(Received for publication, May 10, 1995; and in revised form, June 9, 1995)
From the
Human erythroid anion exchanger AE1 (Band 3) was expressed in the yeast Saccharomyces cerevisiae under the control of the constitutive promoter and transcriptional terminator of the yeast phosphoglycerate kinase gene. AE1 expression in stable yeast transformants was estimated to be approximately 0.7 mg AE1 per liter. Density gradient sedimentation analysis indicated that the AE1 protein was associated with a membrane fraction distinct from plasma membrane, most likely the endoplasmic reticulum. AE1 protein was solubilized from yeast membranes with lysophosphatidyl choline, and the protein, tagged with six histidines at its amino terminus, was purified to 35% homogeneity by metal chelation affinity chromatography. Size-exclusion chromatography in the presence of octaethylene glycol monododecyl ether indicated that the solubilized yeast-expressed AE1, like endogenous erythroid AE1, eluted at a stokes radius of 77 Å, consistent with a dimeric oligomeric state. Binding of partially purified yeast-expressed AE1 to 4-acetamido-4`-isothiocyanostilbene2,2`-disulfonate resin was competitive with the transportable substrate chloride but not the nontransported anion citrate, suggesting that the structure of the anion binding site is preserved. The specific activity of sulfate transport by partially purified yeast AE1 was determined in proteoliposomes to be similar to that of authentic AE1 purified from erythrocyte membranes. These data show that this expression system has the capacity to produce functional mammalian plasma membrane anion exchangers at levels sufficient for biochemical and biophysical analysis.
AE1 (Band 3) belongs to a family of anion exchange proteins that
facilitate the movement of Cl and
HCO
, across the plasma
membrane(1) . Plasma membrane anion-exchange proteins are
widely expressed among mammalian tissues where they participate in the
regulation of intracellular pH and volume(1, 2) .
Three anion-exchanger isoforms have been identified, cloned, and
sequenced: AE1, found in erythrocytes and kidney(3) ; AE2,
found in kidney, stomach, and lymphocytes(4) ; AE3, found in
the brain, retina, and heart(5) . All of these anion-exchange
proteins contain two domains. The highly conserved (70% identity)
membrane domain of approximately 55 kDa spans the bilayer 12-14
times (6) and is responsible for anion-exchange activity (7) . The cytoplasmic domain of 45-110 kDa is more
divergent. In erythrocyte AE1, the cytoplasmic domain anchors the
cytoskeleton to the plasma membranes through interactions with
ankyrin(8) .
AE1 has served as a model for understanding the structure and function of membrane proteins because of its high abundance in the erythrocyte membrane, where it constitutes nearly 50% of the total integral membrane protein(9) . This allowed the early identification of the protein's role in the erythrocyte(10) . Subsequently the protein has been extensively studied using a wide range of methods including nuclear magnetic resonance spectroscopy(11) , chemical modification of specific residues(12) , and electron diffraction(13) . The ability to generate specific point mutant and chimeric proteins has provided important insight into the function of many proteins. However, to study recombinant proteins biochemically and biophysically requires protein expression systems that produce sufficient amounts of functional protein. The availability of suitable overexpression systems is a limiting factor for many mammalian membrane proteins, including AE1. Soluble proteins are readily expressed in bacterial cells, but eukaryotic membrane proteins are not functionally expressed in bacteria because bacteria do not usually target eukaryotic membrane proteins for insertion into the membrane. Eukaryotic membrane proteins expressed in bacteria are often toxic to the cell, resulting in low levels of expression(14, 15) . Furthermore, the thickness of the bacterial inner membrane (25 Å) (16) does not match the hydrophobic region of eukaryotic membrane proteins (30 Å)(17) .
Recombinant plasma membrane anion exchange
proteins have previously been expressed by transient transfection of
human embryonic kidney 293 cells (HEK293)()(18) ,
COS cells(19) , and by cRNA injection of Xenopus laevis oocytes(20, 21) . AE2, expressed in insect cells
using the baculovirus expression system, yielded an undetermined amount
of protein that had a low level of anion-exchange
activity(22) . Insufficient amounts of protein are produced by
these expression systems to permit protein purification and
characterization. In this study, we report the establishment of a yeast
expression system that produces high levels of human AE1 protein. The
yeast-expressed AE1 protein was solubilized in detergent, partially
purified, and reconstituted into proteoliposomes. The reconstituted
protein is structurally and functionally indistinguishable from the
native erythroid protein. A preliminary version of this work has been
published as an abstract(23) .
Figure 1: Yeast expression construct for human AE1. The BamHI fragment of human AE1 (solidblack, with arrow indicating coding direction) was cloned into the expression construct along with a linker encoding a six-histidine sequence. The nucleotide sequence shown indicates the region immediately 5` to the coding sequence and the first 23 codons of the protein. The gray regions represent the 5`- and 3`-untranslated regions from the yeast phosphoglycerate kinase gene. Hashmarked region and solidblack regions, respectively, represent the region required for bacterial propagation (from pBR322) and the sequence coding for the yeast 2 µm origin of replication and leu2 gene. C, ClaI; H3, HindIII; R1, EcoRI.
No
immunoreactive material was observed in membranes prepared from yeast
transformed with vector pMA91 alone (Fig. 2, lane13). The AE1 protein encoded by pJRC16 migrated on
SDS-PAGE with an apparent molecular mass of 75 kDa, which is in good
agreement with the predicted molecular mass of 80 kDa. The expression
level of AE1 in BJ1991(pJRC16) membranes was determined by
densitometric comparison of immunoblots of AE1 from yeast and
erythrocyte membranes (Fig. 2). From the known abundance of AE1
(25% of erythrocyte membrane protein)(9) , yeast-expressed
protein (YAE1) constitutes approximately 1.5% of the membrane protein
in the yeast. Since 1 liter of yeast grown to A = 1.0 yields approximately 44 mg of membrane protein, the
expression level is about 0.7 mg of AE1/liter of culture.
Figure 2: Quantification of AE1 expression in yeast membranes, relative to erythrocyte membranes. Erythrocyte membranes, BJ1991(pJRC16) or BJ1991 membranes were solubilized in SDS and subjected to polyacrylamide gel electrophoresis on a 10% acrylamide gel. The protein was transferred to nitrocellulose, and the immunoblot was processed with anti-AE1 antibody 5-297. Lanes1-6 contain, respectively, 1.0, 0.60, 0.36, 0.22, 0.13, and 0.08 µg of erythrocyte membrane protein. Lanes7-12 contain, respectively, 32, 19, 12, 7, 4, and 2.5 µg of BJ199(pJRC16) membrane protein. Lane13 contains 32 µg of BJ1991 yeast membrane protein.
Erythrocyte AE1 is heterogeneously glycosylated with up to 10 kDa of carbohydrate(48) ; however, this carbohydrate is not required for anion-exchange activity of the protein(49) . Some mammalian membrane proteins functionally expressed in yeast are not glycosylated(50, 51) . To determine whether the yeast-expressed AE1 protein was glycosylated, BJ1991(pJRC16) membranes and human erythrocyte membranes were treated extensively with the enzyme N-glycosidase F, which cleaves the entire carbohydrate structure at the asparagine linkage of N-linked carbohydrates (52) (data not shown). The electrophoretic mobility of YAE1 was unaltered by N-glycosidase F treatment under conditions that resulted in complete deglycosylation of a parallel sample of erythrocyte AE1, suggesting that AE1 expressed in yeast is not glycosylated.
The subcellular location of yeast-expressed AE1 was analyzed by density gradient sedimentation analysis on linear 20-53% sucrose gradients. Fractions from the gradient were analyzed by immunoblotting for AE1 and the yeast plasma membrane marker PMA1, using specific antisera (Fig. 3). YAE1 and PMA1 were separated on the gradient into two distinct, well defined peaks at 48% sucrose and 51% sucrose, respectively. Although this analysis does not exclude the possibility that some AE1 may be present in the plasma membrane, the data suggest that most AE1 is present in other membranes, most likely endoplasmic reticulum.
Figure 3: Separation of yeast membrane microsomes by sucrose gradient centrifugation. Yeast membranes (0.3 ml) were applied to an 11-ml 20% (top) to 53% (bottom) sucrose gradient in a Beckman SW41 rotor and centrifuged for 18 h at 4 °C, 30,000 rpm. Fractions (0.5 ml) were removed from the top of the gradient and analyzed for protein concentration (closedcircles) and sucrose concentration (opensquares). Samples of each fraction (25 µl) were subjected to SDS-polyacrylamide gel electrophoresis on duplicate 10% acrylamide gels, blotted to nitrocellulose, and probed with anti-AE1 antibody 5-297 and an anti-PMA1 antibody (kindly provided by C. Slayman). Insets are the immunoblots stained with these antibodies. Only fractions 13-23 are shown, since no other fractions contained immunoreactive material.
To assess the capacity for
yeast-expressed AE1 to conduct anion exchange, yeast membranes were
reconstituted into vesicles with exogenous lipid(40) . The
vesicles were loaded with 20 mM
[S]SO
, and
assayed for anion exchange by measuring the efflux of radioactive
sulfate in exchange for extravesicular sulfate (40) (Fig. 4). Since AE1 was reconstituted with a large
excess of phospholipid, approximately 80% of sealed vesicles lack an
anion exchanger. Consequently, a large fraction of
[
S]SO
cannot be
transported, and transport ceases after approximately 20% of the total
[
S]SO
has left
the vesicles. Vesicles prepared from BJ1991(pMA91) (vector
alone-transformed) yeast membranes did not mediate measurable
[
S]SO
efflux,
indicating that yeast membrane vesicles provide a suitable null
background for the measurement of anion-exchange activity. By contrast,
vesicles prepared from AE1 expressing BJ1991(pJRC16) yeast mediated
[
S]SO
exchange,
with an initial rate of 6.1
10
cpm
mg of
protein
min
. This
[
S]SO
flux was
inhibited to background levels by 100 µM H
DIDS, a well-characterized inhibitor of anion
exchange in erythrocytes(53) . We conclude that AE1 protein
expressed in yeast is able to carry out anion exchange.
Figure 4:
[S]SO
/SO
anion-exchange assay of reconstituted yeast membranes. Membranes from
vector alone-transformed BJ1991(pMA91) (opencircles)
yeast and YAE1-expressing BJ1991(pJRC16) (opensquares) yeast were reconstituted with exogenous
phosphatidylcholine in the presence of 20 mM
[
S]SO
. After
initiating the anion-exchange assay, at each time point triplicate
samples were removed and pipetted onto a Dowex 1 anion-exchange column,
to remove extravesicular
[
S]SO
.
Radioactivity remaining associated with the vesicles eluted from the
Dowex 1 columns was measured by scintillation counting. BJ1991(pJRC16)
membranes were also assayed in the presence of 100 µM H
DIDS (filledsquares). Data
represent the mean of two independent experiments, each performed in
triplicate.
Figure 5:
Detergent solubilization of
yeast-expressed AE1. BJ1991(pJRC16) membranes in 1 mM EDTA,
0.1% (v/v) 2-mercaptoethanol, 10 mM Tris, pH 8.0, were
resuspended with 2 volumes of this buffer containing: lane
1, no addition; lane2, 2%
CE
; lane3, 2%
C
E
and 4 M urea; lane4, 2% dodecyl-
-D-maltopyranoside; lane5, 2% Mega-9 detergent; lane6, 2%
deoxycholate; lane7, 2% LPC; lane8, 2% SDS; lane9, 2% zwittergent
3-10. Samples were incubated on ice for 10 min and centrifuged
for 4 min at 4 °C, 100,000
g in a Beckman TLA100.2
rotor. The supernatant from each sample was collected, and an equal
fraction was electrophoresed on a 10% acrylamide gel, blotted to
nitrocellulose, and processed as an immunoblot with anti-AE1 antibody
5-297.
To purify AE1 from
yeast, LPC-solubilized yeast membranes were bound to a
Ni-loaded metal chelating resin and eluted with 0.25 M imidazole. Immobilized nickel has an affinity for a sequence
of six histidine residues(54) , such as those introduced near
the amino terminus of YAE1. Approximately 100-fold purification was
achieved following a single cycle of binding and elution from the
column; this was increased to 145-fold after a second cycle (Table 1). Analysis of the fractions on a Coomassie Blue-stained
SDS-polyacrylamide gel (Fig. 6A) indicates that AE1 is
the major protein, constituting 35% of the total protein. This band is
strongly reactive in immunoblots with an AE1 antibody, confirming its
identity as AE1 (Fig. 6B). The major high molecular
weight band observed in these immunoblots is probably dimeric AE1 as it
copurified with YAE1 and, like erythrocyte AE1, increased in amount as
the samples aged(35) .
Figure 6: Purification of yeast-expressed AE1. Membranes from BJ1991(pJRC16) yeast were solubilized and centrifuged, and the supernatant was collected. The supernatant was applied to His-Bind resin; the column was washed with nickel column buffer (10% glycerol, 100 mM sodium chloride, 5 mM imidazole, 0.2% (w/v) LPC, 10 mM sodium phosphate, pH 8.0) and eluted with this buffer containing 250 mM imidazole. The pooled peak of eluting protein was dialyzed against nickel column buffer, rechromatographed on the nickel column, and eluted with nickel column buffer containing 300 mM imidazole. Protein samples were resolved on 7.5% acrylamide gels that were either stained with Coomassie Blue (A) or transferred to nitrocellulose and probed with anti-AE1 antibody 5-297 (B). PanelA, lane1, 20 µg of yeast membrane protein; lane2, 20 µg of the supernatant after solubilization; lane3, 20-µg peak of flow-through fraction from the column; lane4, 2 µg of protein from the peak eluted with 250 mM imidazole elution buffer; lane5, 1-µg peak fraction from second column. PanelB, as in A, but 1 µg of protein/lane.
The oligomeric state of partially
purified, LPC-solublilized YAE1 was determined by size exclusion HPLC
in 0.1% (v/v) CE
and immunological
detection(34) . The elution profile of purified erythrocyte AE1 (Fig. 7, upperpanel) has four peaks, which
have been previously identified as highly associated protein, eluting
at the void volume (5.0 ml), tetrameric AE1 (6.8 ml), dimeric AE1 (7.9
ml), and detergent micelles (10.4 ml)(35) . The majority of
yeast AE1 (Fig. 7, lowerpanel) eluted in two
major peaks, at 6.6 and 8.1 ml, which correspond almost precisely to
the tetramer and dimers of erythroid AE1, respectively. Moreover, since
the NH
-terminal domain of YAE1 lacks the first 182 amino
acid residues of the erythrocyte protein, these results suggest that
the truncated region contributes little to the hydrodynamic behavior of
full-length AE1 in detergent solution.
Figure 7:
Size exclusion HPLC of yeast-expressed and
erythroid AE1 on a TSK 4000SW column. Upperpanel,
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 mM sodium phosphate, pH 7.0. Lowerpanel, a
40-µl sample (8 µg of protein) of YAE1, purified by nickel
affinity chromatography, was chromatographed as above. The elution
position of YAE1 was determined by immunoblotting of the eluted
fractions. Standard proteins were as follows: T, thyroglobulin (R
= 86 Å); F,
ferritin (63 Å); C, catalase (52 Å); A,
aldolase (46 Å). The void volume, V
, was determined from the elution
position of blue dextran 2000 (average molecular weight 2
10
) and the total volume, V
,
was determined from the elution position of
2-mercaptoethanol.
Binding of
detergent-solubilized, partially purified yeast AE1 to immobilized
disulfonic stilbene inhibitor, SITS (38) was used to assess the
structural integrity of the anion binding site (Fig. 8). Since
binding of transportable anions to AE1 in erythrocytes is competitive
with disulfonic stilbenes(53) , the SITSAffi-Gel resin
was incubated with partially purified YAE1 in the presence of either
citrate, a nontransportable anion, or chloride, a transportable anion.
In the presence of citrate, almost all of the AE1 bound to the
SITS-resin and could be eluted with the structurally related inhibitor,
DNDS. By contrast, the presence of chloride significantly attenuated
the binding of AE1 to the resin. These results suggest that the
stilbene disulfonate and anion binding sites of yeast-expressed AE1 are
preserved, supporting the conclusion that the polypeptide is correctly
folded.
Figure 8:
Binding of purified YAE1 to
SITSAffi-Gel resin. Purified yeast AE1 was incubated in the
presence of SITS
Affi-Gel and 0.1% (v/v)
C
E
, 200 mM sodium citrate, pH 8.0 (A), or 0.1% (v/v) C
E
, 100
mM sodium citrate, 100 mM sodium chloride, pH 8.0 (B). The resin was pelleted, washed, and eluted sequentially
with 1 mM DNDS and gel sample buffer. An equal fraction of
each sample was loaded on a 7.5% acrylamide gel and processed as an
immunoblot with anti-AE1 antibody 5-297. Lane1, unbound fraction; lane2,
DNDS-eluted fraction; lane3, sample buffer eluted
fraction.
Figure 9:
[S]SO
/SO
anion-exchange assay of purified, reconstituted AE1 protein. Purified
yeast (10 µg of protein) (squares) or erythroid (5 µg
of protein) (circles) AE1 were reconstituted into soy bean
asolectin liposomes in the presence (filled) or absence (open) of 200 µM DIDS. Vesicles contained 20
mM
[
S]SO
. At each
time point, triplicate samples were removed and pipetted onto a Dowex 1
anion exchange column, to remove extravesicular
[
S]SO
.
Radioactivity remaining associated with the vesicles eluted from the
Dowex 1 columns was measured by scintillation counting. Data represent
the mean of three independent experiments each performed in
triplicate.
This study demonstrates that the human erythrocyte anion
exchanger AE1 can be expressed at high levels in the yeast S.
cerevisiae. Several parameters indicate that the yeast-expressed
AE1 protein is similar to the endogenous erythrocyte anion exchanger in
native membranes. Yeast AE1, like erythroid AE1, had a Stokes radius in
CE
solution consistent with a dimeric
structure and showed anion-specific binding to inhibitor resin. Most
significantly, partially purified AE1 from yeast, reconstituted into
proteoliposomes, mediated sulfate anion exchange with specific activity
similar to erythroid AE1. Together, these data indicate that AE1 has
been functionally expressed in S. cerevisiae and that this
protein has the structural and functional characteristics of native AE1
from erythrocytes. To our knowledge, this is the first example of
purification and functional reconstitution of a mammalian plasma
membrane transport protein overexpressed in yeast.
The production of
sufficient amounts of purified recombinant membrane proteins is a
limiting factor in the study of membrane protein structure in
biochemistry. Other popular expression systems include stable or
transient expression in mammalian cells, Xenopus oocytes, and
expression in baculovirus-infected insect cells. However, none of these
systems assures high levels of expression in the correct membrane,
proper posttranslational modification, or, most importantly,
functionality. For example, expression of AE1 by transient transfection
of human embryonic 293 cells yields about 170 µg of membrane
protein/100 cm of tissue culture dish surface of which AE1
constitutes 0.4%, or 6.8 µg of AE1 protein. (
)To express
the amount of AE1 found in 1 liter of yeast (700 µg) in HEK293
cells would therefore require about 1 m
of tissue culture
dish surface, an impractically large amount. Furthermore, this protein
is retained in the endoplasmic reticulum, where it is glycosylated only
with core carbohydrate(18) . Similarly, AE2 was found at levels
comparable with those in HEK293 cells when expressed in Sf9 insect
cells using baculovirus(22) .
The unicellular eukaryote S. cerevisiae has been used for the expression of heterologous
membrane transporters with mixed success. Plant membrane proteins are
much more readily expressed in S. cerevisiae than are
mammalian membrane proteins, as exemplified by the sheep
Na,K
-ATPase, expressed as 0.1% of
yeast membrane protein (50) and the plant
H
-ATPase found as 40-50% of endoplasmic
reticulum membrane protein(46) . Relative to other mammalian
membrane proteins expressed in yeast, our observation that AE1
constitutes 1.5% of yeast membrane protein represents a high level of
expression. One elegant yeast expression system makes use of yeast sec mutants, which accumulate membrane proteins in
uniform-sized, sealed vesicles of defined orientation. These sec mutants have been used to express the H
-ATPase (55) and P-glycoprotein(51) , which proved useful for
transport assays. Unfortunately, the amount of protein accumulated in
this expression system is well below the mg of protein/liter range
reported here, making it less suitable for biochemical or structural
studies.
In conclusion, the expression system described here provides milligram quantities of functional, recombinant AE1 protein, which will be useful for future biophysical and biochemical characterization. The expression system described here should also be useful for the expression and characterization of other mammalian membrane proteins.