, AE1, in which all five of its cysteine residues have been replaced by serines. We show that this replacement does not alter the protein's oligomeric structure, ankyrin binding ability, or anion exchange activity. Serine was chosen for the substitutions since this is the most conservative mutation possible; cysteine and serine differ only in the sulfur atom of cysteine that is oxygen in serine. Functionally, the two share the ability to form hydrogen bonds. Because serine is similar to cysteine in structure, Cys-Ser mutations would be expected to affect protein function minimally. However, the two residues have differences: they have different volumes (Cys, 109 Å; Ser, 89 Å) (38) , which could alter protein packing; cysteine differs by its ability to form disulfide bonds, its reactivity to certain chemical reagents, and its ionizability (p Kof cysteine = 9.3) (38) .

Our data show that the oligomeric state of AE1Cis indistinguishable from that of wild-type AE1 and that AE1Cis functional in ankyrin binding and anion exchange. Together, these results suggest that the sulfhydryls of AE1 are not required for proper folding during biosynthesis. Since Band 3 tetramers are selectively depleted from detergent extracts of erythrocyte ghosts, it has been suggested that high affinity ankyrin binding either induces Band 3 tetramerization or requires pre-existing tetramers (9) . Our data demonstrate high affinity ankyrin binding to a predominantly dimeric population of AE1 molecules, suggesting that pre-existing AE1 tetramers are not essential for high affinity ankyrin binding. However, it is possible that ankyrin may induce the formation of AE1 tetramers. Since HEK cells do not express immunodetectable ankyrin,() the formation of AE1 tetramers in erythrocytes must be a consequence of interaction with the erythrocyte cytoskeleton. The significance of the AE1 protein that elutes from the void volume of the size exclusion chromatography column is unclear; however, it does not appear to interfere with anion exchange or ankyrin binding activity. Since the protein applied to the size exclusion chromatography column is a solubilized preparation of whole cell membranes, AE1 could be associating with any number of proteins to cause the altered elution position. Since AE1 proteins are retained in the endoplasmic reticulum of HEK cells, it is also possible that some AE1 remains associated with a part of the biosynthetic apparatus, for example with a molecular chaperone. The combination of size exclusion HPLC and immunoblotting to examine oligomeric structure in impure protein preparations and on a microscale may be useful for other proteins that cannot be purified in sufficient quantity to allow spectrophotometric detection.

Cysteines 201 and 317 are in the cytoplasmic domain of AE1. The degree of conservation of the cysteines provides insight into their expected role. Sequence conservation was examined with an alignment of the amino acid sequences of nine anion exchangers.() The region surrounding cysteine 201 is very poorly conserved, and cysteine is found at the homologous position only in mammalian AE1 proteins. However, the region around Cys-317 is better conserved, and cysteine is found in all examined anion exchange proteins, except in chicken AE1, in which the residue is replaced with alanine, and in AE3 proteins, in which it is glycine. Since chicken protein retains ankyrin binding, cysteine is not essential at this position. Our result that serine was sufficient to maintain ankyrin binding at these two positions is consistent with the lack of conservation observed at Cys-217.

The first cysteine residue of the membrane domain is Cys-479. It is at the COOH-terminal end of a sequence (residues 460-479) that is almost fully conserved in all nine anion exchanger sequences. This hydrophobic region is predicted to form transmembrane segment 3. This segment also contains two glutamic acid residues and two serine residues, suggesting that it could be a pore-lining helix. In this case, the ability to form hydrogen bonds is probably essential, so serine is sufficient.

Human AE1 in erythrocytes is palmitylated at Cys-843, at a consensus palmitylation site (15) . Cysteine 843 is conserved in eight out of nine anion exchanger sequences examined; it is not conserved in chicken AE1, in which it is a valine residue. However, the full palmitylation consensus is found only in some AE1 sequences. We have not determined whether or not wild-type AE1 expressed in HEK cells is modified with palmitic acid. However, mouse AE1 was found not to be palmitylated when expressed in X. laevis oocytes (16) . That anion exchange activity is unaltered after mutation of Cys-843 indicates that palmitylation is not essential for anion exchange function by AE1 and is consistent with a recent report on the corresponding mutation in mouse AE1 protein expressed in X. laevis oocytes (16) . In other palmitylated proteins, the role of palmitylation has been examined by mutation of the site to serine or alanine, with varied results. In the -adrenergic receptor, loss of palmitylation results in impaired receptor-G protein coupling (39) . However, in the closely related -adrenergic receptor, mutation of the palmitylated cysteine site has no effect on receptor function (40) . The only general role of palmitylation is to localize a protein segment to the lipid bilayer, where the palmityl chain can partition into the bilayer (41) .

The last cysteine of the membrane domain is Cys-885. It is conserved in all nine anion exchanger sequences examined, except in the AE3 sequences, in which it is an alanine residue. Although the cysteine residue is highly conserved, our data show that serine is also sufficient to maintain AE1 function.

The sulfhydryls of AE1 have been implicated in formation of erythrocyte senescence antigen (42) . Erythrocytes treated with oxidants developed binding sites for autoantibodies. In vivo binding of senescence antibodies results in targeting of the cell to the reticuloendothelial system for degradation (43) . The epitope is lost upon treatment with the reducing agent dithiothreitol, suggesting that some conformational change occurs upon oxidative cross-linking of AE1 cytoplasmic sulfhydryls and that this change is signalled across the membrane to be recognized by extracellular antibodies. Since AE1Chas no sulfhydryls, it may be a useful tool to study the role of sulfhydryls in the formation of senescence antigen.

AE1 was one of the first membrane proteins to be studied intensively, largely because it is easily isolated from erythrocytes, where it constitutes 50% of the integral membrane protein. It was first identified as the erythrocyte anion exchanger in 1974 (44) , and its amino acid sequence was among the first determined for mammalian membrane proteins (45) . Yet, the greatest progress to be made toward determining a structure for the protein has come only recently from a relatively low resolution (20 Å) electron diffraction structure (8) . The development of well ordered two- or three-dimensional crystals, which will improve the resolution of the three-dimensional structure, is a slow difficult process, as evidenced by the fact that only one membrane protein, the bacterial photoreaction center, has had its structure determined at high resolution (46) . Therefore, new approaches are required to attain greater structural information and ultimately to validate the electron diffraction or x-ray diffraction structure.

The availability of a cysteineless AE1 molecule will allow a wide range of structural experiments to be performed using cysteine-specific chemistry. Several such approaches have already been used, primarily on bacterial membrane proteins. The bacterial lactose transport protein, lac permease, was mutated free of its eight cysteine residues, which yielded a functional transporter (47) . After insertion of cysteine residues at novel sites, the protein was probed spectroscopically to determine which transmembrane helices were close to one another in the folded molecule (48) . Other methodologies have been used to study cysteineless mutants of bacteriorhodopsin (49, 50) and the bacterial chemosensory receptor (51, 52) . In mammalian proteins, exposure of surfaces to small, cysteine-reactive, aqueous probes has been used to determine both topology and water-accessible regions, for example an ion permeation channel (53) .

The results presented here show that the functions of the two domains of AE1 are not altered by replacement of the protein's cysteine residues with serines. The cytoplasmic domain retains the ability to bind the cytoskeleton via ankyrin, and the membrane domain is competent to transport anions. The cysteineless AE1 system should be useful for structure-function studies of the protein.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Recipient of a Medical Research Council of Canada postdoctoral fellowship.

Recipient of an American Heart Association postdoctoral fellowship.

**
To whom correspondence should be addressed. Tel.: 415-723-7581; Fax: 415-723-8475.

The abbreviations used are: CE, octaethylene glycol monododecyl ether; HEK, human embryonic kidney; HPLC, high pressure liquid chromatography; Mes, 2-( N-morpholino)ethanesulfonic acid; HDIDS, 4,4`-diisothiocyanodihydrostilbene-2,2`-disulfonate.

I. Sekler, R. S. Lo, and R. R. Kopito, submitted for publication.

J. R. Casey, unpublished data.

Y. Ding, unpublished data.

The anion exchanger sequences aligned were human (17), mouse (54), rat (55), chicken (56), and trout (57) AE1; rat (58) and mouse (59) AE2; and rat (58) and mouse (27) AE3.


ACKNOWLEDGEMENTS

We thank Roger Lo for assistance with anion exchange assays and laboratory members for comments on the manuscript.


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