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INTRODUCTION |
The human erythrocyte anion exchanger 1 (AE1),1 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 (1). 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 (2). Hydropathy analysis of AE membrane domains shows 10 strong peaks of hydrophobicity (1).
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 (3). 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 (4). 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
-helical
transmembrane segment (4). 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 (4).
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 (5). 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 (6) and pyridoxal
phosphate (7). Found here also is the naturally occurring P868L variant
(Band 3 HT), which is characterized by an increased anion transport (8,
9). Our finding highlights the importance of the
Lys851-Pro868 region for the anion transport
function.

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Fig. 1.
Topology model for the membrane domain of
human AE1 protein. Introduced cysteine mutants are labeled
according to their localization, as determined by accessibility to
biotin maleimide and LYIA: black, membrane; gray,
extracellular; hatched, intracellular. The proteolytic sites
are reviewed (1), and further sites are described (6, 34). Proteolytic
sites are as follows: Pa, papain; Pe, pepsin;
T, trypsin; C, chymotrypsin.
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EXPERIMENTAL PROCEDURES |
Materials--
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.
Plasmid Constructs and Site-directed Mutagenesis--
To express
human AE1 in eukaryotic cells, we used the plasmid pRBG4 (10), in which
an AccI-HindIII fragment containing the human AE1
cDNA (11) was cloned into the HindIII and
EcoRI sites, using an AccI/EcoRI
linker (3). 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 (3). 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 (12,
13). 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.
Transient Expression--
HEK cells were transiently transfected
using the calcium phosphate technique, as described (4, 14). 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 (4). The cells were incubated at 37 °C, in a 5%
CO2 incubator, and harvested 48 h
post-transfection.
Topology Assay--
Assays proceeded as described previously
(4). In brief, transfected HEK cells were washed with 10 ml of PBS (140 mM NaCl, 3 mM KCl, 6.5 mM
Na2HPO4, 1.5 mM
KH2PO4), 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.
Surface Processing Assay--
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 mM
acetate 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--
Cell lysis and AE1
immunoprecipitation were performed as described (4). 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 (4). The beads were washed as
described (4) 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.
SDS-PAGE and Immunoblotting--
Samples were electrophoresed on
8.5% acrylamide gel (15) and transferred to PVDF membranes (16).
Biotinylated proteins were detected by incubating the membranes
1.5 h with 10 ml of TBST (0.1% (v/v) Tween 20, 137 mM
NaCl, 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 mM
2-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 (17). 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.
Image Analysis--
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.
Anion Transport Assay--
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 mM
NaHCO3, 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 (18). 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) mM
MTSES 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.
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(Eq. 1)
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RESULTS |
Cysteine Accessibility to Biotin Maleimide--
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.
2A represents a typical
biotinylation profile of the AE1 mutants, and Fig. 2B 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. 2A), consistent with the fact that the
compound only reacts with cysteine residues. All proteins are expressed
to similar levels (Fig. 2B), 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 (4); 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.

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Fig. 2.
Labeling of introduced cysteine mutants of
human AE1, using biotin maleimide. HEK cells were transiently
transfected with cDNA coding for AE1 proteins. The cells were
harvested and incubated with 0.2 mM biotin maleimide, for
10 min at room temperature. After solubilization, AE1 was
immunoprecipitated with a polyclonal anti-AE1 antibody, subjected to
SDS-PAGE, and transferred to PVDF membrane. The data presented show one
representative experiment where all mutants were analyzed together,
except C843*. Cys843 and Cys885 are marked with
an asterisk to indicate that they are found in wild-type AE1
and were reintroduced into AE1C . A, biotin
incorporation was detected using streptavidin-biotinylated horseradish
peroxidase followed by ECL. B, the blot from A
was stripped and probed with the monoclonal anti-AE1 antibody IVF12 to
determine the amount of AE1 present in each lane.
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Fig. 3.
Biotinylation 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.
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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 (17).
Accessibility to LYIA--
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 (4). 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 (19-21). Control extracellular mutants
were Y555C and S643C, which are located in the loop that carries the
external chymotrypsin sites (22) and the loop that is glycosylated in
AE1 (1), 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 (4). This shows
that the assay distinguishes between an intracellular or extracellular
localization of the introduced cysteine residue.

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Fig. 4.
Representative data showing accessibility of
introduced cysteines to lucifer yellow iodoacetamide. HEK cells
were transiently transfected with the vector, pRBG4, or with cDNA
coding for AE1 proteins. The cells were harvested and separated into
two fractions. One was incubated with 0.5 mM LYIA for 10 min at room temperature. Both fractions were then incubated with 0.2 mM biotin maleimide for 10 min at room temperature. After
solubilization, AE1 was immunoprecipitated with a polyclonal anti-AE1
antibody, subjected to SDS-PAGE, and transferred to PVDF membrane. + and refer to the cells that were treated or not with LYIA,
respectively. A, biotin incorporation was detected using
streptavidin-biotinylated horseradish peroxidase followed by ECL.
B, the blot from A was stripped and probed with
the monoclonal anti-AE1 antibody IVF12 to determine the amount of AE1
present in each lane.
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Fig. 5.
Accessibility 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.
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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 of AE1 Mutants--
Anion exchange
activity was measured by monitoring intracellular pH changes associated
with Cl
/HCO3
exchange in
a whole cell fluorescence assay (10). 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.

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Fig. 6.
Anion exchange activity of the AE1
proteins. HEK cells transfected with cDNA coding for AE1
introduced cysteine mutants were grown on glass coverslips. The
coverslips were incubated with the pH-sensitive dye BCECF and then
suspended into a fluorescence cuvette. The cells were perfused with
chloride-free and chloride-containing Ringer's buffers. Anion exchange
activity was measured by following changes in intracellular pH mediated
by inward and outward movement of bicarbonate (4, 37). Anion exchange
activity was measured from the initial slope observed after buffer
changes. Absolute values for the rate of pH change during
alkalinization and acidification were averaged. Transport rates were
corrected for background activity of vector alone transfected cells and
expressed as a percentage of the rate of the AE1C
activity. Data represent mean of 1-3 experiments ± S.E.
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Surface Processing of AE1 Mutants--
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 (4). 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
(14, 23), and no cell-surface expression could be detected (4). Of the
six mutants that were characterized, only F806C was processed to a
lesser extent than AE1C
(73 ± 23%, Table I).
However the reduction in surface processing does not account entirely
for the loss of transport activity. Therefore the low activity of the
F806C mutant is due to a combination of reduced surface processing and
a negative structural change. Since mutants G714C, S725C, S731C, S762C,
and G790C were processed to the same extent as AE1C
,
these mutations impair transport function.
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Table I
Surface processing data
The degree of surface processing for AE1 mutants was determined by
measuring the ability to label cell-surface carbohydrates with biocytin
hydrazide (4). Data are expressed as percentage of the processing of
AE1C . Data are the average of three independent
experiments ± S.E.
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Effect of Sulfhydryl Reagents on Anion Exchange Activity--
The
ability of the membrane-impermeant, cysteine-directed compound MTSES to
inhibit transport was investigated for all functional mutants. Fig.
7 shows that MTSES had no influence on
anion exchange activity of T866C, whereas A858C was inhibited by the
reagent. Residual activity after MTSES treatment was measured for each mutant and is shown in Fig. 8. For most
of the mutants, the incubation of the cells with a 10 mM
concentration of MTSES had no effect on anion exchange activity.
However, S852C and A858C had 72 ± 6 and 56 ± 12% residual
transport activity, after MTSES treatment, respectively. The inhibition
of S852C and A858C was greater after 8 min incubation with 20 mM MTSES, approximately 50% inhibition (data not shown).
The observed level of inhibition is particularly significant, since an
8-min incubation with 0.3 mM DIDS, a potent anion exchange
inhibitor, resulted in 24% residual activity. To confirm the
methanethiosulfonate inhibition data, S852C and A858C were also treated
with 5 mM MTSEA; the residual activities were 78 and 112%,
respectively. In Fig. 1, these mutants are located close together in TM
12 and 13. Clearly, these results define this region as being involved
in anion transport.

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Fig. 7.
Measurement of the residual activity after
treatment with the cysteine-directed,
membrane-impermeant compound, MTSES. HEK cells
transfected with the cDNA coding for T866C (A) and A858C
(B) were grown on glass coverslips. The coverslips were
incubated with the pH-sensitive dye BCECF-AM and then suspended in a
fluorescence cuvette. The cells were perfused either with chloride-free
Ringer's buffer (white bar), chloride-containing Ringer's
buffer (gray bar), or chloride-free Ringer's buffer
containing 10 mM MTSES (black line). Anion
exchange rates were measured from the initial slopes observed after
buffer changes. Residual activity is expressed as a percentage of the
value obtained before MTSES treatment. Both alkalinization and
acidification slopes were taken into consideration to calculate the
residual activity.
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Fig. 8.
Residual anion exchange activity after
incubation of the cells with MTSES. HEK cells expressing AE1
mutants were assayed for anion exchange activity before and after
incubation for 8 min with 10 mM MTSES. Only covalent
effects were measured, because the cells were washed free of MTSES
prior to the second transport assay. Anion exchange rates were measured
from the initial slopes observed after buffer changes, during both the
alkalinization and acidification phases. Residual activity was
expressed as a percentage of the value obtained before MTSES treatment.
The y axis starts at 50% residual activity to emphasize the
difference between mutants. Data represent the mean of 1-3 independent
experiments ± S.E.
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DISCUSSION |
In this paper we have mapped the topology of the second half of
the membrane domain of human AE1, using introduced cysteine mutants and
sulfhydryl reagents. The ability to label with biotin maleimide defines
residues that are located in an aqueous environment, either intra- or
extracellular. Sites that could not be labeled by biotin maleimide were
defined as transmembrane sites, in the plane of the bilayer.
Accessibility to LYIA, a membrane-impermeant compound, defines
aqueous-accessible residues that are located on the extracellular side
of the transmembrane permeability barrier. LYIA may also access aqueous
sites within the plane of the lipid bilayer, if they are part of a
sufficiently large pore. On the basis of the data presented in this
article, we propose a new model for the topology of the membrane domain
of AE1 (Fig. 1). This model contains 13
-helical transmembrane
segments, and not 14, as seen in previous models (1). As well, it
contains one transmembrane region with a non-helical, extended conformation.
The cysteine-directed approach we have used to map the topology of the
membrane domain of AE1 was validated previously in our study of 45 consecutive single cysteine mutants of human AE1 (4). Among these
cysteine mutants, only a stretch of 20 amino acids (TM 8) was not
labeled with biotin maleimide. A similar approach to study topology of
the tetracycline transporter of Escherichia coli used
[14C]N-ethylmaleimide as cysteine labeling
reagent (24, 25). A stretch of 24 consecutive amino acids was not
labeled because it forms an
-helical transmembrane segment (25). As
well, among 27 mutants located in extramembranous loops, either
intracellular or extracellular, only one mutant could not be labeled
with [14C]N-ethylmaleimide (24). Similarly, in
human P-glycoprotein, introduced cysteines located within the plane of
the lipid bilayer or in its immediate vicinity (within two amino acids)
were not labeled using 200 µM biotin maleimide for 30 min; all cysteines in the aqueous phase were significantly labeled
(26). We conclude that in most cases the failure of a cysteine residue
to react with maleimide is due to the localization of that residue in
the plane of the lipid bilayer.
The region studied in this paper begins with Ile684
(following TM 8) and ends at Val911 (the C terminus). In a
previous study, we established that the region
Ile684-Ser690 is located in the cytoplasm (4).
The first three introduced cysteine mutants prepared for the current
study were L708C, G714C, and S725C. Since none of these could be
labeled by biotin maleimide, they are modeled as transmembrane regions.
S731C, G742C, S745C, and A751C define an aqueous-accessible region,
since all of these could be labeled with biotin maleimide. LYIA
accessibility of these mutants indicates an extracellular localization
of the Ala726-Ile761 region. Recently, Popov
et al. (27) showed that AE1 mutants with a single
N-glycosylation site in the loop
Ala726-Ile761 could be
N-glycosylated when expressed in an in vitro
transcription/translation system. Following the 12 + 14 rule described
for the glycosylation of an external loop by the
oligosaccharyltransferase (28), this extracellular aqueous loop appears
to contain the amino acids Trp723-Leu764 and was called the
"T-loop" by the authors (27). To accommodate the data concerning a
low ionic strength internal tryptic site in position Lys743
(17, 22), Popov et al. (27) proposed that this region was folded back into the membrane, with Lys743 close to the
cytosol. Our topology data agrees with Popov et al. (27)
concerning the extracellular localization of the loop Ala726-Ile761. These residues may form an
extracellular loop or the loop could enter the membrane. However, if it
does so, the loop must be in an open, aqueous-accessible structure,
large enough to allow access of LYIA.
Following the T-loop region are seven mutants that are inaccessible to
biotin maleimide, and which we have modeled as part of two
transmembrane segments. Following these transmembrane segments, three
introduced cysteine mutants were made in the
Asp807-His834 region. The
Asp807-His834 region is not predicted to be a
transmembrane region by conventional hydropathy analysis (1), since it
is composed of both hydrophobic and charged amino acids. However, the
data presented here showed that this region did not behave like an
aqueous loop, since only D821C could be labeled by biotin maleimide.
Since Asp821 is LYIA-accessible, it is on the extracellular
side of the permeability barrier. We do not have sufficient data to
determine the conformation of the protein in this region. The length of
the sequence (25 amino acids) is sufficiently long to permit a helical
transmembrane segment. If the region forms a helix, then
Asp821 would have to face an aqueous pore, outside the
permeability barrier, and Lys814 and Thr830
would face away from the pore, inaccessible to biotin maleimide and
LYIA. This region could also form an extended structure, as we have
shown in the model (Fig. 1). Consistent with an extended structure is
the presence of several prolines and glycines (high turn propensity
amino acids).
Our model of topology for the Asp807-His834
region needs to be reconciled with experiments using the monoclonal
antibody Bric132, which recognizes the
Phe813-Tyr824 sequence of human AE1 (21). The
authors have two lines of evidence for an intracellular localization of
this epitope as follows: (i) there is no direct and/or indirect
agglutination of intact human erythrocytes, using this antibody, and
(ii) Bric132 antibody can immunoprecipitate AE1 only in leaky
erythrocytes (29). Preparation of leaky erythrocytes exposes membranes
to low ionic strength medium, and AE1 conformation changes in low ionic
strength medium (17, 30). We propose that the Bric132 epitope is buried
in isotonic conditions, but in low ionic strength solutions, the region
becomes accessible either because it moves or some part of AE1 that
covers it moves out of the way. Consistent with this interpretation,
this region is not accessible to proteases under native conditions, but
after alkaline treatment proteolytic cleavage is observed (6).
Asp821 could form a salt bond that is disrupted under
altered conditions of ionic strength, leading to alternate
conformations. The D821C mutation could induce a similar change of
accessibility; however, the change must not be dramatic since the D821C
mutant has nearly full anion exchange function. An intracellular
localization of Asp821 has also been reported on the basis
of chemical modification studies (31), which underscores the need for
further study of this region.
We have modeled the Leu835-Arg879 region as two
TM, but our data cannot rule out other models. The C terminus of the
region is well defined by the ability to label Cys885 with
biotin maleimide. However, the inability to label any of seven
introduced cysteine residues in this region with biotin maleimide does
not allow us to define the other ends of the helices in this region.
The presence of such a long stretch of amino acids that cannot be
labeled by biotin maleimide suggests that the protein is compactly
folded in the membrane bilayer. Any extramembranous loop may be too
small to be labeled by biotin maleimide.
Our LYIA accessibility data indicate that the C terminus of AE1 is
cytosolic, consistent with previous results (19-21). Since C885*,
K892C, and Y904C could be labeled by biotin maleimide, these residues
are in an aqueous environment. The lack of labeling of the R879C mutant
suggests that the cytosolic C-terminal tail begins somewhere between
Asn880 and Cys885. Previous work using
carboxypeptidase Y digestion and epitope mapping was unable to define
the length of the exposed C-terminal tail (19, 21, 32).
Results obtained by proteolytic digestion of erythrocytes membranes
pretreated with 100 mM NaOH (6, 33, 34) fit remarkably well
with our model (Fig. 1). When placed on our model, all of the
proteolytic sites are found in extramembranous regions, except those
surrounding Asp821. The proteolysis data suggest that
transmembrane segments are insensitive to proteolysis. However, we
propose that the region surrounding Asp821 forms an
extended structure that may be removed from the membrane upon
alkaline treatment. Alkali treatment is known to disrupt protein-protein contacts and denature proteins (33).
We analyzed the effect of the cysteine-directed compound MTSES on the
anion exchange activity of our introduced cysteine mutants. MTSES and
related methanethiosulfonate compounds have been used to probe the
function of introduced cysteine residues in ion channel proteins (35,
36). We infer that inhibition by MTSES results from steric blockage of
a small anion access channel, at the site of the cysteine residue. Our
results identified only two of 21 active mutants, S852C and A858C,
whose anion exchange activity was sensitive to sulfhydryl reagents.
Therefore we interpret S852C and A858C mutations to reside in a
spatially restricted area, possibly part of the anion translocation
channel. The membrane impermeability of MTSES indicates that S852C and
A858C lie outside the transmembrane permeability barrier. These mutants
are located within the last two TM of the anion exchanger, thereby
defining this region as important for the anion transport. This region also contains Lys851, which is the lysine residue that
reacts with DIDS (6) and pyridoxal phosphate (7), two potent inhibitors
of the anion exchange function. As well, this region contains the P868L
mutation found in naturally occurring Band 3 HT variants, which are
characterized by an increased anion transport (8, 9). Taken together, these results show the importance of the
Lys851-Pro868 region for anion transport function.
In this study we have added 27 topological constraints to models of the
C-terminal region of AE1. The lack of labeling with biotin maleimide
has placed limits on the boundaries of TM 9-11. We have confirmed the
presence of an extracellular localization for the
Ala726-Ile761 sequence, consistent with recent
results observed by glycosylation scanning mutagenesis (27). Our data
for the first time define the length of the exposed cytoplasmic
C-terminal tail as between Asn880 and Gln884.
The inability to label the Leu835-Arg879 region
with biotin maleimide indicates that the region has a compact
structure, whose topology cannot be clearly modelled by our data.
However, the presence of two MTSES-sensitive sites in the
Leu835-Arg879 region suggests that these sites
line the translocation channel of AE1. The most unusual region analyzed
was the Asp807-His834 sequence, for which we
could not assign a clear topology. In summary, introduced cysteine
mutants and chemical modification have provided evidence for the
topology model proposed in Fig. 1. Absolute determination of the
topology of AE1 awaits a crystal structure of the protein.