INTRODUCTION

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AE1 (band 3) belongs to a family of anion exchange proteins that facilitate the movement of Cl and HCO₃ across the plasma membrane. Plasma membrane anion exchange proteins are widely expressed among mammalian tissues, where they participate in the regulation of intracellular pH and volume. Three anion exchanger isoforms have been identified, cloned, and sequenced: AE1, found in erythrocytes and kidney; AE2, found in kidney, stomach, and lymphocytes; and AE3, found in the brain, retina, and heart (1). All of these anion exchange proteins contain two domains. The highly conserved (70% identity) membrane domain of approximately 55 kDa spans the bilayer 12-14 times and is responsible for anion exchange activity. The cytoplasmic domain of 45-110 kDa is more divergent. In erythrocyte AE1, the cytoplasmic domain anchors the cytoskeleton to the plasma membrane through interactions with ankyrin (2).

AE1 has served as a model for our understanding of membrane protein structure and function because of its high expression in erythrocytes, where it constitutes 50% of the integral membrane protein (3). AE1 was among the first membrane transport proteins to have its cDNA cloned and sequenced (4). Despite the wealth of information regarding anion exchangers, we still do not know which residues of the protein are involved in the transport process. Jennings and co-workers implicated Glu⁶⁸¹ in the transport process, since labeling this residue with Woodward's reagent K (WRK)¹ and reduction with sodium borohydride resulted in altered anion exchange kinetics (5-7). The functional role of Glu⁶⁸¹ in AE1 was confirmed in mutagenesis experiments of mouse AE1 (8) and extended to the homologous position of mouse AE2, suggesting that the mechanistic role of Glu⁶⁸¹ is conserved among anion exchange proteins (9). WRK chemical modification of AE1 abolishes chloride transport, yet relieves the requirement for proton cotransport during sulfate transport. During sulfate transport in unmodified AE1, a proton, supplied by Glu⁶⁸¹ is cotransported. Sulfate/proton cotransport takes place in both inward and outward directions, which implies that Glu⁶⁸¹ has access to both the intracellular and extracellular sides of the membrane. Taken together, Glu⁶⁸¹ is functionally involved in anion exchange events and may reside at the permeability barrier of AE1. Determination of the location of this residue in the transmembrane region will localize both a part of the transport site and the permeability barrier.

Our current knowledge of AE1 topology comes from both experimental data and hydropathy plots. Hydropathy plots show regions of strong hydrophobicity in the N-terminal half of the membrane domain corresponding to the first seven transmembrane segments of AE1. However, our limited knowledge of the topology of the membrane domain of AE1 has recently been reviewed (10). The site of N-linked glycosylation, Asn⁶⁴² (11), roughly marks the boundary between the first half of the membrane domain, with well defined transmembrane segments, and the second half, with ill defined topology (Fig. 1) (12). A chymotryptic cleavage site in intact erythrocytes has been identified at Tyr⁵⁵³ (13), localizing this residue to the external face. Antibody accessibility studies have mapped the extreme C terminus and two other loops to the cytoplasm (14, 15).

We have chosen to study the topology of the initial portion of the second half of AE1 membrane domain, beginning at the well defined external site of N-glycosylation (from Ser⁶⁴³ to Ser⁶⁹⁰), using introduced cysteine scanning mutagenesis. Mutation of individual amino acids to cysteine represents a minor structural modification, relative to other methods in use to define topology and therefore has some potential advantages. Our goals were to define the membrane topology of an functionally important region of human AE1 anion exchange protein and to validate the use of cysteine scanning mutagenesis to the study of protein topology of mammalian membrane proteins. We have previously constructed a version of human AE1, lacking all cysteines and characterized it as functional (16). This mutant forms the basis for the introduction of cysteine residues at defined sites.

	EXPERIMENTAL PROCEDURES

Top Abstract Introduction Procedures Results Discussion References

Materials-- Restriction endonucleases were from New England Biolabs. ECL chemiluminescent reagent, horseradish peroxidase conjugated to sheep anti-mouse IgG, and Hyperfilm and Immobilon membranes were from Amersham Pharmacia Biotech. Biocytin hydrazide, BCECF-AM, LYIA, biotin maleimide, and stilbene maleimide were from Molecular Probes, Inc. (Eugene, OR). Poly-L-lysine and nigericin were from Sigma. Coverslips were from Fisher.

Construction of Mutant Anion Exchangers-- A human AE1 cDNA construct, called AE1C, in which all five cysteine codons were mutated to serine was previously constructed (16) in the expression vector pRBG4 (18). Individual introduced cysteine codons were cloned into AE1C to yield mutants, each with a unique cysteine codon. Introduced cysteine mutants at amino acids 645-647 were not constructed, because their codons overlap with the SmaI site (nucleotides 2048-2053), used to clone introduced cysteine mutants into AE1C. Mutagenesis was performed using a polymerase chain reaction megaprimer mutagenesis strategy (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 either Vent DNA polymerase (New England Biolabs) or PWO polymerase (Boehringer Mannheim). Mutants were verified by DNA sequencing.

Protein Expression-- Anion exchangers were expressed by transient transfection of human embryonic kidney 293 (HEK293) cells (21), as described previously (22), except that calcium phosphate-precipitated plasmid was added at 2.8 µg of anion exchanger plasmid with 4.2 µg of pRBG4 carrier/100-mm tissue culture dish. Cells were grown at 37 °C in a 5% CO₂ environment in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) containing 5% (v/v) fetal bovine serum (Life Technologies), and 5% (v/v) fetal calf serum (Life Technologies) and harvested 48 h post-transfection.

Antibody Preparation-- A synthetic peptide composed of cysteine followed by the C-terminal 13 amino acids of human AE1 was synthesized, yielding peptide: NH₂-CEGRDEYDEVAMPV-COOH. The peptide was coupled to keyhole limpet hemocyanin and subsequently injected into two rabbits numbered 1657 and 1658. Serum from each rabbit was monitored, on immunoblots containing AE1 protein, until a maximal immune response was observed. Rabbits were sacrificed and exsanguinated, and sera designated 1657 and 1658 were isolated. Both sera have high anti-AE1 titer. Peptide synthesis and antiserum production were completed by SynPep (Dublin, CA).

Immunoprecipitation-- Lysates of whole tissue culture cells were prepared by incubation of cells from one-half of a 100-mm dish, with 250 µl of IPB buffer (1% (v/v) Nonidet P-40, 5 mM EDTA, 0.15 M NaCl, 0.5% (w/v) sodium deoxycholate, 10 mM Tris-HCl, pH 7.5), containing 2 mg/ml bovine serum albumin, 200 µM TPCK, 200 µM TLCK, and 2 mM phenylmethylsulfonyl fluoride, on ice for 15-30 min. Insoluble material was removed by centrifugation for 15 min at 16,000 × g in an IEC Micromax microcentrifuge. The sample was precleared with 2 µl of preimmune rabbit serum and protein A-Sepharose. Each immunoprecipitation used 2 µl of anti-AE1 antibody 1658 and was incubated at 4 °C, overnight, with protein A-Sepharose resin (Amersham Pharmacia Biotech). The resin was washed consecutively with 1 ml each of wash buffer 1 (0.1% Nonidet P-40, 1 mM EDTA, 0.15 M NaCl, 10 mM Tris-HCl, pH 7.5), wash buffer 2 (2 mM EDTA, 0.05% SDS, 10 mM Tris-HCl, pH 7.5), and wash buffer 3 (2 mM EDTA, 10 mM Tris-HCl, pH 7.5). Samples were eluted from the resin by incubation for 5 min at 65 °C with SDS-polyacrylamide gel electrophoresis sample buffer containing 2% (v/v) 2-mercaptoethanol (23).

Chemical Accessibility Assays-- HEK293 cells grown in 100-mm tissue culture dishes were transfected with wild type or mutant AE1 cDNA, as described above. Forty-eight hours post-transfection, cells were harvested by incubation with 1 mg/ml trypsin in PBS (140 mM NaCl, 3 mM KCl, 6.5 mM Na₂HPO₄, 1.5 mM KH₂PO₄) for 5 min at 37 °C. Cells were collected from the plate with a pipette and sedimented by centrifugation for 5 min at 228 × g. Cells were then washed with PBS. After resuspension in 2 ml of PBSCM (PBS buffer containing 0.1 mM CaCl₂ and 1 mM MgCl₂, pH 7.0), cells were divided into two equal samples, labeled + and . To the + sample, 30 µl of 17 mM lucifer yellow iodoacetamide (LYIA) was added, and the sample was incubated for 20 min at room temperature with gentle, occasional mixing. Then 10 µl of 2 mM biotin maleimide (in dimethyl sulfoxide) was added to both the + and  samples, and the samples were incubated with occasional mixing for 15 min at room temperature. Reactions were stopped by the addition of 0.5 ml of 2% (v/v) 2-mercaptoethanol in Dulbecco's modified Eagle's medium and incubated at room temperature for 10 min. Cells were sedimented as above and washed with PBSCM. Sedimented cells were then lysed with 250 µl of IPB containing 2% (w/v) bovine serum albumin, 200 µM TPCK, 200 µM TLCK, and 2 mM phenylmethylsulfonyl fluoride on ice for 15 min. In experiments to measure ability to label with biotin maleimide, samples were prepared as described above for  samples. After immunoprecipitation, samples were electrophoresed on 8% acrylamide gels (23) and transferred to Immobilon membrane (24). Biotinylated proteins were detected by incubation of the blot with 10 ml of 1:2500 diluted streptavidin-biotinylated horseradish peroxidase (Amersham Pharmacia Biotech) in TBSTB buffer (TBST buffer (0.1% (v/v), Tween-20, 137 mM NaCl, 20 mM Tris, pH 7.5), containing 0.5% (w/v) bovine serum albumin). After a 1.5-h incubation, blots were washed with TBST. Blots were visualized using ECL reagent and Hyperfilm (Amersham Pharmacia Biotech).

Cell Surface Processing Assay-- The fraction of anion exchanger protein processed to the cell surface was assessed by labeling intact, whole transfected cells with biocytin hydrazide (26). Transfected HEK293 cells were grown in 100-mm dishes and deplated by incubation for 5 min at 37 °C with 1 mg/ml trypsin in PBS. All steps of the labeling were performed at 4 °C. Cells were resuspended in 2.7 ml of PBSCM. To this was added 300 µl of 100 mM sodium metaperiodate, and cells were incubated for 30 min in the dark with gentle occasional mixing. Cells were washed twice with PBSCM, by centrifugation for 5 min at 228 × g. Cells were resuspended in 1.25 ml of 1 mM biocytin hydrazide, 100 mM sodium acetate, pH 5.5, and incubated for 30 min in the dark with gentle occasional mixing. Cells were washed twice, as above. A whole cell extract was then prepared by solubilization of the cell pellet in IPB buffer containing 2% (w/v) bovine serum albumin, 200 µM TPCK, 200 µM TLCK, and 2 mM phenylmethylsulfonyl fluoride. After AE1 immunoprecipitation (see above), samples were resolved by SDS-polyacrylamide gel electrophoresis on 8% acrylamide gels (23). After transfer to Immobilon, biotinylated proteins were detected by incubation of the blot with 10 ml of 1:2500 diluted streptavidin-biotinylated horseradish peroxidase in TBSTB. After a 1.5-h incubation, blots were washed with TBST, and the image was processed with ECL reagent (see above).

Anion Exchange Assays-- HEK293 cells were grown on top of 7 × 11-mm glass coverslips in 60-mm tissue culture dishes and transfected as described. Two days post-transfection, coverslips were rinsed with serum-free Dulbecco's modified Eagle's medium (Life Technologies) and loaded with BCECF-AM dye by incubation in 4 ml of serum-free Dulbecco's modified Eagle's medium, containing 2 µM BCECF, for 20-30 min, at 37 °C. Coverslips were mounted in a custom-built quartz cuvette, with perfusion capabilities. Intracellular pH was monitored by measuring fluorescence at excitation wavelengths of 440 and 502 nm and emission of 520 nm, in either a Spex Fluorlog spectrofluorometer or a Photon Technologies International RCR/Delta RAM spectrofluorometer. The cuvette was perfused at 3.5 ml/min alternately with Ringer's buffer (5 mM glucose, 5 mM potassium gluconate, 1 mM calcium gluconate, 1 mM MgSO₄, 2.5 mM NaH₂PO₄, 25 mM NaHCO₃, 10 mM Hepes, pH 7.4) containing 140 mM sodium chloride (chloride buffer) or with Ringer's buffer containing 140 mM sodium gluconate (chloride-free buffer). Both buffers were bubbled continuously with air containing 5% carbon dioxide. Intracellular pH was calibrated by the nigericin-high potassium method (27), using three pH values from pH 6.5 to 7.5. Transport rates were determined by linear regression of the initial linear rate of change of pH, using Kaleidagraph software (Synergy Software).

Image Analysis and Data Analysis-- Immunoblots and chemical biotinylation blots were scanned with a Hewlett Packard Scanjet 4C flatbed scanner, calibrated with a Kodak gray scale. Scanned images were quantified using NIH Image 1.60 software. Biotinylation levels were calculated as follows: biotinylation_norm = pixels of biotin signal/pixels of AE1 immunoblot.

norm  LYIA

Molecular Biological Methods-- Plasmid DNA for transfections were prepared using Qiagen columns (Qiagen Inc.). DNA sequencing of plasmids was performed by the Core Facility in the Department of Biochemistry, University of Alberta, with an Applied Biosystems 373A DNA sequencer. All other procedures followed standard protocols (28).

	RESULTS

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Construction of Introduced Cysteine Mutants-- To probe the topology of the region surrounding the functionally essential Glu⁶⁸¹ residue of human AE1 protein, we constructed an array of introduced cysteine mutants. In this array, each residue from the site of AE1 glycosylation (Asn⁶⁴²) to Ser⁶⁹⁰ was individually mutated to cysteine. Each cysteine mutant was cloned into AE1C, an AE1 mutant with all endogenous cysteine codons mutated to serine (16). Fig. 1 shows the sites of cysteine mutants constructed in topologically well defined regions of the protein, to verify the labeling methodology. S555C represents a control site for the extracellular surface of the protein, since it is found between two chymotryptic cleavage sites in intact erythrocytes (13); S595C lies in a hydrophilic region and is separated from S555C by a highly hydrophobic sequence, which defines 595 as an intracellular site; K892C is adjacent to the C terminus of the protein, previously mapped to the inside of the cell (14), thereby defining 892 as intracellular; Ser⁵⁷⁴ is found in the hydrophobic region between Ser⁵⁵⁵ and Ser⁵⁹⁵ and therefore is probably in a transmembrane segment.

View larger version (34K): [in this window] [in a new window]   Fig. 1.   Topology model for membrane domain of human AE1. The model has been truncated at the junction between the cytoplasmic domain and membrane domain, as defined by the sites of high protease sensitivity. T, trypsin; C, chymotrypsin. The branched structure at Asn642 represents N-linked glycosylation. The model is based on data on the accessibility of residues to LYIA (J. Fujinaga, X.-B. Tang, and J. R. Casey, manuscript in preparation) and on data from glycosylation scanning mutagenesis (36).

Labeling Introduced Cysteines with Biotin Maleimide-- To begin to determine the topology of each residue in the introduced cysteine mutant array, we labeled HEK293 cells expressing a single introduced cysteine mutant with the membrane-permeant, cysteine-directed reagent, biotin maleimide. Fig. 2 shows representative data from the labeling experiments at 10 sites throughout the region. AE1C labels with biotin maleimide to a barely detectable level, consistent with the absence of cysteine residues from the construct. Cytosolic control mutants S595C and K892C label to a similar extent to Y555C, which is a control site for the external surface of the protein. Interestingly, mutant S574C does not label with biotin maleimide, suggesting that transmembrane residues are not accessible to biotinylation, under the conditions used in this experiment. Fig. 2B shows the amount of AE1 protein loaded into each lane of the blot in A. It is apparent that all of the mutants express similar amounts of AE1, yet they label differentially with biotin maleimide.

View larger version (30K): [in this window] [in a new window]   Fig. 2.   Labeling human AE1 introduced cysteine mutants with biotin maleimide. AE1C is a cDNA construct encoding a form of human AE1 in which all cysteine codons were replaced by serine codons. Into this cysteineless background, individual cysteine mutants were cloned, to generate mutants each with only a single introduced cysteine codon. HEK293 cells were transfected with cDNA for AE1C and introduced cysteine mutants at sites shown at the top. Cells were harvested and incubated with 2 mM biotin maleimide for 15 min at room temperature. After solubilization, samples were immunoprecipitated with anti-AE1 antibody, subjected to electrophoresis on 8% acrylamide gels, and transferred to PVDF membrane. A, biotin incorporation was detected using horseradish peroxidase-streptavidin and ECL. B, the blot from A was stripped and probed with monoclonal anti-AE1 antibody (IVF12) to detect the level of AE1 expression.

View larger version (28K): [in this window] [in a new window]   Fig. 3.   Biotinylation of introduced cysteine mutants by biotin maleimide. Each introduced cysteine mutant was treated with biotin maleimide, as described. The level of biotin incorporation was quantified by densitometry, and this signal was normalized by the amount of AE1 present in the sample. In each experiment, the level of biotinylation was compared with that of the S643C mutant, whose labeling was set to 100. Data represent the mean of three or four determinations ± S.E. Mutants 645, 646, and 647 were not constructed. The black bars represent the values for control mutants Y555C and K892C. The dashed line represents the mean level of biotinylation for AE1C, the cysteineless mutant (n = 8). At the bottom is a model representing the proposed topological designation for each region of the amino acid sequence.

Relative Aqueous Accessibility of Residues-- The ability to label with biotin maleimide appears to differentiate residues that are extramembraneous from transmembrane residues, as presented in Figs. 2 and 3. In the case of human AE1, we know that the region surrounding Asn⁶⁴² is extracellular, since the site is glycosylated. However, for the introduced cysteine method to be applicable to define regions of unknown topology, a method is required to differentiate the inside of the cell from the outside. Previously this question has been addressed by labeling introduced cysteine mutants, in intact cells, with biotin maleimide and observing the ability to block this labeling by prelabeling with a membrane-impermeant reagent, stilbene maleimide (29). In our hands, however, this reagent behaves as membrane-permeant, when used at 10-200 µM, for 15 min at room temperature. Permeability may have been due to the fact that stilbenes have been reported to be weak substrates of AE1 (30). We therefore chose to use a different membrane-impermeant cysteine-directed reagent, LYIA. This reagent has been used as a fluorescent marker of the pinocytic pathway and is therefore likely to be membrane-impermeant (31).

View larger version (28K): [in this window] [in a new window]   Fig. 4.   Representative data showing accessibility of introduced cysteine residues to lucifer yellow iodoacetamide. HEK293 cells transfected with human AE1 introduced cysteine mutants were incubated for 20 min. at room temperature without () or with (+) 500 µM lucifer yellow iodoacetamide. Cells were then treated with 2 mM biotin maleimide for 15 min at room temperature. A, the amount of biotin incorporated for each introduced cysteine mutant (mutant position shown on top) was detected with horseradish peroxidase-streptavidin and ECL reagent. B, the same blot stripped and probed with anti-AE1 antibody.

View larger version (39K): [in this window] [in a new window]   Fig. 5.   Accessibility of introduced cysteine mutants to lucifer yellow iodoacetamide. Human AE1 introduced cysteine mutants were incubated with or without LYIA and then treated with biotin maleimide, as described. Biotin incorporation was quantified by densitometry and was normalized to the relative expression level of AE1 on the basis of an immunoblot. For each mutant, the normalized level of incorporation of biotin without LYIA prelabeling was divided by biotinylation after prelabeling with LYIA. This ratio represents the relative accessibility of each introduced cysteine site to LYIA. Data represent the mean of three or four determinations ± S.E. The black bars represent the values for control mutants Y555C and K892C. Mutants at amino acids 645-647 were not constructed. At the bottom is a model representing the proposed topological designation for each region of the amino acid sequence. The asterisks represent mutants not analyzed because they did not label with biotin maleimide.

Anion Exchange Activity of AE1 Introduced Cysteine Mutants-- Since introduction of cysteine into sites within AE1 could alter the folding or transport activity of the protein, anion exchange activity was measured for each mutant. In this assay, the alternating chloride gradient from inward to outward results in bicarbonate movement in the opposite direction across the membrane; in chloride-free medium, chloride leaves the cell, bicarbonate enters, and the cell alkalinizes (32). In the present experiments, transport rates were determined from the initial slope of the curve as alkalinization and acidification occur. Fig. 6 shows typical anion exchange data, where AE1C retains a 52% transport rate relative to wild type AE1. This result conflicts with our previous work, which assessed anion exchange activity of AE1 and AE1C reconstituted from microsomes of HEK293 cells expressing human AE1 (16). The impaired transport is explained by a decreased level of cell surface processing of AE1C (see below). Negative control cells transfected with pRBG4 vector only also displayed a variable background level of transport (10-15% of AE1C rate), as seen in Fig. 6C.

View larger version (18K): [in this window] [in a new window]   Fig. 6.   Assay of AE1 anion exchange activity. HEK293 cells transfected with human AE1 (A), AE1C cDNA (B), or pRBG4 vector alone (C) were grown on glass coverslips and then loaded with the pH-sensitive dye, BCECF-AM. Cells were suspended in a fluorescence cuvette, and intracellular pH was monitored, as described under "Experimental Procedures." Cells were perfused with either Ringer's buffer containing 140 mM NaCl or Ringer's buffer with NaCl replaced by 140 mM sodium gluconate. The bar at the top of each panel represents the time period when the cuvette was perfused with chloride-containing (solid bar) or chloride-free buffer (open bar).

View larger version (39K): [in this window] [in a new window]   Fig. 7.   Anion exchange rate of introduced cysteine mutants. Anion exchange activity was assayed as seen in Fig. 5. Transport rates were corrected for the background rate of vector (pRBG4)-transfected cells. Rates are expressed as a percentage of the rate of AE1C ± S.E. Mutants marked with an asterisk were not constructed.

Cell Surface Processing of AE1 Mutants-- Impaired anion exchange activity of introduced cysteine mutants may result from either some effect on the protein's structure or from a reduced processing of the protein to the cell surface. Since the anion exchange assay only measures the functional activity of protein in the plasma membrane, intracellular retention of the protein would appear as nonfunctional protein. Therefore, we measured the cell surface expression of mutants that had low anion exchange activity (Table I). The basis of the cell surface expression assay is to express AE1 mutants in HEK293 cells, oxidize their cell surface carbohydrate with sodium metaperiodate, and react the oxidized carbohydrate with biocytin hydrazide. Thus, only protein at the cell surface incorporates a biotin label. The level of biotin incorporation was quantified relative to the amount of each mutant that was expressed. Surface processing of each AE1 protein was then compared with AE1C. Cell surface expression of AE1C is only 66% as high as wild type AE1. As a negative control, we examined the processing of mouse AE1, shown previously to be retained intracellularly (22), and no cell surface expression could be detected. Among the nonfunctional mutants, only W648C and E681C have reduced processing to the cell surface. However, in neither case is the processing sufficiently impaired to account for the loss of transport activity (Table I). Therefore, reduced levels of processing to the cell surface do not explain loss of transport function observed for the six mutants listed above.

View this table: [in this window] [in a new window]   Table I Cell surface processing of introduced cysteine mutants with low functional activity Mutants marked by an asterisk were functionally inactive. Cell surface processing is expressed as a percentage ± S.E. of the processing of AE1C.

	DISCUSSION

Top Abstract Introduction Procedures Results Discussion References

In this paper, we have used chemical reactivity of introduced cysteine residues to examine the topology of transmembrane segment 8 of human AE1. This region is important to our understanding of AE1 function because it contains Glu⁶⁸¹. Glu⁶⁸¹ may interact with transported anions, since it provides the proton required during sulfate/proton cotransport by AE1. Since the cotransported proton can come from either side of the membrane, Glu⁶⁸¹ may mark the transmembrane permeability barrier. We have developed a quantitative method to examine membrane protein topology that uses the membrane impermeant reagent, lucifer yellow iodoacetamide, as a probe of the environment around introduced cysteine residues. Our findings allow us to identify transmembrane segment 8 as the region from Met⁶⁶⁴ to Gln⁶⁸³, which places Glu⁶⁸¹ within three residues of the intracellular surface of the protein. Use of LYIA allowed us to identify a region from Arg⁶⁵⁶ to Met⁶⁶³ with properties that are consistent with a vestibule region (see below). Consistent with the vestibule model is the observation that sites on the extracellular surface of AE1 were most sensitive to cysteine replacement (Fig. 8).

View larger version (28K): [in this window] [in a new window]   Fig. 8.   Folding models for the transmembrane segment 8 region of AE1. Introduced cysteine mutants were introduced at each position marked by a colored circle. White circles represent mutants that were not constructed. Letters inside circles show the residue present before mutation. Residues are color-coded to show anion exchange activity of introduced cysteine mutants relative to AE1C. A, helical wheel plot of proposed transmembrane region from Met664 to Gln683. B, topology model of the Asn642-Ser690 region of human AE1. The branched, clustered black square structure represents carbohydrate on Asn642. The black box represents the proposed transmembrane region, while the jagged lines with green ovals on the end represent lipid molecules.

Identification of the Transmembrane Region-- On the basis of the inability to label introduced cysteine residues in the Met⁶⁶⁴-Gln⁶⁸³ region, we propose that the region forms transmembrane segment. In studies of the tetracycline transporter, a region of about 20 consecutive introduced cysteine residues that could not be labeled with N-ethylmaleimide was identified as a transmembrane segment (33-35). Our localization of the transmembrane segment is also consistent with recent studies on the introduction of glycosylation sites into AE1 (36), which indicated that a minimum of 14 residues are required between a glycosylation site and the N-terminal start of a transmembrane segment. Asn⁶⁴² is the glycosylation site on human AE1, which places our proposed start of the transmembrane segment 21 residues from the glycosylation site. Experiments using introduced glycosylation sites mapped the N-terminal end of transmembrane segment 8 to Met⁶⁶³, within one residue of our determination (36).

Structure and Proposed Role of Extramembraneous Regions-- The regions of AE1 that are accessible to labeling by biotin maleimide may be structurally subdivided into three parts by their reactivity with LYIA: (i) residues Arg⁶⁵⁶-Met⁶⁶³ show a graded accessibility to LYIA, from high accessibility at Arg⁶⁵⁶ to low accessibility at Met⁶⁶³; (ii) residues Ser⁶⁴³-Leu⁶⁵⁵ show low accessibility to LYIA, with the exception of His⁶⁵¹, which has high accessibility; and (iii) residues Ile⁶⁸⁴-Ser⁶⁹⁰ are poorly accessible to membrane-impermeant LYIA (Fig. 8).

Introduced Cysteines to Determine Membrane Protein Topology-- Accessibility of introduced cysteine residues to LYIA was readily able to differentiate intracellular from extracellular residues. In 45 introduced cysteine mutants analyzed, we found one stretch of 20 residues that did not label with biotin maleimide. Therefore, the inability of biotin maleimide to label a particular cysteine indicates, with reasonable confidence, a transmembrane disposition of that cysteine. It is also possible that a residue deeply buried in the structure of a protein domain would not label. However, extramembraneous introduced cysteine residues that are not maleimide-reactive are probably rare on the basis of the data presented here and evidence from the tetracycline transporter, where only one of 29 extramembraneous introduced cysteine residues was not reactive toward a maleimide (34, 35).

Conclusions-- In this paper, we have developed an introduced cysteine method to determine membrane protein topology and applied this method to residues Ser⁶⁴³-Ser⁶⁹⁰ of human AE1. Central to the method is the use of LYIA to differentiate intracellular from extracellular sites. The method is able to differentiate transmembrane residues from extramembraneous sites by virtue of an inability to label transmembrane cysteines with biotin maleimide. Our method may be widely applicable to the study of membrane protein topology. It has the advantage that structural perturbation of introduced cysteine mutations is milder than in other methods commonly used to determine topology.