From the Department of Clinical Chemistry and
Laboratory Medicine and the ** Department of Molecular
Biology, Graduate School of Medical Sciences, Kyushu University,
Fukuoka 812-8582, Japan and the ¶ Department of Biochemistry,
School of Medical Sciences, University of Bristol,
Bristol BS8 1TD, United Kingdom
Received for publication, November 15, 2002, and in revised form, December 12, 2002
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ABSTRACT |
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We studied the role of the N-terminal region of
the transmembrane domain of the human erythrocyte anion exchanger (band
3; residues 361-408) in the insertion, folding, and assembly of the first transmembrane span (TM1) to give rise to a transport-active molecule. We focused on the sequence around the 9-amino acid region deleted in Southeast Asian ovalocytosis (Ala-400 to Ala-408), which
gives rise to nonfunctional band 3, and also on the portion of the
protein N-terminal to the transmembrane domain (amino acids 361-396).
We examined the effects of mutations in these regions on endoplasmic
reticulum insertion (using cell-free translation), chloride transport,
and cell-surface movement in Xenopus oocytes. We found that
the hydrophobic length of TM1 was critical for membrane insertion and
that formation of a transport-active structure also depended on the
presence of specific amino acid sequences in TM1. Deletions of 2 or 3 amino acids including Pro-403 retained transport activity provided that
a polar residue was located 2 or 3 amino acids on the C-terminal side
of Asp-399. Finally, deletion of the cytoplasmic surface sequence
G381LVRD abolished chloride transport, but not
surface expression, indicating that this sequence makes an essential
structural contribution to the anion transport site of band 3.
The red cell anion exchanger (band 3, AE1) is composed of two
distinct domains: a C-terminal transmembrane domain predicted to span
the membrane up to 14 times and a N-terminal cytosolic domain (1-4).
The transmembrane domain mediates chloride-bicarbonate exchange,
whereas the cytosolic domain associates peripheral proteins with
the membrane.
Southeast Asian ovalocytosis
(SAO)1 is caused by the
heterozygous presence of an abnormal band 3 (band 3 SAO), which has a deletion of 9 amino acid residues (amino acids 400-408) at the boundary between the cytosolic domain and the first
transmembrane-spanning segment (5-7). SAO is prevalent in areas where
malaria is endemic (8) and is thought to confer some protection against
cerebral malaria (9). Homozygosity for band 3 SAO is probably lethal (10).
Band 3 SAO is expressed at the surface of SAO red cells, but the mutant
protein has no anion transport function and does not bind the anion
transport inhibitor di-isothiocyanatodihydrostilbene either reversibly
or irreversibly (11-13). Changes in di-isothiocyanatodihydrostilbene binding (14), N-glycosylation (15), and blood group antigen expression (16, 17) indicate that the SAO deletion results in the
misfolding of the transmembrane domain. The Transmembrane segment 1 (TM1) of SAO band 3 does not insert into
microsomes in an alkali-stable fashion, unlike TM1 of normal band 3 (19). However, TMs 1-3 and larger fragments of SAO band 3 are
incorporated stably into microsomal membranes. Lys-430, which is
located in the first extracellular loop of band 3 between TM1 and TM2,
is labeled with eosin maleimide in normal band 3, but not in SAO band 3 (18). These observations suggest that the TM1-5 region of band 3 SAO
is misfolded. However, other data indicate that the gross topology and
structure of band 3 SAO between TMs 5 and 6 and TMs 7 and 8 is similar
to normal band 3 (15, 19). Recent studies show that band 3 SAO has a
dominant structural effect on normal band 3 in SAO membranes, altering
the structure of all the normal band 3 molecules (20). The structural
changes are transmitted throughout the normal band 3 molecules. The
structures of domains encompassing TMs 1-5, TMs 13 and 14, the fourth
and sixth extracellular loops, and the fourth, fifth, and sixth
cytoplasmic loops are all altered (20).
The SAO deletion is situated within the region of band 3 that acts as a
signal sequence for insertion of TM1 into the endoplasmic reticulum
(ER) membrane. A NMR study of synthetic peptides encompassing the
region of the SAO deletion showed that the SAO deletion leads to
formation of an In this study, we performed an extensive mutational analysis of the
region of band 3 within and around the SAO region. Our results
highlight three critical factors controlling both the insertion of TM1,
and the assembly of a transport-active band 3 molecule. First, the
hydrophobic length of TM1 is critical for correct insertion of TM1 into
membranes, and specific amino acid sequences in TM1 are critical for
the formation of a transport-active structure. Second, individual amino
acid residues at the interface between the cytoplasmic domain and TM1
are not essential for band 3 activity, and deletions of two or three
amino acids, which include Pro-403, retain transport activity provided
that a polar residue is located 2 or 3 amino acids C-terminal to
Asp-399. Third, we show that mutation of the basic cluster
R387RR to AAA has no effect on band 3 chloride transport.
Surprisingly, deletion of the sequence G381LVRD more distal
from TM1 at the cytoplasmic membrane surface abolished anion transport.
We suggest that this sequence (in conjunction with other cytoplasmic
loops of band 3) makes an essential contribution to the structure of
the anion transport site of band 3.
DNA Constructs--
DNA fragments encoding amino acids 1-518
and 375-518 (with an additional N-terminal methionine to act as
translational initiator) of band 3 and the reporter domain of mutant
prolactin, either without (PL) or with an N-glycosylation
site (gPL) (22), were amplified by PCR. The band 3 fragments were
digested with NcoI and XhoI, and the prolactin
fragment was digested with XhoI and XbaI. The
fragments were subcloned into the pCITE2b vector (Novagen) using
NcoI and XbaI to create the band 3-prolactin
chimeras. The SAO and mutant constructs were made using the previously
described method of overlap extension (23). Constructs containing
internal N-glycosylation sites either between transmembrane
spans 3 and 4 (375-484-G loop-485-518-PL, with an additional
N-terminal methionine to act as translational initiator) or between
transmembrane spans 1 and 2 (1-432-G loop-433-518-PL) were made by
inserting a fragment encoding amino acids Ile-627 to Pro-660 of band 3 (the fourth extracellular loop including Asn-642, the
N-glycosylation site of band 3; termed the G loop) between
Leu-484 and Glu-485 or between Arg-432 and Asn-433 respectively. The
construct containing a trileucine insertion within the central region
of transmembrane span 1 (417LLL), was made by inserting a
fragment encoding the insertion into 1-432-gPL SAO between Leu-417 and
Ser-418.
The constructs pBSXG1.b3 and pBSXG.GPA have been described previously
(24). They contain the cDNAs encoding human intact red cell band 3 and glycophorin A (GPA) respectively, flanked by the 5'- and
3'-noncoding regions of Xenopus In Vitro Transcription--
In vitro transcription
was carried out essentially as described previously (24, 25). For
cell-free translation and the topology assay, each plasmid was
linearized with ScaI and then transcribed with T7 RNA
polymerase. For expression into Xenopus oocytes, the
BSXG-based plasmids were linearized with HindIII and
transcribed using the mMessage mMachine kit (Ambion) to produce capped cRNA.
Cell-free Translation and Topology Assay--
Rabbit
reticulocyte lysate (26) and canine pancreatic microsomes (27) were
prepared as previously described. The canine pancreatic microsomes were
washed with 25 mM EDTA and treated with staphylococcal
nuclease (Roche Molecular Biochemicals) to remove endogenous mRNA.
Cell-free translation of cRNA was performed in the presence of canine
pancreatic microsomes using the cell-free system containing 20%
reticulocyte lysate and 15.5 kBq/µl EXPRE35SS protein
labeling mix (PerkinElmer Life Sciences). Microsomal membranes
were isolated from the translation mixture by ultracentrifugation through a high salt cushion (0.5 M sucrose, 30 mM potassium HEPES (pH 7.4), 5 mM potassium
acetate, 500 mM magnesium acetate; 200,000 × g for 10 min). Endoglycosidase H treatment was performed as previously described (28). Samples were separated by SDS-PAGE, and the
gel image was visualized using a phosphorimager (FLA2000, Fuji).
Quantitation was performed using MacBAS software (Fuji).
Expression of Protein in Xenopus Oocytes--
The methods used
for isolation of Xenopus oocytes, injection with cRNA,
chloride transport assay, and chymotrypsin assay have been described
previously (24, 29, 30). For the oocyte chymotrypsin assay, samples
were separated by SDS-PAGE (31) and subjected to Western blotting as
described in the figure legends.
Insertion of TM1 of Band 3 and SAO Band 3 into ER Membranes Using
Cell-free Expression into Microsomes
Fig. 1A shows a "bubble
diagram" representation of the N-terminal side of the transmembrane
domain of band 3. SAO band 3 lacks the 9-amino acid region at the
cytoplasmic face of TM1 (shaded in Fig. 1A). We
have already reported that the first transmembrane domain (TM1) of SAO
band 3 does not insert into the ER membrane independently, using a
construct encompassing both the cytoplasmic domain of band 3 and TM1 in
a cell-free translation system (19). However, the inability of TM1 of
SAO band 3 to insert into membranes may affect the topology of the
adjacent C-terminal TM segments. We made constructs corresponding to
the region of band 3 from TM1 to TM4 and inserted a loop containing an
N-glycosylation site (the fourth extracellular loop of band
3), termed the G loop, between TM3 and TM4 (Fig. 1B). This
construct also contained the reporter domain of prolactin lacking an
N-glycosylation site (PL) at its C terminus. The constructs
were expressed in the reticulocyte lysate cell free translation system
in the presence of microsomes. As reported (22), the G loop in the
construct corresponding to normal band 3 was efficiently glycosylated
(Fig. 1C, lane 1), implying that TM3
and TM4 integrate into the membrane with the expected orientation. The
same result was obtained when the SAO mutation was present (Fig.
1C), indicating that the membrane topologies of TM3 and TM4
are not grossly affected by the SAO mutation, because the loop between
TM3 and TM4 is translocated into the ER lumen irrespective of whether
TM1 and TM2 are properly inserted into the membrane (Fig.
1D).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helical content of band
3 SAO is similar to normal band 3 (15, 18), but the transmembrane
helices of band 3 SAO are disorganized while remaining folded (18).
-helix with only 8 contiguous hydrophobic residues,
too short to stably insert into the ER membrane (19). The study also
indicated that Pro-403 was at the N-terminal end of the
-helix
forming TM1 of normal band 3. The region immediately preceding Pro-403
also had helical character. Spectroscopic studies on synthetic peptides
incorporated into either micelles or liposomes also indicate that the
region encompassing the SAO deletion has a high degree of structure
(21).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-globin. For functional studies each of the mutations was also prepared in pBSXG1.b3, so that
the mutant band 3 proteins could be expressed in Xenopus oocytes.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
TM3 and TM4 integrate into membrane in both
wild-type band 3 and SAO band 3. A, diagram showing the
proposed topology of TMs 1-4 of band 3. The region corresponding to
that deleted in SAO band 3 is shaded. B,
constructs were made corresponding to both wild-type band 3 and SAO
band 3 TMs 1-4 with an N-glycosylation sequence loop (the
fourth extracellular loop of band 3 containing an
N-glycosylation site; termed the G loop) between TM3 and TM4
(375-484-G loop-485-518-PL, with an additional N-terminal methionine
to act as translational initiator). C, gel showing
N-glycosylation of the constructs. Constructs were expressed
by cell-free translation in the presence of canine pancreatic
microsomes. Both the wild-type band 3 and SAO band 3 constructs were
glycosylated well (arrowhead). Upon endoglycosidase H
treatment, the glycosylated bands were shifted down and became single
bands (arrow). D, diagram showing possible
membrane topologies. In both wild-type band 3 and SAO band 3, TM3 and
TM4 integrate into microsomal membranes, implying that the topology of
the TM segments C-terminal to the SAO deletion was not affected by it.
N and C termini are indicated in the diagram by N and
C, respectively.
ER Insertion of SAO Region Mutants
To examine the portions of the 9-residue SAO deletion that are essential for membrane insertion, we made series of N- and C-terminal deletions within the SAO region (constructs -A400 to -PQVLAA in Table I) in a fragment containing the N terminus and TM1 of normal band 3 fused at the C terminus to a prolactin reporter (gPL) containing an N-glycosylation site (Fig. 2A). Glycosylation at the gPL site will occur if TM1 takes up a transmembrane topology with the correct orientation. Fig. 2C shows the results of transmembrane insertion experiments for some of the mutants, and the percentage of the construct that was glycosylated (i.e. the percentage of the construct inserted into ER membranes) of all the mutants is summarized in Table II (Insertion column). Deletion of up to six residues at the N-terminal side of the SAO deletion (-AFSPQV) gave high levels of glycosylation, but when seven residues were deleted (-AFSPQVL), the glycosylation decreased. Deletion of eight residues (-AFSPQVLA) completely abolished glycosylation (Fig. 2C, Table II).
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It is possible that TM2 or more distant TM spans may facilitate integration of TM1 of SAO band 3 into the membrane. To examine this possibility, constructs were made corresponding to a region encompassing the N terminus of band 3 and TM spans 1-4, with the G loop inserted between TM1 and TM2, and fused at the C terminus with the prolactin reporter lacking an N-glycosylation site (Fig. 2B). When the construct corresponding to normal band 3 was expressed, it was efficiently glycosylated as previously reported (4). The constructs corresponding to band 3 SAO and band 3 deletions in the SAO region (Fig. 2D) displayed a parallel trend of N-glycosylation to the constructs containing TM1 only (73, 44, and 32% for the TM1-4 constructs compared with 86, 58, and 7% for the TM1 constructs of -AFSPQV, -AFSPQVL, and -AFSPQVLA respectively (Table II)). Although deletion of eight residues (-AFSPQVLA) gave a less marked decrease in glycosylation than the corresponding construct that only included TM1, these results strongly suggest that the membrane insertion of TM1 and TM2 is dependent solely upon the translocation initiation function of TM1. They also suggest that the topogenesis of TM1 + TM2 and of TM3 + TM4 is independent.
Studies of the C-terminal deletion series constructs (shown in Fig.
3A) showed that deletion of up
to two residues from the C-terminal side of the SAO region led to no
change in the insertion of TM1 (Fig. 3C and Table II).
Deletion of three residues (-LAA) reduced insertion, and deletion of
four or five residues abolished insertion (Table II). ER insertion
returned upon deletion of six residues (Fig. 3C, Table II).
These results suggested that, for efficient TM1 insertion, the length
of the continuous sequence of hydrophobic residues was important. This
was particularly clear in the case of -VLAA and -QVLAA, as Pro-403
would have been brought too near to the membrane and the turn
propensity or helix-breaking action of this residue would not allow a
sufficiently long membrane-spanning helical region to be formed. Helix
formation and membrane insertion would be allowed if Pro-403 was
absent, as in the deletion -PQVLAA, because the helix could now extend
at the N-terminal side with the addition of the sequence -AFS- (Table
I) so that the hydrophobic helical length is the same as in the -AA
mutant, which is able to insert into the ER membrane. To confirm that
the length of hydrophobic sequence of TM1 was essential for membrane
insertion, we added a trileucine sequence to the central region of TM1
of SAO band 3. The construct comprised the N terminus and TM1 of SAO
band 3, with three leucine residues inserted consecutively between
amino acids 417 and 418, fused at the C terminus (residue 432) to the
prolactin reporter, which contained an N-glycosylation site
(417LLL, see Fig. 3B). Glycosylation of this
construct occurred at a similar level to the construct corresponding to
normal band 3 (Fig. 3D), indicating that it took up the
transmembrane orientation as normal band 3. This result adds support to
the view that the inability of band 3 SAO TM1 to integrate into the
membrane is the result of the insufficient length of its hydrophobic
sequence.
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We also constructed several mutants within and around the SAO region to analyze their effects on ER insertion and band 3 anion transport function (the substitution; single, double, or triple deletion; insertion; and N-terminal truncation mutants shown in Table I). For ER insertion studies, all the mutations were prepared in the normal 1-432-gPL construct represented in Fig. 2A, so that the mutant TM1 region of band 3 was fused to a prolactin reporter containing an N-glycosylation site. All the mutant constructs were efficiently glycosylated because they maintained a sufficient length of hydrophobic residues, indicating that the mutant TM1s took up the same transmembrane orientation as normal TM1 in ER membranes (results summarized in Table II).
Expression of SAO Region Mutants in Xenopus Oocytes
We also examined the effects of the mutations on the anion
transport function and surface expression of band 3 using constructs that expressed whole band 3 (amino acids 1-911) containing each mutation. In vitro transcribed mRNA was co-injected into
Xenopus oocytes with mRNA corresponding to erythrocyte
GPA (known to enhance the surface presentation of band 3 in oocytes
(Ref. 30)), and the band 3-specific anion transport was estimated from
the stilbene-sensitive chloride uptake induced in the oocytes (Fig.
4). Chloride transport of all the mutants
was measured for ~15 oocytes/experimental sample, and in duplicate
sets of experiments using different batches of oocytes. Relevant
positive and negative controls were also measured in each experiment to
allow the normalization of the data sets. Because reduction in
transport activity in this assay could reflect impaired movement of the
mutant constructs to the cell surface, as well as a reduction in their
intrinsic transport activity, we also measured the proportion of band 3 that moved to the oocyte surface using a protease accessibility assay
(30). Band 3 has a single site susceptible to extracellular
chymotrypsin cleavage, which is located between TM5 and TM6. Intact
oocytes expressing the mutant band 3 were treated with chymotrypsin,
and the 35-kDa C-terminal band 3 fragment generated by extracellular
cleavage of band 3 at the oocyte surface was separated from the
uncleaved band 3 located within the cells using SDS-PAGE.
Immunoblotting with a monoclonal antibody was used to detect and
estimate the relative proportions of 35-kDa (surface band 3) and intact
band 3 (internal band 3) in the cells (Fig.
5). Data for all the mutants studied are
summarized in Table II. Although each lane in the blots on Fig. 5
represents the protein from 5 oocytes of the same batch, the protease
accessibility assays for each mutant were repeated at least once on a
different batch of oocytes, to allow for batch variability. The values
for the percentage of band 3 mutant at the surface of the oocyte (Table
II) are representative values from two experiments on separate batches
of oocytes, except for wild-type band 3 and SAO band 3, where these
values are presented as an average of six separate experiments. As
previously reported, SAO band 3 showed no anion transport activity
(Fig. 4A; Ref. 12). However, although ~27% of normal band
3 reached the oocyte surface, only ~11% of SAO band 3 was
surface-located (Fig. 5A, Table II). Although it has
previously been reported that similar proportions of band 3 and SAO
band 3 reach the surface of Xenopus oocytes (12),
consistently lower amounts of SAO band 3 were found to reach the oocyte
surface than normal band 3 in six separate experiments.
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No anion transport activity was observed for any of the constructs where insertion of TM1 into ER membranes was greatly reduced on cell free expression (-AFSPQVL, -AFSPQVLA, -LAA, -VLAA, and -QVLAA; Figs. 4B and 5 (B and C)). However, no anion transport activity was also observed for some constructs that displayed a high level of membrane insertion (-AFSP, -AFSPQ, -AFSPQV, and -AA; Figs. 4B and 5 (B and C)). All the mutants that displayed high levels of anion transport also displayed high levels of oocyte surface expression, as expected. Some mutants that did not show anion transport in oocytes were either not present at the cell surface in significant amounts (-AFSP, -AFSPQ, -VLAA; Fig. 5, B and C), or present at a reduced level similar to that of SAO band 3 (-AFSPQVL, -PQVLAA; Fig. 5, B and C). However, other mutants that did not yield anion transport activity in oocytes were found to be present at the cell surface at similar levels to normal band 3 (-AFSPQV, -AA; Fig. 5, B and C). Although the inability of TM1 to insert into ER membranes explains why SAO band 3 was nonfunctional, it does not explain why SAO region mutants that both inserted into ER membranes and reached the oocyte surface were nonfunctional.
We analyzed further the role of the SAO region of band 3 on band 3 function, by preparing the groups of band 3 mutants summarized below and shown in Table I. We examined the effect of the following mutations on anion transport and cell surface movement in oocytes, and the results are summarized in Table II.
Substitution Mutants-- Several glycine and alanine substitutions were made within the SAO region (A400G, F401G, S402G, etc.; Fig. 5E).
Single Deletion Mutants-- These mutants were -A400 (Fig. 5B), -P403, -Q404, -V405, and -A408 (Fig. 5C).
Double and Triple Deletion Mutants-- These mutants were -FS, -SP, etc., and -FSP, -SPQ, etc. (Fig. 5D).
Neither the glycine substitutions nor the alanine substitutions resulted in any significant reduction in anion transport, suggesting no single residue within the SAO region is critical for anion transport function. All the glycine and alanine substitutions moved at least as efficiently as normal band 3 to the oocyte surface. Both A400G and S402G were present at the oocyte surface at markedly higher levels than normal band 3, and, in oocytes expressing S402G, it appeared that essentially all the band 3 was at the oocyte surface (Fig. 5E). This may have been because of low expression of the construct, where all the expressed band 3 could traffic to the cell surface. Alternatively, it is possible that the internal population of mutant band 3 was unstable, and rapidly degraded. Although -V405 showed slightly reduced band 3 activity, and -A408 had only 24% of normal band 3 activity, all the other single deletion mutants (-A400, -P403, -Q404) were fully functional, and expressed at high levels at the oocyte surface. These results suggest that, apart from Ala-408, no single residue in this region is critical for correct band 3 function. The large reduction in chloride transport seen upon deletion of Ala-408 is probably a result of the reduction in length of the hydrophobic portion of TM1, the maintenance of which appears to be critical for band 3 activity.
The double deletion mutants -AF and -FS showed high levels of anion transport function, but mutants -SP and -PQ had much reduced levels of anion transport (both 11% of normal band 3) and mutants -QV and -AA had no anion transport function (Table II; Fig. 4B). All the double deletion mutants moved to the surface of the oocytes, although the amount of -QV was reduced (15 and 10% compared with 27% for normal band 3; Table II and Fig. 5 (B-D)). The triple deletions -SPQ and -LAA had no transport activity, although they were present at the cell surface. Mutants -AFS and -PQV had high levels of anion transport (84 and 70% of normal band 3, respectively), and -FSP had partial anion transport function (24% of normal band 3). Both mutants -FSP and -SPQ showed levels of surface expression similar to that of SAO band 3, whereas mutants -AFS, -PQV, and -LAA showed normal levels of surface expression (Table II; Figs. 4B and 5 (B-D)). Analysis of these results indicated that the region PQV was very important for band 3 function. Deletion in any combination of the first three residues of the SAO region (AFS) still gave rise to a high level of anion transport function. Any multiple deletion that included Pro-403, or residues immediately C-terminal to it, except for -PQV, led to either a reduced activity (-SP and -PQ), or no activity (-QV and -SPQ).
The isolated membrane domain of band 3 (residues 361-911 as defined by
protease cleavage studies) retains anion transport function both in red
cells and Xenopus oocytes (1). The region between residue
361 and the SAO region (starting at Ala-400) that is located at the
cytoplasmic surface of the membrane might also be important for the
structure and function of band 3. As shown in Figs. 1A and
6, the N-terminal side of the SAO region
contains a cluster of positive charges: R387RR. We
substituted all three arginine residues for alanine in the mutant
R387RR AAA (Table I), and found it was fully functional
and expressed at high levels at the oocyte surface (Table II).
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We also prepared a series of N-terminal truncation mutants of the membrane domain (Table II) and analyzed their function (Figs. 4B and 5F). Truncation of the protein to residue 380 (construct 381-911) gave rise to similar anion transport levels to that of intact band 3. Further truncation (386-911, 391-911, and 396-911) completely abolished anion transport. All constructs up to and including 386-911 were expressed at high levels at the oocyte surface, showing that the absence of anion transport of 386-911 was not caused by lack of surface expression. Surface expression was reduced somewhat for constructs 391-911 and 396-911 (14 and 10%, and 14 and 12%, compared with 27% for intact band 3).
To examine the role of the region between amino acids 386 and 400, we
created an insertion series, where residues E, SE, LSE, and ELSE were
inserted between Asp-399 and Ala-400 (at the N-terminal side of the SAO
region; see Table I)). All four insertion mutants were fully functional
and expressed at the surface of the oocyte (Table II). However, the
surface expression of mutants +SE, +LSE, and +ELSE was somewhat reduced
(12 and 15%, 15 and 18%, and 14 and 23%, compared with 27% for
normal band 3). This result implies that the link between amino acids
381-386 and the SAO region is flexible enough to accommodate an
insertion of at least four amino acids and still give rise to a
transport-competent molecule, but the longer insertions impair the
ability of band 3 to traffic to the cell surface.
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DISCUSSION |
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The deletion that leads to the loss of anion transport activity of SAO band 3 is located at the cytoplasmic surface of TM1 and includes both residues in TM1 and the surface region of the protein. We have carried out a mutagenesis study of both these regions, and the interface between them, to help understand the role of these portions of the molecule in the biosynthesis, folding, and transport activity of the protein.
Both the Hydrophobic Length and Sequence of TM1 Are Critical for Band 3 Function-- The ER insertion studies using cell-free translation clearly show that reduction of the hydrophobic length of normal TM1 in the series -AA, -LAA, and -VLAA progressively decreases the ability of TM1 to integrate into the ER membrane. This is accompanied by the loss of anion transport activity and, in the case of -VLAA, almost complete loss of movement to the cell surface. Although restoration of the hydrophobic helix length by the insertion of three leucine residues into the SAO TM1 (417LLL) restored the ability of TM1 to insert into the membrane, it did not restore transport activity. Clearly the amino acid sequence within the hydrophobic portion of TM1 is important for the folded protein to take up a transport active structure. The observation that -A408 efficiently inserted into membranes but had reduced transport activity further confirms that integration of TM1 into the membrane is not sufficient for full transport activity. Interestingly the additional removal of Pro-403 in the -PQVLAA construct substantially restored membrane insertion but not transport. Pro-403 probably initiates the TM1 helix (19), and the deletion of Pro-403 would allow the extension of the hydrophobic helix on the N-terminal side by the adjacent hydrophobic-compatible residues AFS. In the absence of Pro-403, the N-terminal end of the membrane-embedded portion of TM1 is delimited by Asp-399 and the polar sequence on the N-terminal side. Consistent with this, the deletion series -AFSP to -AFSPQVL and the construct -PQVLAA all insert into the membrane because they contain a sufficiently long hydrophobic segment in TM1 extending from Asp-399, whereas -AFSPQVLA does not insert because the hydrophobic segment has length reduced to that of the hydrophobic segment of the helix initiated by Pro-403 in -VLAA and -QVLAA, which also insert poorly.
Role of the Boundary between the Cytoplasmic Domain and
TM1--
The sequence around P403Q forms the boundary between the end
of the cytoplasmic surface region and start of TM1 (19). Surprisingly, neither point mutation of Pro-403, Gln-404, and the surrounding residues nor deletion of Pro-403, Gln-404, or Val-405 had any major
effect on membrane insertion or transport activity. The progressive
deletions from -A400 to -AFS on the N-terminal side of Pro-403, and
the -FS double deletion also had no effect, suggesting that the AFS
sequence does not have an important role in the structural and
functional properties of the protein. However, extension of the
deletion to include Pro-403 (-AFSP, -AFSPQ) abolished transport activity and greatly reduced cell surface movement without affecting membrane insertion. The triple and double deletions including Pro-403
(-FSP, -SPQ, -PQV, -SP, and -PQ) gave interesting results. In all
these mutants, the removal of Pro-403 moves the boundary of the
hydrophobic region of TM1 to Asp-399. Although -PQV and -FSP had
substantial transport activity (the poor cell surface presentation of
-FSP probably underestimates its intrinsic transport activity), -SPQ
had no transport activity and impaired movement to the cell surface.
-PQV and -FSP differ from -SPQ only by single residue substitutions
of hydrophobic for polar residues (Val Ser and Phe
Gln at
residues +3 and +2, respectively, from Asp-399). The -SP and -PQ
mutants, which also retain some activity, have polar amino acids at
residue +3 to Asp-399, and inspection of Table II shows that, among the
constructs lacking Pro-403 that insert into membranes, only those with
a polar residue at +2 or +3 to Asp-399 show activity, whereas those
with hydrophobic residues at both these positions are
transport-inactive. This suggests that, in the absence of Pro-403,
interactions (presumably hydrogen bonding) of the polar residues +2 or
+3 to Asp-399 with other regions of the molecule enable the protein to
form a transport-active structure.
A Cytoplasmic Surface Region on the N-terminal Side of the Membrane Domain Is Important for Anion Transport Activity-- The cytoplasmic surface region immediately N-terminal to the SAO mutation was able to accommodate the progressive series of insertions +E to +ELSE without deleterious effects on transport function or membrane insertion but displayed a decreasing efficiency of cell surface movement. We found that mutation of the basic amino acid cluster R387RR to AAA showed no effect on membrane insertion, cell surface movement, or anion transport activity. This was a surprising observation because the positively charged residues at the cytoplasmic side of a transmembrane segment are thought to be important for its topology and structure (32), and this cluster of positive charges is highly conserved in the bicarbonate transporter sequence superfamily which contains band 3 (Fig. 6). A series of N-terminal truncations of the membrane domain was tested to examine the role of other regions of this cytoplasmic surface portion of band 3. These experiments showed that the deletion of the sequence -GLVRD- (residues 381-385) adjacent to R387RR resulted in the loss of anion transport activity but retention of cell surface movement. The deleted region, which is 17 amino acids distant from the point where TM1 enters the membrane, is also strongly conserved within the bicarbonate transporter superfamily (Fig. 6). Previous studies have suggested that band 3 contains subdomains comprising TM1-5, TM6-8, and TM9-12 that interact with each other (33-35). The presence of these extensive interactions has been confirmed by studies demonstrating that the SAO deletion causes structural perturbations throughout band 3 that were particularly evident in the C-terminal portion of the molecule (20). These experiments suggested that the cytoplasmic region of band 3 adjacent to the SAO region interacts with the cytoplasmic loops between TM8 and TM9 (loop 8-9), loop 10-11, and loop 12-13. The present results suggest that the sequence -G381LVRD- on the N-terminal side of the SAO deletion probably participates in this association between loop 8-9, loop 10-11, and loop 12-13 and that these loops may together form part of the active transport site in band 3.
In summary, we draw three major conclusions from this study on the role
of different portions of the sequence of band 3 surrounding the SAO
deletion on band 3 structure and function. First, the hydrophobic
length of TM1 is important for its membrane insertion and the formation
of a transport-active structure depends on specific amino acid
sequences in TM1. Second, individual amino acid residues at the
interface between the cytoplasmic domain and TM1 are not essential for
band 3 transport activity, and deletions of two or three amino acids
that include Pro-403 retain transport activity, provided that a polar
residue is located 2 or 3 amino acids C-terminal to Asp-399. We infer
that hydrogen bonding to this polar residue can maintain band 3 in a
transport-competent structure. Finally, we show that mutation of the
basic cluster R387RR to AAA had no effect on band 3 function, whereas deletion of the sequence G381LVRD more
distal from TM1 at the membrane surface surprisingly abolished anion
transport. We suggest that this sequence (in conjunction with some of
the other cytoplasmic loops of band 3) makes an essential contribution
to the structure of the anion transport site of band 3.
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FOOTNOTES |
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* This work was supported in part by a grant from the Wellcome Trust (to M. J. A. T.) and by a grant from the Ministry of Education, Science, Sports and Culture of Japan (to N. H.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ These authors contributed equally to this work.
Recipient of a Wellcome Prize fellowship.
To whom correspondence should be addressed. Tel.:
44- 1179288271; Fax: 44-1179288274; E-mail:
m.tanner@bristol.ac.uk.
Published, JBC Papers in Press, December 12, 2002, DOI 10.1074/jbc.M211662200
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ABBREVIATIONS |
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The abbreviations used are: SAO, Southeast Asian ovalocytosis; TM, transmembrane span; ER, endoplasmic reticulum; GPA, glycophorin A; PL, prolactin; gPL, prolactin with an N-glycosylation site; DNDS, 4,4'-dinitro-2,2'-stilbene disulfonate.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Tanner, M. J. (1993) Semin. Hematol. 30, 34-57[Medline] [Order article via Infotrieve] |
2. | Reithmeier, R. A., Landolt-Marticorena, C., Casey, J. R., Sarabia, V. E., and Wang, J. (1993) Soc. Gen. Physiol. Ser. 48, 161-168[Medline] [Order article via Infotrieve] |
3. | Wang, D. N. (1994) FEBS Lett. 346, 26-31[CrossRef][Medline] [Order article via Infotrieve] |
4. |
Popov, M.,
Tam, L. Y., Li, J.,
and Reithmeier, R. A.
(1997)
J. Biol. Chem.
272,
18325-18332 |
5. | Jarolim, P., Palek, J., Amato, D., Hassan, K., Sapak, P., Nurse, G. T., Rubin, H. L., Zhai, S., Sahr, K. E., and Liu, S. C. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 11022-11026[Abstract] |
6. | Mohandas, N., Winardi, R., Knowles, D., Leung, A., Parra, M., George, E., Conboy, J., and Chasis, J. (1992) J. Clin. Invest. 89, 686-692[Medline] [Order article via Infotrieve] |
7. | Schofield, A. E., Reardon, D. M., and Tanner, M. J. (1992) Nature 355, 836-838[CrossRef][Medline] [Order article via Infotrieve] |
8. | Serjeantson, S., Bryson, K., Amato, D., and Babona, D. (1977) Hum. Genet. 37, 161-167[Medline] [Order article via Infotrieve] |
9. | Genton, B., al-Yaman, F., Mgone, C. S., Alexander, N., Paniu, M. M., Alpers, M. P., and Mokela, D. (1995) Nature 378, 564-565[CrossRef][Medline] [Order article via Infotrieve] |
10. |
Liu, S. C.,
Jarolim, P.,
Rubin, H. L.,
Palek, J.,
Amato, D.,
Hassan, K.,
Zaik, M.,
and Sapak, P.
(1994)
Blood
84,
3590-3591 |
11. | Schofield, A. E., Tanner, M. J., Pinder, J. C., Clough, B., Bayley, P. M., Nash, G. B., Dluzewski, A. R., Reardon, D. M., Cox, T. M., Wilson, R. J., et al.. (1992) J. Mol. Biol. 223, 949-958[Medline] [Order article via Infotrieve] |
12. | Groves, J. D., Ring, S. M., Schofield, A. E., and Tanner, M. J. (1993) FEBS Lett. 330, 186-190[CrossRef][Medline] [Order article via Infotrieve] |
13. | Chernova, M. N., Jarolim, P., Palek, J., and Alper, S. L. (1995) J. Membr. Biol. 148, 203-210[Medline] [Order article via Infotrieve] |
14. |
Okubo, K.,
Kang, D.,
Hamasaki, N.,
and Jennings, M. L.
(1994)
J. Biol. Chem.
269,
1918-1926 |
15. |
Sarabia, V. E.,
Casey, J. R.,
and Reithmeier, R. A.
(1993)
J. Biol. Chem.
268,
10676-10680 |
16. | Booth, P. B., Serjeantson, S., Woodfield, D. G., and Amato, D. (1977) Vox Sang. 32, 99-110[Medline] [Order article via Infotrieve] |
17. |
Smythe, J. S.,
Spring, F. A.,
Gardner, B.,
Parsons, S. F.,
Judson, P. A.,
and Anstee, D. J.
(1995)
Blood
85,
2929-2936 |
18. |
Moriyama, R.,
Ideguchi, H.,
Lombardo, C. R.,
Van Dort, H. M.,
and Low, P. S.
(1992)
J. Biol. Chem.
267,
25792-25797 |
19. | Chambers, E. J., Bloomberg, G. B., Ring, S. M., and Tanner, M. J. (1999) J. Mol. Biol. 285, 1289-1307[CrossRef][Medline] [Order article via Infotrieve] |
20. | Kuma, H., Abe, Y., Askin, D., Bruce, L. J., Hamasaki, T., Tanner, M. J., and Hamasaki, N. (2002) Biochemistry 41, 3311-3320[CrossRef][Medline] [Order article via Infotrieve] |
21. | Kuma, H., Inoue, K., Fu, G., Ando, S., Lee, S., Sugihara, G., and Hamasaki, N. (1998) J. Biochem. (Tokyo) 124, 509-518[Abstract] |
22. |
Ota, K.,
Sakaguchi, M.,
Hamasaki, N.,
and Mihara, K.
(1998)
J. Biol. Chem.
273,
28286-28291 |
23. | Ho, S. N., Hunt, H. D., Horton, R. M., Pullen, J. K., and Pease, L. R. (1989) Gene (Amst.) 77, 51-59[CrossRef][Medline] [Order article via Infotrieve] |
24. |
Groves, J. D.,
and Tanner, M. J.
(1992)
J. Biol. Chem.
267,
22163-22170 |
25. | Sakaguchi, M., Tomiyoshi, R., Kuroiwa, T., Mihara, K., and Omura, T. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 16-19[Abstract] |
26. | Jackson, R. J., Campbell, E. A., Herbert, P., and Hunt, T. (1983) Eur. J. Biochem. 131, 289-301[Medline] [Order article via Infotrieve] |
27. | Walter, P., and Blobel, G. (1983) Methods Enzymol. 96, 84-93[Medline] [Order article via Infotrieve] |
28. | Ota, K., Sakaguchi, M., von Heijne, G., Hamasaki, N., and Mihara, K. (1998) Mol. Cell 2, 495-503[Medline] [Order article via Infotrieve] |
29. | Groves, J. D., and Tanner, M. J. (1994) J. Membr. Biol. 140, 81-88[Medline] [Order article via Infotrieve] |
30. |
Groves, J. D.,
and Tanner, M. J.
(1995)
J. Biol. Chem.
270,
9097-9105 |
31. | Laemmli, U. K. (1970) Nature 227, 680-685[Medline] [Order article via Infotrieve] |
32. | von Heijne, G., and Gavel, Y. (1988) Eur. J. Biochem. 174, 671-678[Abstract] |
33. | Groves, J. D., and Tanner, M. J. (1999) Biochem. J. 344, 699-711[CrossRef][Medline] [Order article via Infotrieve] |
34. | Hamasaki, N., Okubo, K., Kuma, H., Kang, D., and Yae, Y. (1997) J. Biochem. (Tokyo) 122, 577-585[Abstract] |
35. | Hamasaki, N., Kuma, H., Ota, K., Sakaguchi, M., and Mihara, K. (1998) Biochem. Cell Biol. 76, 729-733[CrossRef][Medline] [Order article via Infotrieve] |