Topology of the Membrane Domain of Human Erythrocyte Anion Exchange Protein, AE1
1999; Elsevier BV; Volume: 274; Issue: 10 Linguagem: Inglês
10.1074/jbc.274.10.6626
ISSN1083-351X
AutoresJocelyne Fujinaga, X. Charlene Tang, Joseph R. Casey,
Tópico(s)Cellular transport and secretion
ResumoAnion exchanger 1 (AE1) is the chloride/bicarbonate exchange protein of the erythrocyte membrane. By using a combination of introduced cysteine mutants and sulfhydryl-specific chemistry, we have mapped the topology of the human AE1 membrane domain. Twenty-seven single cysteines were introduced throughout the Leu708-Val911 region of human AE1, and these mutants were expressed by transient transfection of human embryonic kidney cells. On the basis of cysteine accessibility to membrane-permeant biotin maleimide and to membrane-impermeant lucifer yellow iodoacetamide, we have proposed a model for the topology of AE1 membrane domain. In this model, AE1 is composed of 13 typical transmembrane segments, and the Asp807-His834region is membrane-embedded but does not have the usual α-helical conformation. To identify amino acids that are important for anion transport, we analyzed the anion exchange activity for all introduced cysteine mutants, using a whole cell fluorescence assay. We found that mutants G714C, S725C, and S731C have very low transport activity, implying that this region has a structurally and/or catalytically important role. We measured the residual anion transport activity after mutant treatment with the membrane-impermeant, cysteine-directed compound, sodium (2-sulfonatoethyl)methanethiosulfonate) (MTSES). Only two mutants, S852C and A858C, were inhibited by MTSES, indicating that these residues may be located in a pore-lining region. Anion exchanger 1 (AE1) is the chloride/bicarbonate exchange protein of the erythrocyte membrane. By using a combination of introduced cysteine mutants and sulfhydryl-specific chemistry, we have mapped the topology of the human AE1 membrane domain. Twenty-seven single cysteines were introduced throughout the Leu708-Val911 region of human AE1, and these mutants were expressed by transient transfection of human embryonic kidney cells. On the basis of cysteine accessibility to membrane-permeant biotin maleimide and to membrane-impermeant lucifer yellow iodoacetamide, we have proposed a model for the topology of AE1 membrane domain. In this model, AE1 is composed of 13 typical transmembrane segments, and the Asp807-His834region is membrane-embedded but does not have the usual α-helical conformation. To identify amino acids that are important for anion transport, we analyzed the anion exchange activity for all introduced cysteine mutants, using a whole cell fluorescence assay. We found that mutants G714C, S725C, and S731C have very low transport activity, implying that this region has a structurally and/or catalytically important role. We measured the residual anion transport activity after mutant treatment with the membrane-impermeant, cysteine-directed compound, sodium (2-sulfonatoethyl)methanethiosulfonate) (MTSES). Only two mutants, S852C and A858C, were inhibited by MTSES, indicating that these residues may be located in a pore-lining region. The human erythrocyte anion exchanger 1 (AE1), 1The abbreviations used are: AE1, anion exchanger 1; AE1C−, cysteineless AE1 protein; BCECF-AM, 2′,7′-bis(2-carboxyethyl)-(5 and 6)-carboxyfluorescein, acetoxymethyl ester; biotin maleimide, 3-(N-maleimidylpropionyl)biocytin; DIDS, 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid, disodium salt; DMEM, Dulbecco's modified Eagle media; ECL, enhanced chemiluminescence; HEK, human embryonic kidney; HT, high transport; LYIA, lucifer yellow iodoacetamide, dipotassium salt; MTSEA, 2-(aminoethyl) methanethiosulfonate, hydrobromide; MTSES, sodium (2-sulfonatoethyl)methanethiosulfonate); PBS, phosphate-buffered saline; PVDF, polyvinylidene fluoride; PAGE, polyacrylamide gel electrophoresis; TM, transmembrane segment. also called Band 3, facilitates the electroneutral exchange of Cl− and HCO3− across the plasma membrane. Three anion exchanger isoforms are known that show differences in their tissue expression as follows: AE1 is found in erythrocytes and kidney; AE2 is in a wide variety of tissue; and AE3 is in the brain, retina, and heart (1Reithmeier R.A.F. Chan S.L. Popov M. Konings W.N. Kaback H.R. Lolkema J.S. Handbook of Biological Physics. 2. Elsevier Science Publishers B.V., Amsterdam1996Google Scholar). All members of the anion exchanger family consist of two domains, an N-terminal cytoplasmic domain and a 55-kDa C-terminal membrane domain. The membrane domain is highly conserved (70% overall identity), spans the lipid bilayer 12–14 times, and is able to mediate anion transport by itself (2Grinstein S. Ship S. Rothstein A. Biochim. Biophys. Acta. 1979; 507: 294-304Crossref Scopus (183) Google Scholar). Hydropathy analysis of AE membrane domains shows 10 strong peaks of hydrophobicity (1Reithmeier R.A.F. Chan S.L. Popov M. Konings W.N. Kaback H.R. Lolkema J.S. Handbook of Biological Physics. 2. Elsevier Science Publishers B.V., Amsterdam1996Google Scholar). Our goal is to map the topology of the human AE1 membrane domain, using substituted cysteine mutants and sulfhydryl-specific chemistry. Individual cysteine residues were introduced into a cysteineless form of AE1, called AE1C− that was previously shown to be fully functional (3Casey J.R. Ding Y. Kopito R.R. J. Biol. Chem. 1995; 270: 8521-8527Crossref PubMed Scopus (54) Google Scholar). Two sulfhydryl-directed compounds were used to characterize the introduced cysteine mutants as follows: (i) membrane-permeant biotin maleimide that covalently labels cysteine residues with a biotin group that is readily detected by streptavidin-biotin chemistry, and (ii) LYIA, a membrane-impermeant reagent, used to block the biotinylation of cysteines by the former compound. Introduced cysteine mutants were incubated with biotin maleimide, with or without a preincubation of the cells with LYIA. Biotinylation signals obtained for these mutants were interpreted as follows: no labeling with biotin maleimide implies localization to a hydrophobic membrane environment, biotinylation signal prevented by the preincubation with LYIA implies an extracellular localization, and biotinylation signal unaffected by LYIA implies an intracellular localization. Previously we have used this methodology to map the topology of the Ser643-Ser690 region of AE1 (4Tang X.-B. Fujinaga J. Kopito R. Casey J.R. J. Biol. Chem. 1998; 273: 22545-22553Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). As expected, AE1C− was not labeled by the sulfhydryl-directed compound biotin maleimide. In the 45 mutants that were made between residues Ser643 and Ser690, we found a stretch of 20 amino acids (Met664-Gln683) that were not labeled, implying that these residues formed an α-helical transmembrane segment (4Tang X.-B. Fujinaga J. Kopito R. Casey J.R. J. Biol. Chem. 1998; 273: 22545-22553Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). By contrast, introduced cysteine residues in the aqueous phase, either intracellular or extracellular, were labeled by biotin maleimide. A preincubation of the cells with the membrane-impermeant compound LYIA had little effect on the biotinylation of intracellular cysteines but impaired the biotinylation of most of the extracellular cysteine residues (4Tang X.-B. Fujinaga J. Kopito R. Casey J.R. J. Biol. Chem. 1998; 273: 22545-22553Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). In this article, we used the same approach to study the topology of the second half of the membrane domain of human AE1. Twenty-seven single cysteine mutants of human AE1 were constructed between residues Leu708 and Val911. Analysis of these mutants formed the basis for a topology model of human AE1 membrane domain (Fig. 1). To identify amino acids important for the anion transport function, we also measured the anion transport activity of the introduced cysteine mutants, using a whole cell fluorescence assay (5Lindsey A.E. Schneider K. Simmons D.M. Baron R. Lee B.S. Kopito R.R. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5278-5282Crossref PubMed Scopus (142) Google Scholar). Mutants G714C, S725C, and S731C had low activity, indicating that this region has a structurally and/or catalytically important role. Furthermore, we analyzed the residual activity after cell treatment with the cysteine-directed compound MTSES. Of the 20 mutants analyzed, the compound significantly inhibited only two, S852C and A858C. This region contains Lys851, which is the site of labeling by the anion transport inhibitors 4,4′-diisothiocyanodihydrostilbene-2,2′-disulfonate (6Okubo K. Kang D. Hamasaki N. Jennings M.L. J. Biol. Chem. 1994; 269: 1918-1926Abstract Full Text PDF PubMed Google Scholar) and pyridoxal phosphate (7Kawano Y. Okubo K. Tokunaga F. Miyata T. Iwanaga S. Hamasaki N. J. Biol. Chem. 1988; 263: 8232-8238Abstract Full Text PDF PubMed Google Scholar). Found here also is the naturally occurring P868L variant (Band 3 HT), which is characterized by an increased anion transport (8Salhany J.M. Schopfer L.M. Kay M.M. Gamble D.N. Lawrence C. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 11844-11848Crossref PubMed Scopus (19) Google Scholar,9Bruce L.J. Kay M.M.B. Lawrence C. Tanner M.J.A. Biochem. J. 1993; 293: 317-320Crossref PubMed Scopus (61) Google Scholar). Our finding highlights the importance of the Lys851-Pro868 region for the anion transport function. DMEM and all the tissue culture reagents were from Life Technologies, Inc. Biotin maleimide, biocytin hydrazide, BCECF-AM, DIDS, LYIA were from Molecular probes. MTSES and MTSEA were from Toronto Research Chemicals. Poly-l-lysine, nigericin,N-p-tosyl-l-lysine chloromethyl ketone,N-tosyl-l-phenylalanine chloromethyl ketone, sodium m-periodate, and bovine serum albumin were from Sigma. Phenylmethylsulfonyl fluoride was supplied by ICN. Protein A-Sepharose CL4B was from Amersham Pharmacia Biotech. The ECL immunoblot detection reagents, streptavidin-biotinylated horseradish peroxidase and anti-mouse IgG-conjugated horseradish peroxidase, were from Amersham Pharmacia Biotech. The PVDF membrane was from Millipore. To express human AE1 in eukaryotic cells, we used the plasmid pRBG4 (10Lee B.S. Gunn R.B. Kopito R.R. J. Biol. Chem. 1991; 266: 11448-11454Abstract Full Text PDF PubMed Google Scholar), in which an AccI-HindIII fragment containing the human AE1 cDNA (11Lux S.E. John K.M. Kopito R.R. Lodish H.F. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 9089-9093Crossref PubMed Scopus (279) Google Scholar) was cloned into the HindIII andEcoRI sites, using an AccI/EcoRI linker (3Casey J.R. Ding Y. Kopito R.R. J. Biol. Chem. 1995; 270: 8521-8527Crossref PubMed Scopus (54) Google Scholar). The vector pJRC9 coding for the wild-type human AE1 was previously mutated to give rise to the vector pJRC26, where all five cysteine codons were replaced by serine codons (3Casey J.R. Ding Y. Kopito R.R. J. Biol. Chem. 1995; 270: 8521-8527Crossref PubMed Scopus (54) Google Scholar). Individual introduced cysteine codons were cloned in this cysteineless background to yield mutants, each with a unique cysteine codon. Mutagenesis was performed using a megaprimer polymerase chain reaction methodology (12Sarkar G. Sommer S.S. BioTechniques. 1990; 8: 404-407PubMed Google Scholar,13Ho S.N. Hunt H.D. Horton R.M. Pullen J.K. Pease L.R. Gene (Amst.). 1989; 77: 51-59Crossref PubMed Scopus (6833) Google Scholar). 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. HEK cells were transiently transfected using the calcium phosphate technique, as described (4Tang X.-B. Fujinaga J. Kopito R. Casey J.R. J. Biol. Chem. 1998; 273: 22545-22553Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 14Ruetz S. Lindsey A.E. Ward C.L. Kopito R.R. J. Cell Biol. 1993; 121: 37-48Crossref PubMed Scopus (77) Google Scholar). Briefly, 1.5 × 106 cells were seeded in a 100-mm Petri dish, in 10 ml of DMEM containing 5% fetal bovine serum, 5% calf serum. After 4–6 h the cells were transfected with 890 μl of a precipitate made as described (4Tang X.-B. Fujinaga J. Kopito R. Casey J.R. J. Biol. Chem. 1998; 273: 22545-22553Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). The cells were incubated at 37 °C, in a 5% CO2 incubator, and harvested 48 h post-transfection. Assays proceeded as described previously (4Tang X.-B. Fujinaga J. Kopito R. Casey J.R. J. Biol. Chem. 1998; 273: 22545-22553Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). In brief, transfected HEK cells were washed with 10 ml of PBS (140 mm NaCl, 3 mm KCl, 6.5 mmNa2HPO4, 1.5 mmKH2PO4), pH 7.5, and allowed to lift up in 4 ml of PBS for 10 min at room temperature. The cells were collected, centrifuged (1000 rpm, 5 min), and resuspended in 2 ml of PBSCM (PBS supplemented with 0.1 mm CaCl2 and 1 mm MgCl2). Cell suspension (1 ml) was transferred into two tubes. One tube (labeled +) was supplemented with 50 μl of a LYIA solution (6.5 mg/ml in H2O). Samples were incubated for 10 min at room temperature. Biotin maleimide (10 μl of a 10.4 mg/ml solution in Me2SO) was then added, and cells were incubated for an additional 10 min, with occasional resuspension. The reaction was terminated by addition of 500 μl of 2% (v/v) 2-mercaptoethanol in DMEM (containing serum and antibiotics). After 5–10 min at room temperature, the cells were centrifuged and washed with 1 ml of PBSCM. The tube labeled minus was processed similarly, except that the preincubation step with the membrane-impermeant compound was omitted. The cells were lysed and AE1 immunoprecipitated, as described in a later section. Transfected HEK cells were washed twice with 10 ml of PBS and allowed to lift up in 5 ml of PBS for 10 min. The cells were centrifuged (2000 rpm, 5 min) and resuspended in 5 ml of PBSCM containing 10 mm NaIO4 and incubated for 30 min in the dark, at 4 °C, with occasional gentle resuspension of the cells. The cells were washed twice with PBSCM, resuspended in 1 ml of 100 mm sodium acetate, pH 5.5, and transferred to an Eppendorf tube. The suspension was supplemented with 250 μl of 10 mm biocytin hydrazide in 100 mmacetate buffer, pH 5.5. The biotinylation was carried on for 30 min, at 4 °C, in the dark, with occasional resuspension of the cells. Washing the cells twice with PBSCM stopped the reaction. The cells were lysed in IPB buffer, and AE1 was immunoprecipitated. Cell lysis and AE1 immunoprecipitation were performed as described (4Tang X.-B. Fujinaga J. Kopito R. Casey J.R. J. Biol. Chem. 1998; 273: 22545-22553Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). Briefly, the cells were lysed on ice in IPB buffer (1% (v/v) Nonidet P-40, 5 mm EDTA, 150 mm NaCl, 0.5% (w/v) sodium deoxycholate, 10 mm Tris-HCl, pH 7.5) supplemented with 2 mg/ml bovine serum albumin, 0.1 mm phenylmethylsulfonyl fluoride, 0.2 mm N-tosyl-l-phenylalanine chloromethyl ketone, and 0.1 mm N-p-tosyl-l-lysine chloromethyl ketone. After centrifugation, the supernatant was cleared up with 1.5 μl of non-immune serum and protein A beads. AE1 was immunoprecipitated overnight at 4 °C, using 1.5 μl of the anti-human AE1 antibody 1657 and protein A beads. Antibody 1657 was produced by injecting rabbits with a synthetic peptide corresponding to the last 13 amino acids of human AE1 (4Tang X.-B. Fujinaga J. Kopito R. Casey J.R. J. Biol. Chem. 1998; 273: 22545-22553Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). The beads were washed as described (4Tang X.-B. Fujinaga J. Kopito R. Casey J.R. J. Biol. Chem. 1998; 273: 22545-22553Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar) and resuspended in 30 μl of SDS-PAGE sample buffer (4% (w/v) SDS, 20% (v/v) glycerol, 2% (v/v) 2-mercaptoethanol, 0.001% (w/v) bromphenol blue, 130 mm Tris-HCl, pH 6.8). Samples were heated at 65 °C for 4 min and centrifuged at 9000 rpm for 1 min prior to SDS-PAGE. Samples were electrophoresed on 8.5% acrylamide gel (15Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207233) Google Scholar) and transferred to PVDF membranes (16Towbin H. Staehelin T. Gordon J. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 4350-4354Crossref PubMed Scopus (44924) Google Scholar). Biotinylated proteins were detected by incubating the membranes 1.5 h with 10 ml of TBST (0.1% (v/v) Tween 20, 137 mmNaCl, 20 mm Tris-HCl, pH 7.5), containing 0.5% (w/v) bovine serum albumin and 3 μl of streptavidin-biotinylated horseradish peroxidase. The membranes were washed with TBST and developed using ECL reagent followed by exposure to x-ray film. To probe blots for AE1 expression, the membranes were stripped by incubation for 10 min at 50 °C in 100 mm2-mercaptoethanol, 62.5 mm Tris-HCl, pH 6.8. The membranes were washed with TBST and then incubated overnight at 4 °C in 10 ml of TBST containing 5% (w/v) non-fat dry milk and 3 μl of the mouse monoclonal anti-human AE1 antibody, IVF12. The IVF12 antibody was a kind gift from Dr. Michael Jennings (17Jennings M.L. Anderson M.P. Monaghan R. J. Biol. Chem. 1986; 261: 9002-9010Abstract Full Text PDF PubMed Google Scholar). The membranes were washed and incubated 1.5 h with 10 ml of TBST containing 5% (w/v) non-fat dry milk and 3 μl of an antibody anti-mouse IgG, horseradish peroxidase conjugate. After washing, the membranes were developed using ECL reagents followed by exposure to x-ray film. Films were scanned with a Hewlett-Packard scanner ScanJet 4C calibrated with a Kodak gray scale. The quantification of the signals was performed using the program NIH Image 3.60. Biotinylation signals were normalized to the amount of AE1 present in each sample by dividing the pixels of biotinylated AE1 by the pixels of the corresponding immunoblots as follows: biotinylationnorm = pixels biotin signal/pixels AE1 immunoblot. Each mutant was normalized to the biotinylation level observed for the mutant Y555C, treated in parallel, and electrophoresed on the same gel as follows: relative biotinylation = (biotinylationnormmutant/biotinylationnormY555C) × 100%. LYIA accessibility was expressed as the ratio: LYIA accessibility = biotinylationnorm-LYIA/biotinylationnorm+LYIA. To measure the anion exchange activity of AE1, HEK cells were grown on 6 × 11-mm glass coverslips and transfected. Cells were rinsed with serum-free DMEM and loaded with the pH-sensitive dye, BCECF-AM (2 mm final concentration), for 30 min. The coverslip was then suspended in a fluorescence cuvette with perfusion capabilities. Experiments were performed in a Photon Technologies International RCR fluorimeter, using excitation wavelengths 440 and 503 nm and emission wavelength 520 nm. The cuvette was perfused alternately with Ringer's buffer (5 mm glucose, 5 mm potassium gluconate, 1 mm calcium gluconate, 1 mm MgSO4, 2.5 mm NaH2PO4, 25 mmNaHCO3, 10 mm Hepes, pH 7.4) containing 140 mm sodium chloride (chloride containing buffer) or 140 mm sodium gluconate (chloride-free buffer). Both buffers were bubbled continuously with air containing 5% carbon dioxide. At the end of the experiment a pH/fluorescence standard curve was determined using the nigericin high potassium pH clamp method (18Thomas J.A. Buchsbaum R.N. Zimniak A. Racker E. Biochemistry. 1979; 18: 2210-2218Crossref PubMed Scopus (1766) Google Scholar). For the inhibition assays, anion exchange activity was measured as described above. The same coverslip was then incubated with chloride-free Ringer's buffer containing 10 (or 20) mmMTSES or 5 mm MTSEA. To remove non-covalent methanethiosulfonates, cells were washed for 300–500 s with chloride-free Ringer's buffer and anion exchange was then assayed again. Transport rates were determined by linear regression of the initial rate of change of pH, using the Kaleidagraph program. The MTSES inhibition data were expressed as residual activity after MTSES treatment and were calculated as shown in Equation 1.Residual activity=transport rate after MTSES treatmenttransport rate before MTSES treatment×100%Equation 1 The membrane-permeant compound biotin maleimide reacts covalently with sulfhydryls to introduce a biotin group, which then can be readily detected on a blot, using streptavidin-biotinylated horseradish peroxidase followed by chemiluminescence. Fig.2 A represents a typical biotinylation profile of the AE1 mutants, and Fig. 2 B shows the amount of AE1 in each sample. HEK cells that express AE1C− had no biotinylation signal on the blot at the position of AE1 (Fig. 2 A), consistent with the fact that the compound only reacts with cysteine residues. All proteins are expressed to similar levels (Fig. 2 B), but the reactivity of each individual cysteine residue with biotin maleimide varies greatly. Fig.3 quantifies the biotinylation signal of each mutant relative to the Y555C mutant. Of the 27 mutants, 19 have no significant biotinylation signal (L708C, G714C, S725C, S762C, A767C, L775C, S781C, G790C, S801C, F806C, K814C, T830C, G838C, C843* (where the asterisk indicates endogenous cycteine found in wild-type AE1), S852C, A858C, T866C, R871C, and R879C). Previously we have established that introduced cysteine residues located in the bilayer region are not labeled with biotin maleimide (4Tang X.-B. Fujinaga J. Kopito R. Casey J.R. J. Biol. Chem. 1998; 273: 22545-22553Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar); therefore, we propose that these unlabeled amino acids localize to the bilayer region, as modeled in Fig. 1. Among the biotin-labeled mutants, defined as aqueous-accessible, the reactivity of each individual cysteine to biotin maleimide can vary by a factor of 10 (Fig. 3). Intracellular (Y892C) and extracellular (Y555C) control mutants were readily labeled by biotin maleimide, indicating that differences in reactivity were not related to their intra- or extracellular localization but rather to their exposure to the aqueous environment.Figure 3Biotinylation of introduced cysteine mutants by biotin maleimide. Each introduced cysteine mutants was treated with biotin maleimide as described. Biotin incorporation and AE1 expression level were quantified by densitometry. The biotinylation signals were normalized by the amount of AE1 present in each lane. In each experiment, the level of biotinylation was compared with that of the Y555C mutant, whose labeling was set to 100. Data represent mean of 4–5 independent experiments ± S.E.View Large Image Figure ViewerDownload (PPT) Biotinylation data for the Y904C mutant are not shown, because this mutant is not recognized by the mouse monoclonal antibody IVF12 we are using to normalize the biotinylation signals. This lack of reactivity is interesting, since it defines the epitope recognized by antibody IVF12, which previously was known only to be somewhere in the last 20 kDa of AE1 (17Jennings M.L. Anderson M.P. Monaghan R. J. Biol. Chem. 1986; 261: 9002-9010Abstract Full Text PDF PubMed Google Scholar). To determine the transmembrane topology of AE1-introduced cysteine residues, we measured the ability to block biotin maleimide labeling by prior incubation with the membrane-impermeant compound LYIA (4Tang X.-B. Fujinaga J. Kopito R. Casey J.R. J. Biol. Chem. 1998; 273: 22545-22553Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). In these experiments, biotin incorporation was measured for cells treated with and without LYIA, giving rise to "+" and "−" samples, respectively. After scanning, the signals were quantified and normalized for the amount of AE1 present in each lane, and the LYIA accessibility factor was calculated as the −/+ ratio. Preincubation with LYIA blocked biotinylation of extracellular cysteines, whereas labeling of intracellular cysteines was unaffected. For an extracellular cysteine the accessibility factor was high (the higher the factor, the more accessible to LYIA), whereas it was close to 1 for a cytosolic residue. Fig. 4 shows a typical LYIA accessibility experiment, and the mean of four independent experiments ± S.E. is shown in Fig. 5. In the particular experiment shown in Fig. 4, D821C had an uncharacteristically low labeling with biotin maleimide, but the average value for several experiments is seen in Fig. 5. Control mutants with a known cytosolic localization were S595C and K892C, the former being present in the cytosolic loop between TM 6 and 7, whereas the latter is present in the cytoplasmic C-terminal tail (19Lieberman D.M. Nattriss M. Reithmeier R.A.F. Biochim. Biophys. Acta. 1987; 903: 37-47Crossref PubMed Scopus (9) Google Scholar, 20Lieberman D.M. Reithmeier R.A.F. J. Biol. Chem. 1988; 263: 10022-10028Abstract Full Text PDF PubMed Google Scholar, 21Wainwright S.D. Mawby W.J. Tanner M.J.A. Biochem. J. 1990; 272: 265-272Crossref PubMed Scopus (20) Google Scholar). Control extracellular mutants were Y555C and S643C, which are located in the loop that carries the external chymotrypsin sites (22Jennings M.L. Seldin D.W. Giebisch G. The Kidney: Physiology and Pathophysiology. 2nd Ed. Raven Press, Ltd., New York1992: 503-535Google Scholar) and the loop that is glycosylated in AE1 (1Reithmeier R.A.F. Chan S.L. Popov M. Konings W.N. Kaback H.R. Lolkema J.S. Handbook of Biological Physics. 2. Elsevier Science Publishers B.V., Amsterdam1996Google Scholar), respectively. There was a clear difference between inside and outside residues; the LYIA accessibility factors for the inside controls were 1.4 and 1.5 for the K892C and S595C mutants, respectively (Fig. 5). The ratios obtained for the outside controls were always higher, typically around 5.0 for the Y555C mutant (Fig. 5), which is similar to values found previously for the S643C mutant (4Tang X.-B. Fujinaga J. Kopito R. Casey J.R. J. Biol. Chem. 1998; 273: 22545-22553Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). This shows that the assay distinguishes between an intracellular or extracellular localization of the introduced cysteine residue.Figure 5Accessibility of introduced cysteines 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 and AE1 expression level were quantified by densitometry. The biotinylation signals are normalized by the amount of AE1 present in each lane. For each mutant, the biotin incorporation obtained without LYIA prelabeling was divided by the biotin incorporation observed with the preincubation step (−/+ ratio). This ratio represents the relative accessibility of each introduced cysteine to LYIA. Data represent mean of 4–5 independent experiments ± S.E.View Large Image Figure ViewerDownload (PPT) Mutants S731C, G742C, S745C, A751C, and D821C had LYIA accessibility similar to the outside control Y555C (Figs. 4 and 5). In contrast, cysteine residues present in position C885* and K892C were relatively insensitive to LYIA treatment, as expected due to the intracellular localization of the C-terminal tail of AE1. On the basis of LYIA accessibility data, residues S731, G742, S745, A751, and D821 were mapped to an extracellular site (gray circles in Fig. 1), whereas C885* and K892C were mapped to intracellular sites and (hatched circles in Fig. 1). Anion exchange activity was measured by monitoring intracellular pH changes associated with Cl−/HCO3− exchange in a whole cell fluorescence assay (10Lee B.S. Gunn R.B. Kopito R.R. J. Biol. Chem. 1991; 266: 11448-11454Abstract Full Text PDF PubMed Google Scholar). The anion exchange activity of cells expressing human AE1 proteins was well above the background, typically 6.5 times higher than vector-transfected cells. The activity of AE1C− was around 0.2 pH/min. Anion exchange activity of each AE1 mutant was expressed relative to AE1C− (Fig.6). Six of the 27 mutated AE1s retained less than 40% of the activity of AE1C−: G714C, S725C, S731C, S762C, G790C, and F806C (Fig. 6). Three mutants with highly reduced transport activity were found in the region Gly714-Ser731, which may indicate that this region is important for anion transport function. The anion exchange assay measures transport across the plasma membrane, so that only proteins present in the plasma membrane are assayed. Impaired anion exchange activity may therefore result either from mutation of a functionally or structurally important amino acid, or intracellular retention of AE1 proteins. To distinguish between these possibilities, cell-surface expression was analyzed for the mutants that had reduced anion exchange activity. In the cell-surface expression assay, we measured the biotinylation of oxidized cell-surface carbohydrates by a membrane-impermeant compound, biocytin hydrazide (4Tang X.-B. Fujinaga J. Kopito R. Casey J.R. J. Biol. Chem. 1998; 273: 22545-22553Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). The level of biotin incorporation was normalized to the amount of AE1 present in each sample. Results were then expressed relative to AE1C−, as seen in Table I. As a control, we examined the processing of an aberrantly processed mutant of mouse AE1, shown previously to be retained intracellularly (14Ruetz S. Lindsey A.E. Ward C.L. Kopito R.R. J. Cell Biol. 1993; 121: 37-48Crossref PubMed Scopus (77) Google Scholar, 23Chernova M.N. Humphreys B.D. Robinson D.H. Stuart-Tilley A.K. Garcia A.M. Brosius F.C. Alper S.L. Biochim. Biophys. Acta. 1997; 1329: 111-123Crossref PubMed Scopus (26) Google Scholar), and no cell-surface expression could be detected (4Tang X.-B. Fujinaga J. Kopito R. Casey J.R. J. Biol. Chem. 1998; 273: 22545-22553Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). Of the six
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