The N-terminal Region of the Transmembrane Domain of Human Erythrocyte Band 3

RESIDUES CRITICAL FOR MEMBRANE INSERTION AND TRANSPORT ACTIVITY*

Tomotake KankiDagger §, Mark T. Young§||, Masao Sakaguchi**, Naotaka HamasakiDagger , and Michael J. A. TannerDaggerDagger

From the Dagger  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

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 alpha -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).

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 alpha -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 alpha -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).

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.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 beta -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.

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.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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).


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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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Table I
SAO region mutants constructed
The sequence shown for wild-type band 3 encompasses amino acids 387-420.ER insertion was measured using constructs containing amino acids 1-432 of band 3 fused to an N-glycosylation acceptor sequence (1-432-gPL series; see "Experimental Procedures" and Fig. 2A). Anion transport and surface presentation were measured using full-length band 3 constructs.


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Fig. 2.   Membrane insertion of TM1 of N-terminal deletion series mutants. A, diagrams of the N-terminal deletion series constructs used. B, diagram of the TM1-4 constructs used (1-432-G loop-433-518-PL). C, gel showing glycosylation of the N-terminal deletion series constructs. Constructs were expressed by cell-free translation in the presence of canine pancreatic microsomes. Membranes were purified by centrifugation through a high salt cushion and analyzed by SDS-PAGE. The percentage of insertion into the ER of each mutant is shown below the lane for that mutant. D, using the cytoplasmic domain and TM1 to TM4 of wild-type band 3, SAO band 3 and the SAO region mutants, we made the 1-TM1-G loop-TM4-PL constructs, where the G loop was inserted between TM1 and TM2. Constructs were expressed by cell-free translation in the presence of canine pancreatic microsomes and analyzed by SDS-PAGE. The percentage of insertion into the ER of each mutant is shown below the lane for that mutant.

                              
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Table II
Summary of the results of ER insertion, anion transport, and surface presentation of the SAO region mutants
Data for each mutant are represented as a percentage of the band 3 control for ER insertion (percentage of insertion) and anion transport (percentage of transport; shown with the standard error of the measurements), and percentage of total expression for the amount at the surface (percentage at surface). All ER insertion data were measured using constructs containing amino acids 1-432 of band 3 fused to an N-glycosylation acceptor sequence (1-432-gPL series; see "Experimental Procedures" and Fig. 2A). Anion transport and surface presentation were measured using constructs based on full-length band 3, except for the series of progressive N-terminal truncations of the membrane domain (361-911 to 396-911), where the portions of band 3 indicated by the residue numbers preceded by an N-terminal initiator methionine were expressed. In most cases data from two separate experiments are shown separated by a comma. NT indicates mutants not tested for ER insertion. *, anomalously high surface percentage caused by either low expression or internal degradation of protein (discussed under "Results").

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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Fig. 3.   Membrane insertion of C-terminal deletion series and 417LLL. A, diagrams of the C-terminal deletion series constructs used. B, diagram of the 417LLL construct (1-432-gPL-SAO+LLL) used. C, gel of glycosylation of C-terminal deletion series mutants. The mutants were analyzed in the same manner as the N-terminal deletion series mutants in Fig. 2C. The percentage of insertion into the ER of each mutant is shown below the lane for that mutant. D, gel of glycosylation of the 417LLL construct (containing a trileucine insertion into the central region of TM1 (1-432-gPL-SAO+LLL)), showing that restoring the hydrophobic length of TM1 restores ER insertion. The percentage of insertion into the ER of each mutant is shown below the lane for that mutant.

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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Fig. 4.   36Cl influx assay of SAO band 3 mutants. Oocytes were injected with the molar equivalent of 10 ng of cRNA/oocyte of full-length band 3 or band 3 mutant, with 1 ng of cRNA/oocyte of GPA. Chloride influx over a 1-h period was measured using groups of 12-15 oocytes either in Barth's saline or in Barth's saline containing 400 µM DNDS as described previously (24). A, bars represent chloride influx expressed in nanomoles of Cl-, and error bars show the standard error of the influx measurements. The graph shows the chloride transport of the controls (band 3, SAO band 3, and uninjected oocytes) with (black bars) and without (clear bars) DNDS. B, bars represent DNDS-sensitive chloride influx (influx without DNDS minus influx in the presence of DNDS) expressed as a percentage of control (band 3) for selected mutants. Error bars show the standard error of the measurements.


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Fig. 5.   Western blotting of band 3 from chymotrypsin-treated oocytes. Oocytes were co-injected with 10 ng of cRNA/oocyte of full-length band 3 or band 3 mutant with 1 ng of cRNA/oocyte of GPA, to facilitate the expression of the band 3 mutants to the surface of the oocytes (24). Oocytes were allowed to express protein for 48 h. Groups of 30 oocytes were treated with chymotrypsin for 1 h at 4 °C as described previously (24). Groups of 10 oocytes were homogenized with 30 µl of radioimmunoprecipitation assay buffer (10 mM Tris-HCl, pH 7.2, 150 mM NaCl, 1% (w/v) Triton X-100, 0.1% (w/v) SDS) and centrifuged twice at 13,000 rpm for 5 min to remove cell debris and yolk granules. The homogenate was separated on 12% polyacrylamide gels according to the method of Laemmli (31). Band 3 and band 3 fragments were visualized by Western blotting using the monoclonal antibody BRIC 155 (against the C terminus of B3). Each lane represents the protein from 5 oocytes. The proportion of band 3 that was cleaved by chymotrypsin at the surface of the oocytes to yield the 35-kDa C-terminal portion detected by BRIC 155 was determined using scanning densitometry, and is shown in Table II (Surface column). The table shows duplicate results for nearly all the constructs studied. Each lane is labeled at the top with the name of the construct that it represents, and the whole protein bands and 35-kDa fragment are indicated. Most lanes also show a third band, which runs at ~55 kDa. This band is assumed to be the membrane domain of band 3. It is likely that there is some internal cleavage of band 3 between the membrane and cytoplasmic domains during expression in the oocyte. Composite gels are indicated with a thick black line between the composites. A, comparison of non-chymotrypsin-treated and chymotrypsin-treated band 3 and SAO band 3 (SAO). B, N-terminal deletion series of the SAO region (-A400 to -AFSPQVL). C, C-terminal deletion series of the SAO region (-A408 to -PQVLAA). D, double and triple deletion mutants. E, substitution mutants. Note that, for samples A400G and S402G, there is a much lower amount of whole band 3 present in these samples compared with the 35-kDa surface band 3 fragment. In these samples, there is a smaller band present (thought to be a proteolytic cleavage product of the membrane domain of band 3). This may be the result of enhanced degradation of mutant protein in the oocyte. F, N-terminal truncation mutants of the membrane domain of band 3 (361-911 to 396-911) where the portions of band 3 indicated by the residue numbers preceded by an N-terminal initiator methionine were expressed.

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 right-arrow AAA (Table I), and found it was fully functional and expressed at high levels at the oocyte surface (Table II).


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Fig. 6.   Sequence alignment of band 3 (AE1) and other members of the anion exchanger superfamily at the N-terminal side of the transmembrane domain. The putative region of band 3 TM1 is indicated, and the SAO region is shown in bold. The region of homology corresponding to amino acids 381-385 is shown in bold italics, and the adjacent R387RR region is shown in bold. Sequence similarity is shown at the bottom of the alignment, with asterisks (*) indicating amino acid identity, colons (:) indicating a high degree of homology, and dots (.) indicating a lesser degree of homology.

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.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 right-arrow Ser and Phe right-arrow 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.

    FOOTNOTES

* 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.

Dagger Dagger 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

    ABBREVIATIONS

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.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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