From the
AE1 (Band 3), a ≅110-kDa integral plasma membrane protein,
facilitates the electroneutral movement of Cl
AE1 is the best studied member of a family of plasma membrane
anion exchange proteins that are widely distributed across mammalian
tissues. These proteins include AE1 (Band 3) expressed in erythrocytes
and kidney; AE2 found in kidney, lymphocytes, and stomach; and AE3
found in brain, heart, and retina
(1) . All three anion
exchangers function to exchange the physiological substrates
Cl
Anion exchangers are bifunctional proteins that have two clearly
separated domains: a highly conserved 55-kDa membrane domain that is
sufficient to mediate anion exchange and a more divergent
45-110-kDa cytoplasmic domain
(2) . In erythrocytes, the
cytoplasmic domain of AE1 is required to anchor the plasma membrane to
the spectrin-actin cytoskeleton via interaction with ankyrin
(3) . AE1 transports a wide range of small anions including
Cl
An important aspect of
AE1 structure is its oligomeric state. Early studies indicated that AE1
could be cross-linked covalently to dimers by oxidation of its
cytoplasmic cysteine residues
(5) . A wide range of techniques
including electron microscopy, radiation inactivation, spectroscopy,
cross-linking, and hydrodynamics have been used to examine the
oligomeric structure of AE1, with the conclusion that AE1 is a mixture
of homodimers and homotetramers in the erythrocyte membrane and in
detergent solution
(6) . Monomeric AE1 can be observed only
after protein denaturation with dimethylmaleic anhydride or SDS
(7) . Furthermore, the electron diffraction structure of AE1 has
2-fold symmetry
(8) , suggesting, together with the above data,
that the oligomeric unit of native AE1 in the membrane is minimally a
homodimer.
The affinity of ankyrin binding to AE1 is dependent upon
AE1 oligomeric structure. The population of AE1 released from
erythrocyte membranes by C
Human AE1 protein contains five cysteine residues at amino
acids 201, 317, 479, 843, and 885. As seen schematically in
Fig. 1
, two of these cysteines are found in the cytoplasmic
domain of AE1, and three are in the membrane domain. We have mutated
each of these cysteines to serine to examine their role in the function
of AE1.
One cysteine
residue of human AE1, Cys-843, has been shown to be
post-translationally modified by palmitylation in human erythrocytes
(15) . It has been suggested that palmitylation could serve to
localize a residue to the inner leaflet of the plasma membrane and
thereby stabilize a transmembrane helix. However, palmitylation of the
residue corresponding to Cys-843 in mouse AE1 was recently shown not to
be required for anion exchange activity when expressed in Xenopus
laevis oocytes
(16) .
In this study, we have examined
the role of native sulfhydryls in AE1 function and oligomeric
structure. Our data demonstrate that the sulfhydryls of AE1 have no
role that cannot be replaced by serine. The availability of such a
mutant will facilitate future structure-function studies of AE1 that
employ chemistry of engineered -SH groups.
Expression of AE1 and AE1C
As seen in Fig. 3( upper panel), the
elution profile of purified erythrocyte Band 3 protein has four peaks.
These peaks were previously identified as highly associated AE1
protein, which elutes at the void volume (5.03 ml); tetrameric AE1
(6.75 ml); dimeric AE1 (7.85 ml); and detergent micelles (10.35 ml)
(9) . Fig. 3( lower panel) shows that
both wild-type AE1 and AE1C
Fig. 4
shows the binding of
the 43-kDa ankyrin fragment to AE1 and AE1C
We have constructed and expressed a version of the plasma
membrane anion exchange protein, AE1, in which all five of its cysteine
residues have been replaced by serines. We show that this replacement
does not alter the protein's oligomeric structure, ankyrin
binding ability, or anion exchange activity. Serine was chosen for the
substitutions since this is the most conservative mutation possible;
cysteine and serine differ only in the sulfur atom of cysteine that is
oxygen in serine. Functionally, the two share the ability to form
hydrogen bonds. Because serine is similar to cysteine in structure,
Cys-Ser mutations would be expected to affect protein function
minimally. However, the two residues have differences: they have
different volumes (Cys, 109 Å
Our data show that the oligomeric state of
AE1C
Cysteines 201 and 317 are in the
cytoplasmic domain of AE1. The degree of conservation of the cysteines
provides insight into their expected role. Sequence conservation was
examined with an alignment of the amino acid sequences of nine anion
exchangers.
The first cysteine residue
of the membrane domain is Cys-479. It is at the COOH-terminal end of a
sequence (residues 460-479) that is almost fully conserved in all
nine anion exchanger sequences. This hydrophobic region is predicted to
form transmembrane segment 3. This segment also contains two glutamic
acid residues and two serine residues, suggesting that it could be a
pore-lining helix. In this case, the ability to form hydrogen bonds is
probably essential, so serine is sufficient.
Human AE1 in
erythrocytes is palmitylated at Cys-843, at a consensus palmitylation
site
(15) . Cysteine 843 is conserved in eight out of nine anion
exchanger sequences examined; it is not conserved in chicken AE1, in
which it is a valine residue. However, the full palmitylation consensus
is found only in some AE1 sequences. We have not determined whether or
not wild-type AE1 expressed in HEK cells is modified with palmitic
acid. However, mouse AE1 was found not to be palmitylated when
expressed in X. laevis oocytes
(16) . That anion
exchange activity is unaltered after mutation of Cys-843 indicates that
palmitylation is not essential for anion exchange function by AE1 and
is consistent with a recent report on the corresponding mutation in
mouse AE1 protein expressed in X. laevis oocytes
(16) .
In other palmitylated proteins, the role of palmitylation has been
examined by mutation of the site to serine or alanine, with varied
results. In the
The last cysteine of the membrane domain is Cys-885.
It is conserved in all nine anion exchanger sequences examined, except
in the AE3 sequences, in which it is an alanine residue. Although the
cysteine residue is highly conserved, our data show that serine is also
sufficient to maintain AE1 function.
The sulfhydryls of AE1 have
been implicated in formation of erythrocyte senescence antigen
(42) . Erythrocytes treated with oxidants developed binding
sites for autoantibodies. In vivo binding of senescence
antibodies results in targeting of the cell to the reticuloendothelial
system for degradation
(43) . The epitope is lost upon treatment
with the reducing agent dithiothreitol, suggesting that some
conformational change occurs upon oxidative cross-linking of AE1
cytoplasmic sulfhydryls and that this change is signalled across the
membrane to be recognized by extracellular antibodies. Since
AE1C
AE1 was one of the first membrane proteins to be studied
intensively, largely because it is easily isolated from erythrocytes,
where it constitutes 50% of the integral membrane protein. It was first
identified as the erythrocyte anion exchanger in 1974
(44) , and
its amino acid sequence was among the first determined for mammalian
membrane proteins
(45) . Yet, the greatest progress to be made
toward determining a structure for the protein has come only recently
from a relatively low resolution (20 Å) electron diffraction
structure
(8) . The development of well ordered two- or
three-dimensional crystals, which will improve the resolution of the
three-dimensional structure, is a slow difficult process, as evidenced
by the fact that only one membrane protein, the bacterial photoreaction
center, has had its structure determined at high resolution
(46) . Therefore, new approaches are required to attain greater
structural information and ultimately to validate the electron
diffraction or x-ray diffraction structure.
The availability of a
cysteineless AE1 molecule will allow a wide range of structural
experiments to be performed using cysteine-specific chemistry. Several
such approaches have already been used, primarily on bacterial membrane
proteins. The bacterial lactose transport protein, lac permease, was mutated free of its eight cysteine residues, which
yielded a functional transporter
(47) . After insertion of
cysteine residues at novel sites, the protein was probed
spectroscopically to determine which transmembrane helices were close
to one another in the folded molecule
(48) . Other methodologies
have been used to study cysteineless mutants of bacteriorhodopsin
(49, 50) and the bacterial chemosensory receptor
(51, 52) . In mammalian proteins, exposure of surfaces
to small, cysteine-reactive, aqueous probes has been used to determine
both topology and water-accessible regions, for example an ion
permeation channel
(53) .
The results presented here show
that the functions of the two domains of AE1 are not altered by
replacement of the protein's cysteine residues with serines. The
cytoplasmic domain retains the ability to bind the cytoskeleton via
ankyrin, and the membrane domain is competent to transport anions. The
cysteineless AE1 system should be useful for structure-function studies
of the protein.
We thank Roger Lo for assistance with anion exchange
assays and laboratory members for comments on the manuscript.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
and
HCO3- across the erythrocyte membrane and serves as the primary
attachment site for the erythrocyte spectrin-actin cytoskeleton. In
this investigation, we have characterized the role of native cysteines
in the function of AE1. We have constructed a mutant version of human
AE1 (AE1C
) in which all five cysteines of AE1 were
replaced with serines. Wild-type and AE1C
cDNAs were
expressed by transient transfection of human embryonic kidney cells.
Two of the mutated cysteines in AE1C
are in a region
involved in ankyrin binding, and ankyrin binding has previously been
shown to be sensitive to the oxidation state of these cysteines.
However, the K
values for ankyrin binding
by AE1 and AE1C
were indistinguishable, suggesting
that AE1 cysteines are not essential components of the ankyrin-binding
site. Using size exclusion chromatography, both AE1 and
AE1C
were found to associate as a mixture of dimers
and high molecular mass complexes. The rate of anion exchange by
AE1C
, as measured in a reconstituted microsome
sulfate transport assay, was indistinguishable from that by AE1 and was
inhibited by 4,4`-diisothiocyanodihydrostilbene-2,2`-disulfonate. We
conclude that the cysteines of AE1 are not required for the anion
exchange or cytoskeletal binding roles of the protein.
and HCO3- across the plasma membrane. This
anion exchange process contributes to the regulation of intracellular
pH, [Cl
], and volume and is involved in
proton, bicarbonate, and chloride secretion in some epithelial tissues.
, HCO3-, H
PO4-, and
SO42-
(4) . Sulfate is frequently used to measure anion
exchange activity because it is transported 10
times more
slowly than Cl
, allowing measurement of sulfate
transport on a longer time scale
(4) .
E
(
)
detergent is purely homodimeric, suggesting that the
tetrameric fraction remains bound to the cytoskeleton via ankyrin
binding
(9) . Furthermore, using time-resolved phosphorescence
anisotropy, two populations of AE1 were resolved, one smaller and more
mobile and the other larger and immobilized
(10) , which was
interpreted to mean that AE1 tetramers bind to ankyrin. Taken together,
it is likely that AE1 tetramers form the high affinity ankyrin-binding
site.
Figure 1:
Folding model of human AE1 protein
showing positions of cysteine residues. The dashed lines represent the lipid bilayer. The stars represent the five
cysteine residues at amino acids 201, 317, 479, 843, and 885. The Y on the protein represents the single chain of N-linked
carbohydrate on the protein. The site that was shown to be palmitylated
in erythrocyte Band 3 protein (15) is marked by the unfilled star.
The regions of the cytoplasmic domain that are involved in
ankyrin binding have been mapped to amino acids 190-203 and
317-359 because antibodies directed against these sequences block
ankyrin binding
(11, 12, 13) . Chemical
modification or oxidative cross-linking of Cys-201 and Cys-317 has been
shown to impair ankyrin binding
(11) , suggesting that they
either are directly essential for ankyrin binding or are in a region
that is essential for ankyrin binding. The ability to cross-link
Cys-201 and Cys-317 suggests that they are close to each other in the
protein's folded structure
(11) . Since they can also be
cross-linked intermolecularly between subunits of an AE1 dimer
(5) , Cys-201 and Cys-317 may be oriented along the dimeric
interface. Kidney AE1, which lacks the amino-terminal 79 amino acids of
AE1, was recently shown not to bind ankyrin
(14) . Furthermore,
the presence of ankyrin impaired the ability to phosphorylate two
tyrosine residues within this amino-terminal region
(12) .
Together, these data suggest that the NH-terminal region is
essential for ankyrin binding, although the region containing the two
cytoplasmic cysteine residues may also play a role.
Human AE1 Expression Construct
A human AE1 cDNA
(pHB3)
(17) on an AccI- HindIII fragment was
cloned into the HindIII and EcoRI sites of expression
vector pRBG4
(18) using an AccI- EcoRI linker.
The resulting human AE1 expression construct was called pJRC9 and
contains 27 bases of 5`-untranslated sequence and the full coding
sequence. Expression vector pRBG4 contains the cytotomegalovirus
immediate early gene promoter
(18) .
Site-directed Mutagenesis
Mutagenesis was
performed using a polymerase chain reaction megaprimer mutagenesis
strategy as described
(19, 20) . Polymerase chain
reaction primers were designed using the Primers program (Whitehead
Institute for Medical Research). Polymerase chain reaction was
performed using an ERICOMP thermal cycler and Vent DNA polymerase (New
England Biolabs Inc.). Sequences generated by polymerase chain reaction
were sequenced in their entirety to ensure that no polymerase errors
were introduced. The human cysteineless AE1 mutant was constructed by
cloning the mutant regions of each individual Cys-Ser mutant
consecutively into pJRC9. The construct coding for human cysteineless
AE1 cDNA in the pRBG4 expression vector was called pJRC26. The
cysteineless protein encoded by pJRC26 was called
AE1C.
Protein Expression and Membrane Isolation
Anion
exchangers were expressed by transient transfection of human embryonic
kidney (HEK) 293 cells
(21) as described previously
(22) , except that calcium phosphate-precipitated plasmid was
added at 12 µg of plasmid/150-mm tissue culture plate. Eight 150-mm
dishes of cells were harvested by scraping from the plate, and cells
were washed twice with 140 m
M NaCl, 10 m
M Tris/HCl,
pH 7.5. Cells were swollen in 10 ml of lysis buffer (1 m
M phenylmethylsulfonyl fluoride, 10 m
M Tris/HCl, pH 7.5)
for 20 min at 4 °C and then lysed by 50 strokes in a Dounce
homogenizer on ice. The lysate was diluted to 20 ml with lysis buffer
and made to 10% (w/v) sucrose by the addition of 50% (w/v) sucrose.
Nuclei were sedimented by centrifugation at 500 g for
5 min. The resulting supernatant was centrifuged at 4000
g for 5 min. The two pellets were pooled, extracted with 25 ml of
lysis buffer containing 10% (w/v) sucrose, and centrifuged at 4000
g for 5 min. This supernatant was pooled with the
previous supernatant and made to 25 m
M KCl and 10 m
M Hepes/NaOH, pH 7.4. The membrane extract was centrifuged at 35,000
rpm in a Beckman Ti-45 rotor for 50 min. This membrane pellet was
resuspended in 0.5 ml of 7% (w/v) sucrose, 25 m
M KCl, 1 m
M MgCl
, 15 m
M Hepes/NaOH, pH 7.4, by 10 gentle
strokes in a Dounce homogenizer. Microsomal membranes were used either
fresh or frozen as aliquots in liquid nitrogen. Erythrocyte ghost
membranes and purified Band 3 protein were isolated as described
(23) .
Size Exclusion HPLC
Membranes (1.4 mg of protein)
were suspended in 1 m
M dithiothreitol, 5 m
M sodium
phosphate, pH 8, and centrifuged for 30 min at 35,000 rpm in a Beckman
Ti-50 rotor. Membranes were solubilized by resuspension in 200 µl
of 2% (v/v) CE
, 0.1% (v/v) 1 m
M dithiothreitol, 5 m
M sodium phosphate, pH 8.0. After 20
min of incubation on ice, samples were centrifuged for 5 min at 45,000
rpm in a Beckman TLA-100 rotor. Supernatants were subjected to size
exclusion HPLC on a 0.75
30-cm TSK 4000SW column (Beckman
Instruments) eluted with 0.1
M sodium chloride, 0.1% (v/v)
C
E
, 5 m
M sodium phosphate, pH 7.0, as
described previously
(9) . Flow rate was 0.5 ml/min using a
Beckman 114M pump. The column was calibrated with protein standards
(Pharmacia Biotech Inc.) that were shown not to bind detergent
(24) . To assay the elution position of the anion exchange
protein, 30-s fractions of column eluate were collected, made to 1%
(w/v) SDS, and dot-blotted onto nitrocellulose membrane. Dot blots were
processed as immunoblots as described below and quantified using a
Scanjet Plus scanner (Hewlett-Packard Co.) and Image 1.44 software
(National Institutes of Health, Bethesda, MD).
Ankyrin Binding Assay
A truncated 43-kDa fragment
of ankyrin was expressed in Escherichia coli, purified
(25) , and I-labeled as described
(26) .
Radioiodinated ankyrin was diluted with unlabeled ankyrin to a specific
activity of 2
10
cpm/µg. Ankyrin binding assays
were performed as described
(14) . Briefly, frozen HEK cell
membranes were thawed, and aliquots (30 µg of protein) were added
to
I-labeled ankyrin in 150 µl of binding buffer (1
mg/ml bovine serum albumin, 90 m
M NaCl, 1 m
M EDTA,
0.5 m
M dithiothreitol, 0.02% (v/v) Tween 20, 10 m
M sodium phosphate, pH 7.5) and incubated for 2 h at room
temperature. The suspension was then loaded onto 0.25 ml of binding
buffer containing 20% (w/v) sucrose in a 0.4-ml microtest tube (4
43 mm; Eppendorf North America, Inc.) and centrifuged at 16,000
rpm in an SS34 rotor for 20 min. Tubes were then frozen in dry ice. The
bottom of the tube was cut off with a razor blade, and the pelleted
membranes were subjected to
-counting in a Beckman
-5500B
counter.
Sulfate Transport Assay
The reconstitution method
was as described.(
)
In a typical time course, 150
µg (
20 µl) of membrane protein was added to 500 µl of
2
reaction buffer (40 m
M sodium sulfate, 4 m
M MgCl
, 20 m
M Mes/KOH, pH 6.0), 40 µCi of
[
S]sulfuric acid (DuPont NEN), and 500 µl of
sonicated soybean phosphatidylcholine (Sigma) in water (20 mg/ml). To
fuse the lipid vesicles with the microsomes (forming larger, less leaky
vesicles), the mixture was frozen in liquid nitrogen, thawed at room
temperature, and sonicated twice for 1 s each time in a G112SP1G bath
sonicator (Laboratory Supplies Co., Hickesville, NY). Extravesicular
[
S]SO42- was removed on a 10-ml spin
column of Sephadex G-50 (fine) equilibrated with 1
reaction
buffer. The eluate from the column was placed into an ice-cold glass
tube. H
DIDS-treated samples also contained 160 µ
M H
DIDS (Molecular Probes, Inc.) in the reconstitution
mixture and in 1
reaction buffer. Transport was initiated by
placing the glass tube in a water bath at 25 °C. For each time
point, samples in triplicate were removed from the tube and pipetted
onto an ice-cold 1.5-ml column of Dowex 1 resin equilibrated with 0.1
M sucrose. The columns were rapidly washed with 2
0.75
ml of ice-cold 0.1
M sucrose. The radioactivity associated
with each eluate was then measured by scintillation counting.
Electrophoresis and
Immunoblotting
SDS-polyacrylamide gel electrophoresis was
performed
(28) , and proteins were transferred to nitrocellulose
as described
(29) . Immunoblots were blocked by incubation for
30 min in antibody buffer (5% (w/v) nonfat dry milk, 137 m
M NaCl, 20 m
M Tris/HCl, pH 7.6). Blots were incubated with
anti-AE1 antibody 5-297, an antipeptide antibody raised against a
synthetic peptide corresponding to the COOH-terminal 12 amino acids of
mouse Band 3 protein
(30) . Conditions were 10 ml/blot 1:2500
diluted antibody in antibody buffer for 2 h at room temperature,
followed by 10 ml/blot 1:2500 diluted donkey anti-rabbit IgG conjugated
to horseradish peroxidase (Amersham Corp.) incubated for 1 h at room
temperature. Blots were visualized using Renaissance chemiluminescent
reagent (DuPont NEN) and Hyperfilm (Amersham Corp.).
Molecular Biological Methods
Plasmids for
transfections were prepared using QIAGEN columns (QIAGEN Inc.). DNA
sequencing of plasmids was performed with Sequenase 2.0 (U. S.
Biochemical Corp.) following the manufacturer's instructions. All
other procedures followed standard protocols
(31) .
Analytical Methods
Protein concentrations were
determined with bicinchoninic acid reagent (Sigma) in the presence of
SDS. Amino acid sequences were aligned with Intelligenetics software.
-Fig. 2 shows
an immunoblot of wild-type AE1 and AE1C
. In the
experiment shown, equal amounts of transfected HEK cell membrane
protein were loaded, yet slightly more AE1 is observed in the lane
containing the cysteineless mutant. This higher expression level was
not observed consistently, so the mutations in AE1C
do not influence biosynthetic levels. The recombinant proteins
have a faster electrophoretic mobility than erythrocyte Band 3 because
erythrocyte AE1 is glycosylated with up to 10 kDa of heterogeneous
carbohydrate
(32) . Since AE1 expressed in HEK cells is retained
in the endoplasmic reticulum, it receives only core carbohydrate
(22) .
Oligomeric Structure of AE1 Proteins
One measure of AE1
native structure is its oligomeric state. Several lines of evidence
suggest that in the native membrane and in detergent solution, the
minimal oligomeric unit of AE1 is the homodimer
(6) . Size
exclusion HPLC has been shown to resolve Band 3 oligomers and has
revealed that the protein is a mixture of dimers and tetramers in
CE
solution
(9) . In size exclusion
HPLC experiments, the elution position of protein is monitored, usually
spectrophotometrically, and the Stokes radius of the eluted molecule
can be determined after appropriate calibration of the column. Since
AE1 in HEK cell membranes is expressed at
1% of the level of Band
3 in the erythrocyte, isolation of sufficient pure recombinant AE1 to
allow spectrophotometric detection of protein elution is not possible.
Therefore, to determine the oligomeric state of AE1 protein expressed
in HEK cells, we combined size exclusion HPLC with detection of eluted
protein by immunoblots. In the experiment shown in Fig. 3,
C
E
-solubilized HEK cell membranes were applied
to the size exclusion HPLC column. Fractions of the eluate were applied
to nitrocellulose by ``dot blotting,'' and the nitrocellulose
was processed as a conventional immunoblot. The amount of AE1 eluting
in each fraction was then quantitated relative to standard amounts of
AE1.
from HEK cell membranes
elute as two peaks; the first is at the void volume of the column and
therefore represents a large protein complex, while the second elutes
close to the position of dimeric erythrocyte AE1 protein. Although the
peak positions of AE1 and AE1C
do not coincide, the
elution position of AE1C
is not consistent with that
of the tetramer. In some preparations, wild-type AE1 eluted as a single
major peak at a position corresponding to dimeric protein; however, in
no preparation of AE1C
was the protein peak at the
void volume absent. The oligomeric state of AE1 expressed in HEK cells
is predominantly dimeric, but the protein also associates into very
large complexes. Oligomeric structure is not grossly altered in
AE1C
.
Figure 3:
Resolution of AE1 oligomers by size
exclusion HPLC. Upper panel, shown is the elution
profile monitored at 215 nm of purified erythrocyte AE1 protein (6
µg) applied to a TSK 4000SW column eluted with 0.1
M sodium chloride, 0.1% (v/v) CE
, 5 m
M sodium phosphate, pH 7.0. Lower panel, the
elution positions of AE1 (
) and AE1C
(
)
from C
E
-solubilized HEK cell membranes (20
µl) were determined by immunoblotting of the eluted protein. Shown
at the bottom are the elution positions of the following standard
proteins: thyroglobulin ( T; Stokes radius = 86
Å), ferritin ( F; 63 Å), catalase ( C; 52
Å), and aldolase ( A; 46 Å). The void volume
( V
) was determined from the elution position of
blue dextran 2000 (average M
= 2
10
), and the total volume ( V) was determined from
the elution position of 2-mercaptoethanol.
Ankyrin Binding to AE1 Proteins
Erythrocyte Band 3
protein (AE1) anchors the cytoskeleton to the membrane via interactions
between the cytoplasmic domain of Band 3 and ankyrin
(3) . Since
cysteines 201 and 317 reside in the predicted ankyrin-binding region of
AE1, one possible consequence of the mutation of AE1 cysteines to
serines is to alter the binding of ankyrin. To examine the role of
these cysteine residues in ankyrin binding, AE1 proteins were expressed
in HEK cells, and the binding of a radioiodinated truncated form of
ankyrin was measured. In this system, AE1 has an inside-out orientation
(22) such that ankyrin may interact with the cytoplasmic
surface of AE1 in the binding assay. Full-length ankyrin has a
molecular mass of 206 kDa and contains 24 repeats of a 33-amino acid
sequence that has affinity for the cytoplasmic domain of AE1
(33) . In the ankyrin binding assay used here, a 43-kDa
truncated ankyrin molecule, with 12 33-amino acid AE1-binding repeats,
was used because it is more readily overexpressed and purified, yet is
able to displace bound full-length ankyrin from erythrocyte membranes
with similar affinity
(25) . We have recently demonstrated that
AE1 expressed in HEK cells retains the ability to bind the 43-kDa
fragment of ankyrin
(14) .
. The
observed binding is saturable (Fig. 4 A) and from Scatchard
analysis (Fig. 4 B) resolves into high and low affinity
components. In Fig. 4 A, a single line can be drawn
through the binding data from the two protein samples. The Scatchard
plot
(34) of the same data shows that the data cluster around a
common line, indicating that the binding affinity for ankyrin (curve
slope = -1/ K
) and the
binding capacity of the membranes (from the x intercept) are
not altered by mutating the cysteines to serines. The high affinity
component has a K
of 14 n
M and
represents 76 pmol of binding sites/mg of membrane protein, while the
low affinity component has a K
of 106
n
M and represents 155 pmol of binding sites/mg of membrane
protein. The binding affinity observed in this assay is consistent with
the K
of 5-10 n
M found for
the interaction of native ankyrin with AE1 in erythrocyte membranes
(35, 36) . However, the density of AE1 protein in the
microsomal membranes is much lower than in the erythrocyte membrane,
which has 49 nmol of binding sites/mg of membrane protein
(37) or 644 times the density of binding sites. Together, these
data suggest that the cysteine residues of AE1 can be replaced by
serine without altering the affinity of AE1 for ankyrin. Anion Exchange Activity of AE1 and AE1C
-The role
of the membrane domain of AE1 is to transport anions across the plasma
membrane. Thus, the best criterion of whether mutation of AE1 cysteines
to serines interferes with the protein's native state is the
effect upon anion exchange activity. Anion exchange proteins will
transport a range of anions
(4) . The assay for anion exchange
activity makes use of the ability of AE1 to transport
SO42-.
In this assay, microsomal membranes were
reconstituted with exogenous soybean phosphatidylcholine and loaded
with [
S]SO42-. Exchange for extravesicular
nonradioactive SO42- resulted in a decrease in vesicular
[
S]SO42- as a function of time.
Figure 4:
Ankyrin binding by HEK cell membranes
expressing AE1 and AE1C proteins. A, net
ankyrin binding by membranes (30 µg of membrane protein/point)
containing wild-type AE1 protein (
) or AE1C
protein (
). The binding by an equivalent amount of
membranes from vector alone (pRBG4)-transfected HEK cells was
subtracted from each value. B, Scatchard plot (34) of the
binding of
I-labeled ankyrin to membranes isolated from
HEK cells transfected with wild-type AE1 (
), AE1C
(
), and the pRBG4 vector alone (
). The units of
bound and free ankyrin are picomoles of ankyrin/milligram of membrane
protein and picomoles/milliliter, respectively. Error bars that cannot
be seen are smaller than the data point.
Fig. 5
shows [S]SO42-/SO42-
exchange mediated by vesicles prepared from HEK cells transfected with
AE1 or AE1C
cDNA. From Fig. 5, the half-times
of [
S]SO42- efflux for AE1 and
AE1C
were both
2.5 min. Transport in AE1 and
AE1C
vesicles could be inhibited by H
DIDS
to a similar extent. To test whether the anion exchange assay was
sensitive to the number of functional AE1 molecules, microsomes were
prepared from HEK cells transfected with either pJRC9 (AE1 cDNA) or
pRBG4 (vector alone). Microsomes were mixed at a range of ratios, and
the initial rate of anion exchange was proportional to the amount of
AE1 protein present over the protein range in which transport
experiments were performed here.
(
)
In Fig. 5, the
two initial slopes (over the first 5 min) are not different from one
another within the error of the experiment (AE1, 3.1%
min
; AE1C
, 2.8%
min
), suggesting that the anion exchange activity of
AE1C
is not compromised by the cysteine mutations. In
control experiments, vesicles prepared from cells transfected with the
pRBG4 vector alone had anion exchange activity similar to that seen in
Fig. 5
for AE1 vesicles assayed in the presence of
H
DIDS.
Human embryonic kidney cells were
originally chosen for expression of anion exchange proteins because of
their very low level of background anion exchange activity
(18) ; therefore, the activity seen in Fig. 5is not due
to an endogenous anion exchanger. The slight increase in intravesicular
[
S]SO42- as a function of time was
observed reproducibly in H
DIDS-treated samples, although
the explanation for the observation is unclear. We conclude that the
anion exchange function of the AE1 membrane domain is not influenced by
mutation of the protein's cysteine residues to serines.
Figure 5:
[S]SO42-/SO42-
anion exchange assay of AE1 proteins. Microsomal membranes from
transfected HEK cells were reconstituted with exogenous
phosphatidylcholine in the presence of
[
S]SO42-. At each time point, triplicate
samples were removed and pipetted onto a Dowex 1 anion exchange column
to remove effluxed [
S]SO42-. Radioactivity
remaining associated with the vesicles eluted from the Dowex 1 columns
was measured by scintillation counting. Shown is the efflux of
intravesicular [
S]SO42- mediated by HEK
cell membranes expressing wild-type AE1 (200 µg of protein/time
course) ( A) and AE1C
(120 µg of
protein/time course) ( B) in the absence (
) and presence
(
) of 160 µ
M H
DIDS. The amount of
protein in each assay was normalized to contain the same amount of
anion exchange protein based on the intensity of staining on an
immunoblot probed with anti-AE1 antibody 5-297. Error bars that cannot
be seen are smaller than the data point. Initial time points represent
3-4
10
cpm.
; Ser, 89
Å
)
(38) , which could alter protein packing;
cysteine differs by its ability to form disulfide bonds, its reactivity
to certain chemical reagents, and its ionizability
(p K
of cysteine = 9.3)
(38) .
is indistinguishable from that of wild-type AE1
and that AE1C
is functional in ankyrin binding and
anion exchange. Together, these results suggest that the sulfhydryls of
AE1 are not required for proper folding during biosynthesis. Since Band
3 tetramers are selectively depleted from detergent extracts of
erythrocyte ghosts, it has been suggested that high affinity ankyrin
binding either induces Band 3 tetramerization or requires pre-existing
tetramers
(9) . Our data demonstrate high affinity ankyrin
binding to a predominantly dimeric population of AE1 molecules,
suggesting that pre-existing AE1 tetramers are not essential for high
affinity ankyrin binding. However, it is possible that ankyrin may
induce the formation of AE1 tetramers. Since HEK cells do not express
immunodetectable ankyrin,
(
)
the formation of AE1
tetramers in erythrocytes must be a consequence of interaction with the
erythrocyte cytoskeleton. The significance of the AE1 protein that
elutes from the void volume of the size exclusion chromatography column
is unclear; however, it does not appear to interfere with anion
exchange or ankyrin binding activity. Since the protein applied to the
size exclusion chromatography column is a solubilized preparation of
whole cell membranes, AE1 could be associating with any number of
proteins to cause the altered elution position. Since AE1 proteins are
retained in the endoplasmic reticulum of HEK cells, it is also possible
that some AE1 remains associated with a part of the biosynthetic
apparatus, for example with a molecular chaperone. The combination of
size exclusion HPLC and immunoblotting to examine oligomeric structure
in impure protein preparations and on a microscale may be useful for
other proteins that cannot be purified in sufficient quantity to allow
spectrophotometric detection.
(
)
The region surrounding cysteine 201
is very poorly conserved, and cysteine is found at the homologous
position only in mammalian AE1 proteins. However, the region around
Cys-317 is better conserved, and cysteine is found in all examined
anion exchange proteins, except in chicken AE1, in which the residue is
replaced with alanine, and in AE3 proteins, in which it is glycine.
Since chicken protein retains ankyrin binding, cysteine is not
essential at this position. Our result that serine was sufficient to
maintain ankyrin binding at these two positions is consistent with the
lack of conservation observed at Cys-217.
-adrenergic receptor, loss of
palmitylation results in impaired receptor-G protein coupling
(39) . However, in the closely related
-adrenergic receptor, mutation of the palmitylated
cysteine site has no effect on receptor function
(40) . The only
general role of palmitylation is to localize a protein segment to the
lipid bilayer, where the palmityl chain can partition into the bilayer
(41) .
has no sulfhydryls, it may be a useful tool to
study the role of sulfhydryls in the formation of senescence antigen.
E
, octaethylene
glycol monododecyl ether; HEK, human embryonic kidney; HPLC, high
pressure liquid chromatography; Mes,
2-( N-morpholino)ethanesulfonic acid; H
DIDS,
4,4`-diisothiocyanodihydrostilbene-2,2`-disulfonate.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.