©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Co-expressed Complementary Fragments of the Human Red Cell Anion Exchanger (Band 3, AE1) Generate Stilbene Disulfonate-sensitive Anion Transport (*)

Jonathan D. Groves , Michael J. A. Tanner (§)

From the (1) Department of Biochemistry, School of Medical Sciences, University of Bristol, Bristol BS8 1TD, United Kingdom

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We have constructed cDNA clones encoding various portions of the human red cell anion transporter (band 3), a well characterized integral membrane protein with up to 14 transmembrane segments. The biosynthesis, stability, cell surface expression, and functionality of these band 3 fragments were investigated by expression from the cRNAs into microsomal membranes using the reticulocyte cell-free translation system and in Xenopus oocytes. Co-expression of the pairs of recombinants encoding the first 8 and last 6 transmembrane spans (8+6) or the first 12 and last 2 spans (12+2) of band 3 generated stilbene disulfonate-sensitive anion transport in oocytes. When the pairs of fragments 8+6 or 12+2 were co-expressed with glycophorin A (GPA), translocation to the plasma membrane of the fragment corresponding to the first 12 or the first 8 transmembrane spans was greater than in the absence of GPA. Only the fragment encoding the first 12 transmembrane spans showed GPA-dependent translocation when expressed in the absence of its complementary fragment. A truncated form of band 3 encoding all 14 transmembrane spans but lacking the carboxyl-terminal 30 amino acids of the cytoplasmic tail did not induce anion transport activity in oocytes and was not translocated to the plasma membrane but appeared to be degraded in oocytes. Our results suggest that there is no single signal for the insertion of the different transmembrane spans of band 3 into membranes and that the integrity of the loops between transmembrane spans 8-9 or 12-13 is not essential for anion transport function. Our data also suggest that a region of transmembrane spans 9-12 of band 3 is involved in the process by which GPA facilitates the translocation of band 3 to the surface.


INTRODUCTION

The human erythrocyte anion transporter (band 3, AE1) is the most abundant integral membrane protein in red cells (reviewed in Refs. 1-6). This 911-amino acid protein comprises two domains that are structurally and functionally distinct: the amino-terminal 43-kDa cytoplasmic domain binds cytoskeletal and other peripheral membrane proteins, whereas the carboxyl-terminal 52-kDa integral membrane domain is responsible for red cell anion exchange. A combination of hydropathy analysis on the amino acid sequence, proteolysis, and various chemical and antibody binding studies suggest that the polypeptide chain traverses the lipid bilayer up 14 times (reviewed in Refs. 2-4, 6 and 7).

Our current knowledge of the three-dimensional structure of integral membrane proteins is very limited in comparison with the wealth of data that is available for soluble proteins. Most of the structures that have been obtained at high resolution (with the exception of the bacterial porins) have indicated that these proteins are based upon bundles of hydrophobic transmembrane -helices, and the transmembrane spans of band 3 have been shown to be almost entirely -helical in conformation (8) . A low resolution (20 Å) two-dimensional model for structure of the membrane domain of band 3 obtained from the study of two-dimensional crystals indicated the presence of a mobile subdomain in this region of the protein (9) . However, a recent three-dimensional map of the membrane domain suggests that this mobile region is made up from the portions of the membrane domain located in the cytoplasm (10) .

In this paper, we describe the expression of two pairs of cRNAs representing fragments of band 3 and show that, while the individual fragments are not competent for transport, the pairs of fragments are able to assemble to form a structure that carries out anion transport with the stilbene-disulfonate sensitivity that is characteristic of band 3.


MATERIALS AND METHODS

Construction of Band 3 Fragments

The cDNA clones encoding human band 3 (pBSXG1.b3), the band 3 membrane domain (pBSXG1.b3 mem) and glycophorin A (pBSXG.GPA) have been previously described (11) . In these constructs, the protein-coding cDNA is flanked by the 5`- and 3`-untranslated regions of Xenopus -globin. cDNAs encoding fragments of band 3 truncated at the amino or carboxyl terminus of the protein were prepared from pBSXG1.b3 and pBSXG1.b3 mem by PCR() as detailed below. In each case, the pairs of oligonucleotide primers were designed to anneal to the band 3 cDNA with a calculated Tof approximately 56-60 °C.

The recombinants encoding fragments containing the amino-terminal portion of the protein ( i.e. truncated at the carboxyl terminus) were prepared by incorporating a translation termination codon into the coding sequence of band 3 and deleting the region of the cDNA on the 3`-side of this termination codon. Each antisense PCR primer contained the sequence 5`-GGCGGTAACCTCA-3` (which includes a BstEII restriction enzyme site and a termination codon), followed by the particular 17-20-nucleotide sequence, which perfectly matches the band 3 cDNA encoding the 5-7 amino acids of band 3 immediately prior to the required carboxyl-terminal truncation. For example, the antisense primer used to prepare construct b3(1:12), which is carboxyl-terminally truncated after residue Tyrin the loop between transmembrane spans 12 and 13, was as follows.

On-line formulae not verified for accuracy

On-line formulae not verified for accuracy

On-line formulae not verified for accuracy

For each of these PCR reactions, the sense primer was 5`-TCCCTGGCCCTGCCCTT-3`, which anneals to the portion of the band 3 cDNA encoding amino acid residues 856-861 and which is upstream of the BstEII site in the 3`-untranslated region of -globin. After amplification, the cDNAs were digested with BstEII, recircularized, and cloned into Escherichia coli strain TG2.

There combinantsen coding fragments containing the carboxyl-terminal portion of the protein ( i.e. truncated at the amino terminus) were prepared by inserting a methionine codon and deleting there gion of the band 3 cDNA upstream of this protein initiator codon. Eachsense PCR primer contained the sequence 5`-CCGCCATGG-3` (which includes an NcoI restriction enzyme site and a methionine codon), followed by the particular 15-20-nucleotide sequence, which perfectly matched the portion of band 3 cDNA encoding the first 5-6 amino acids of band 3 that would follow in-frame with the initiator methionine of the amino-terminally truncated protein. For example, the sense primer used to prepare construct (13:14), which initiates at residue 825 in the loop between transmembrane spans 12 and 13, was as follows.

On-line formulae not verified for accuracy

On-line formulae not verified for accuracy

On-line formulae not verified for accuracy

On-line formulae not verified for accuracy

For each of these PCR reactions, the antisense primer 5`-TAGTCTGTGGCTGTTGCCT-3` was used. This anneals to the portion of the band 3 cDNA encoding amino acid residues 46-41 of the protein and downstream of the NcoI site of pBSXG1.b3, which includes the codon corresponding to the initiator methionine of band 3. In each case, the amplified cDNA was digested with NcoI, recircularized, and cloned.

The COOH-terminally truncated mutant b3(1:14*) was constructed from pBSXG1.b3 by site-directed mutagenesis (Amersham Sculptor kit) using the oligonucleotide 5`-AGGAACGTGTAGCTTCAGT-3`, which mutates the GAG codon that corresponds to amino acid Gluto a TAG terminator codon.

The sequences of cDNA constructs were verified using either a DuPont Genesis 2000 automated sequencer or by manual double-stranded dideoxy sequencing using Sequenase (U. S. Biochemical Corp.) according to the manufacturer's protocols.

Preparation of cRNAs and Expression of Band 3 Fragments

Protocols for synthesis of cRNA, cell-free translation using rabbit reticulocyte lysate with canine pancreatic microsomes, inhibition of N-glycosylation using the acceptor tripeptide N-benzoyl-Asn-Leu-Thr- N-methylamide (NLT peptide), purification of microsomes, trypsin treatment of microsomally expressed proteins, oocyte isolation and cRNA injection, S amino acid labeling and immunoprecipitation of proteins, chymotrypsin treatment of oocytes, and chloride transport assay methods were all as previously detailed (11, 12, 13) . SDS-PAGE was by the method of Laemmli (14) or using the tricine buffer system of Schägger and von Jagow (15) .


RESULTS

Preparation of cDNAs

The PCR was used to delete various portions of the coding sequence from pBSXG1.b3 and pBSXG1.b3 mem cDNAs (11) , which encode, respectively, for the entire human band 3 (b3) and the membrane domain alone of band 3 (b3 mem, amino acids 360-911 of band 3). The amino- and carboxyl-terminal truncations were each positioned in a loop that was thought to be located in the cytoplasm in the model of 14 transmembrane spans for the band 3 monomer (4, 7, 10) . Band 3 fragments were prepared as ``complementary'' pairs, which together contained all 14 putative transmembrane spans of the entire band 3 membrane domain. The protein fragments derived from the expression of these recombinants are illustrated in Fig. 1, which also gives the exact locations of the truncations and nomenclature used for each fragment. For example, fragment b3(1:12) is truncated after amino acid residue 824 and includes the cytoplasmic domain and transmembrane spans 1-12; likewise, the complementary fragment (13:14) initiates with a methionine residue followed by amino acid residues 825-911 and includes transmembrane spans 13 and 14 and the carboxyl terminus of the protein.

The band 3 membrane domain fragments mem(1:8) and mem(1:12) were similar to the intact band 3 mutants b3(1:8) and b3(1:12), except that in these constructs the cytoplasmic domain of band 3 (amino acid residues 1-359) was replaced by a single initiator methionine residue.

Characterization of Band 3 Fragments by Cell-free Translation

For initial characterization of the fragments, cRNA was prepared from each of the fragment cDNAs and translated in the rabbit reticulocyte lysate cell-free system. Analysis by SDS-PAGE and autoradiography demonstrated that the fragments gave the expected Min each case (Fig. 2).

Cell-free translation of the amino-terminal fragments b3(1:12) and b3(1:8) in the presence of canine pancreatic microsomes resulted in two closely spaced bands on SDS-PAGE, with the higher molecular weight band predominating (Fig. 2 a). Inclusion of the N-linked glycosylation acceptor NLT peptide at 30 µ M in the translation mixture caused the intensity of the lower band to increase and the upper band to decrease in each case, corresponding to a 50-75% inhibition of core N-glycosylation, similar to the effect of this peptide on intact band 3 (12) . Cell-free translation of each fragment in the absence of microsomes resulted in a single band on SDS-PAGE, running in the same position as the lower (unglycosylated) band obtained in the presence of microsomes. These results demonstrate that the amino-terminal fragments are all N-glycosylated at a single site, similarly to intact band 3.


Figure 2: Expression of band 3 fragments in the rabbit reticulocyte lysate cell-free system. Various band 3 fragment cRNAs (50 ng) were translated individually (in 6.25-µl reaction mixtures), as described in Ref. 11. For each cRNA, parallel translations were carried out in the presence or absence of canine pancreatic microsomes and in the presence or absence of the N-glycosylation acceptor NLT peptide at 30 µ M final concentration, as described in Ref. 12. Samples (0.5 µl) of each translation were analyzed by SDS-PAGE either on 9% Laemmli gels (for larger fragments) ( a) or 10% Tricine gels (for smaller fragments) ( b), followed by autoradiography.



Cell-free translation of the carboxyl-terminal fragments (13:14) and (9:14) resulted in a single band on SDS-PAGE, the position of which was not affected by the addition of NLT peptide or by the absence of microsomes from the translation (Fig. 2 b). These results confirm that, as expected, the carboxyl-terminal fragments do not contain an N-glycosylation site.

A lower yield of each of the four fragment proteins was obtained from cell-free translation in the absence of microsomes. This suggests that the integration of nascent proteins into microsomes is important for efficient protein synthesis. A similar effect is observed in the absence of microsomes on translation of intact band 3 (data not shown).

Trypsin Treatment of Intact Band 3 and the Band 3 Fragments

Intact band 3 was synthesized from the cRNA (in the absence of GPA cRNA) and incorporated into microsomes using the reticulocyte cell-free translation system. Microsomes were then isolated and treated with trypsin at 0, 3, or 10 µg/ml (Fig. 3 a). When the band 3 in microsomes was treated with trypsin either at 3 or at 10 µg/ml (Fig. 3 a, lanes 3 and 5) the amino-terminal cytoplasmic domain (running at 43 kDa) was cleaved from the carboxyl-terminal membrane domain (running at 47-51 kDa). Very small quantities of intact band 3 and of a 75-kDa fragment were visible after treatment with 3 µg/ml trypsin, but these were degraded fully with 10 µg/ml trypsin. Fragments with apparent molecular masses of approximately 32, 26, and 21 kDa were obtained at both trypsin concentrations, in addition to smaller fragments that ran at the SDS-PAGE gel front.


Figure 3: Trypsin digestion of band 3 and the band 3 fragments expressed in microsomes by the rabbit reticulocyte lysate cell-free system. a, band 3 cRNA (0.5 µg) was translated (in a 50-µl reaction mixture) and microsomes were purified by Airfuge centrifugation. The pelleted membranes were resuspended and divided into six portions (8 µl each), and 1 µl of 10% (w/v) Triton X-100 was added to each of three tubes. Trypsin was then added (to a final concentration of 0, 3, or 10 µg/ml) to both detergent-treated and untreated samples (10 µl, final volume), and all six tubes were incubated at 4 °C for 1 h, as described in Ref. 11. b, equimolar amounts of the various band 3 fragment cRNAs were cotranslated with GPA cRNA. The amounts of cRNA (in 30-µl reaction mixtures) were 45 ng, band 3 and b3(1:12); 37.5 ng, b3(1:8); 15 ng, (13:14), (9:14), and GPA. Microsomes were purified and then treated with trypsin at 5 µg/ml, as described above. Samples were separated by SDS-PAGE on 10% Laemmli gels ( a) or 4-20% Laemmli ``Ready-Gels'' (Bio-Rad) ( b), followed by autoradiography. The apparent molecular weights of the various tryptic digestion products and residual undigested proteins are detailed in the text. Key: mem, 44-51-kDa band 3 membrane domain; cyt, 43-kDa band 3 cytoplasmic domain; A, 25-kDa glycophorin A monomer. Other numbers indicate the apparent molecular mass (kDa) of the various proteolytic fragments.



Immunoprecipitation by the monoclonal antibody BRIC 155 (which reacts with the carboxyl terminus of band 3) was used to probe for this region of the polypeptide in the various tryptic fragments. When samples were treated either with 3 or 10 µg/ml trypsin (Fig. 3 a, lanes 9 and 11), the polypeptide corresponding to the entire band 3 membrane domain was the major proteolytic product that was immunoprecipitated. A small amount of immunoreactive material migrated at the gel front, probably representing very small peptide fragments containing the carboxyl terminus of the protein. This indicates that the other fragments (observed in Fig. 3 a, lanes 3 and 5) do not contain the carboxyl terminus of band 3. In the immunoprecipitations obtained after treatment with 3 µg/ml trypsin (Fig. 3 a, lane 9), traces of both intact band 3 and the 75-kDa fragment were visible, indicating that both of these products contained the carboxyl terminus. The 75-kDa polypeptide therefore corresponds to band 3 cleaved at a site within the amino-terminal cytoplasmic domain.

A parallel set of microsomal membranes containing the protein were treated with the detergent Triton X-100 prior to trypsin digestion to examine the susceptibility of solubilized band 3 to proteolysis. In the detergent-treated samples that had been digested either with 3 or 10 µg/ml trypsin, the 47-kDa band 3 membrane domain and the products running at approximately 32 and 26 kDa were not visible (Fig. 3 a, lanes 4 and 6), but the 21-kDa fragment was not affected by prior solubilization with Triton X-100. Immunoprecipitation with BRIC 155 confirmed that the band 3 membrane domain was degraded by the trypsin under these conditions in the presence of detergent (Fig. 3 a, lanes 10 and 12). The 43-kDa cytoplasmic domain was more resistant to proteolysis in the presence of Triton X-100 than the membrane domain and was only digested partially by 10 µg/ml trypsin in the presence of detergent (Fig. 3 a, lane 6). These results indicate that detergent solubilization causes the membrane domain of band 3 to become susceptible to digestion by trypsin at various sites that are normally cryptic to the enzyme when the protein is present in microsomal membranes.

In a subsequent experiment, each of intact band 3, b3(1:12), b3(1:8), (9:14), and (13:14) was translated and incorporated into microsomes from their respective cRNAs using the cell-free system. GPA cRNA was co-expressed with the band 3 fragment in each of the translations. Although we have shown previously that co-expression of GPA with band 3 does not affect sensitivity to trypsin and hence the overall structure of the intact protein (11) , we could not exclude the possibility that GPA might influence the initial folding of protein fragments. Furthermore, it is clear that the presence or absence of GPA does affect the eventual conformation and functional activity of the mature protein in the red cell (16) . A trypsin concentration of 5 µg/ml was selected for this analysis of the topography and folding of the newly synthesized amino- and carboxyl-terminal fragments on the basis of the results of the experiment shown in Fig. 3 a. The products of trypsin digestion were analyzed by SDS-PAGE and autoradiography (Fig. 3 b). GPA was not digested by trypsin, and the GPA monomer may be seen in each lane running at approximately 25 kDa.

The carboxyl-terminally truncated band 3 fragments b3(1:12) and b3(1:8) both yielded proteolysis products that were in common with the trypsin-treated intact protein, migrating at positions corresponding to approximately 43 kDa (demonstrated by immunoprecipitation with BRIC 170, which reacts with the amino terminus of band 3, to be the cytoplasmic domain (data not shown) and at 26 kDa (Fig. 3 b, lanes 1-3). Superimposed upon this pattern was a ``ladder'' of distinctive proteolysis products running on the gel at apparent molecular masses as follows: intact band 3, 51 kDa (demonstrated to include the carboxyl terminus of band 3 by immunoprecipitation with BRIC 155; data not shown); b3(1:12), 41 kDa; and b3(1:8), 32 kDa. These proteolysis products correspond in size to the intact membrane domain portion of each carboxyl-terminally truncated fragment, resulting from cleavage after amino acid residue 359. The band 3 and b3(1:12) lanes also contained another proteolysis product that was not present in b3(1:8), running at approximately 35 kDa. This probably represents the proteolysis product from the trypsin cleavage site at amino acid 360 to that after residue 742. This tentative identification is supported by the presence of faint bands at lower apparent molecular weights in both band 3 and b3(1:12), which may represent the polypeptide from amino acid 743 to the carboxyl terminus. Very small amounts of residual undigested proteins may be seen running on the gel at apparent molecular masses as follows: band 3, 97 kDa; b3(1:12), 86 kDa; and b3(1:8), 70 kDa.

Trypsin treatment of the amino-terminally truncated fragment (9:14) yielded a major band migrating at approximately 24 kDa (Fig. 3 b, lane 6) that was slightly more intense than the equivalent band derived from intact band 3 (Fig. 3 b, lane 7) and may contain amino acids 743-911. Trypsin digestion of intact band 3 yielded a product migrating at approximately 28 kDa, which was of similar mobility to the undigested fragment (9:14). Other bands were obtained migrating with apparent molecular masses of 17 kDa in the case of (13:14) and at about 21 kDa in (9:14) and intact band 3.

The trypsin susceptibility of both the amino- and carboxyl-terminally truncated fragments is comparable with that of intact band 3, and these fragments are therefore likely to be folded in a manner similar to that in the intact protein. Furthermore, in the chymotrypsin cleavage experiment described in Fig. 8(below), fragments b3(1:12) and b3(1:8) were cleaved at the same site as intact band 3, with no indication of cleavage at additional sites. These observations, together with the analysis of the N-glycosylation of the fragments (Fig. 2), provide further evidence that both the amino- and carboxyl-terminally truncated band 3 fragments insert into the membrane with the correct topography and form tightly folded structures.


Figure 8: Immunoprecipitation of band 3 fragments from chymotrypsin-treated oocytes. Oocytes were co-injected with an equimolar quantity of either band 3 cRNA or of one or more band 3 fragment cRNAs, in the presence ( even-numbered lanes) or absence ( odd-numbered lanes) of 1.5 ng of GPA cRNA. The band 3-derived cRNAs injected were (per oocyte) lanes 1-4, 4.5 ng of b3(1:12); lanes 3 and 4, 1.5 ng of (13:14); lanes 5-8, 3.75 ng of b3(1:8); lane 7 and 8, 1.5 ng (9:14); lanes 9 and 10, 4.5 ng of band 3; lanes 11 and 12, 4.5 ng of b3(1:14*). Oocytes were pulse labeled with S-labeled amino acids for 24 h at 20 °C and then chased with unlabeled amino acids for a further 24 h. Groups of 10 oocytes were treated with chymotrypsin for 1 h at 4 °C and then immunoprecipitated with BRIC 170 (against the amino-terminal domain of band 3) as detailed in Ref. 11. Each lane represents immunoprecipitated protein from 10 oocytes, separated by SDS-PAGE on 9% Laemmli gels. Fluorographs were exposed for 18-48 h. The proportion of each band 3 fragment that was cleaved by chymotrypsin (to yield the 60-kDa amino-terminal portion of band 3) was determined by scanning densitometry of the SDS-PAGE fluorographs. Expressed protein was estimated from the areas under the peaks after allowing for the different amounts of radioactivity incorporated into the various cleaved and uncleaved fragments, based on their methionine and cysteine contents. In a parallel immunoprecipitation using BRIC 155 (directed against the COOH terminus of band 3), oocytes injected with intact band 3 and GPA cRNAs showed 42% cleavage of band 3 by chymotrypsin, which is comparable with the result obtained using BRIC 170 ( lane 10). nd indicates cleavage was too low to be determined ( lanes 11 and 12).



Expression of Band 3-mediated Anion Transport from Pairs of Band 3 Fragments

Xenopus oocytes were injected with cRNAs encoding various amino- and carboxyl-terminally truncated recombinant fragments of band 3, either individually or by co-injection of the complementary pairs of cRNAs, which together encode the entire protein sequence of the membrane domain. In each case, the cRNA coding for GPA was also co-expressed, as this has been shown to increase the amount of band 3 translocated to the plasma membrane of oocytes by up to 5-fold (11, 13) . After 24 h of incubation of the oocytes to allow the proteins to be expressed, the influx of Clinto the oocytes was measured over a 1-h period in either the presence or absence of the band 3-specific inhibitor DNDS (Fig. 4). The difference between the chloride influx values in the absence and presence of inhibitor (the DNDS-sensitive chloride influx) was used to provide an estimate of the band 3-specific anion transport in each case.

None of the truncated fragments b3(1:12), b3(1:8), (9:14), or (13:14) individually exhibited a measurable level of DNDS-sensitive chloride uptake into the oocytes (Fig. 4 a). However, co-injection of the pair of cRNAs encoding fragments b3(1:8) and (9:14) and the pair of fragments b3(1:12) and (13:14) did give rise to DNDS-sensitive chloride transport (significant at the 0.1% level in t tests). In the case of b3(1:12) and (13:14), the level of DNDS-sensitive transport induced in the oocytes was almost as high as that obtained by injection of an equal weight of the cRNA encoding intact band 3. These results show that these two pairs of band 3 fragments are able to complement each other to generate a protein structure that is competent for anion transport, which shows the stilbene disulfonate sensitivity characteristic of intact band 3.


Figure 4: Chloride influx into oocytes expressing band 3 fragments. Oocytes were injected with 1.5 ng ( a) or 5 ng ( b) of the band 3-derived cRNAs indicated, together with 1.5 ng of GPA (9 µl each) cRNA. Chloride influx (over 60 min) was measured 24 h after injection using groups of 12-15 oocytes, either in Barths saline ( hatched bars) or in Barths saline containing 400 µ M DNDS ( shaded bars), as described in Ref. 11. The bar shows the standard error of the chloride influx in each case. The DNDS-sensitive chloride influx may be derived from the difference between the mean values in the presence and absence of DNDS.



Co-expression of Pairs of Overlapping Recombinant Band 3 Fragments

We also co-injected oocytes with cRNAs encoding b3(1:12) and (9:14) at a higher concentration (5 ng each cRNA/oocyte) together with GPA cRNA. When anion transport activity was measured in the oocytes 24 h after injection, the level of chloride influx in the absence of DNDS was about 1.7 times greater than in the presence of DNDS (significant at the 5% level in a t test) (Fig. 4 b). Although this amount of chloride transport activity obtained with b3(1:12) and (9:14) was not as high as that observed with the complementary fragments b3(1:8) and (9:14) under the same conditions, these overlapping fragments (which duplicate transmembrane helices 9-12, amino acid residues 696-824) may be able to complement each other (poorly) to restore a low level of DNDS-sensitive chloride transport activity.

Effect of the Amino-terminal Cytoplasmic Domain and GPA on Band 3-mediated Anion Transport Activity by Co-expressed Fragments

The amino-terminal cytoplasmic domain of band 3 is structurally and functionally distinct from the membrane domain and is not essential for the expression of band 3-mediated anion transport in oocytes (11, 17) . We co-injected oocytes with the pairs of cRNAs encoding either mem(1:8) and (9:14) or b3(1:8) and (9:14) and with the pairs of cRNAs encoding either mem(1:12) and (13:14) or b3(1:12) and (13:14). When GPA cRNA was also co-injected, all four sets of fragments expressed a moderate level of DNDS-sensitive anion transport activity in comparison with the equivalent intact band 3 or band 3 membrane domain controls (Fig. 5). This confirms that the cytoplasmic domain is not involved in the process of folding and association of the band 3 fragments to generate the functional protein at the plasma membrane.

Fig. 5 also shows that although a modest level of DNDS-sensitive chloride transport was obtained with the intact band 3 and intact band 3 membrane domain controls in the absence of GPA, a very low level of DNDS-sensitive chloride influx was given by all of the four pairs of fragments when GPA was not co-expressed. To investigate whether GPA is essential for the expression of anion transport activity of the fragments, higher concentrations (>8-fold) of the cRNAs for b3(1:12) and (13:14) and for b3(1:8) and (9:14) were expressed in oocytes in the absence of GPA cRNA (Fig. 6). A measurable level of DNDS-sensitive anion transport was obtained under these conditions by co-expression of b3(1:12) and (13:14) (significant at the 0.1% level); however, even using these high concentrations of cRNA, no significant band 3-mediated chloride transport was detected in the oocytes when the pair of fragments b3(1:8) and (9:14) was expressed without GPA.


Figure 5: The effect of the band 3 cytoplasmic domain on the DNDS-sensitive chloride influx into oocytes expressing band 3 fragments. Oocytes were injected with equimolar quantities of cRNAs encoding either band 3 or the band 3 membrane domain (b3 mem) or various fragments of band 3 or b3 mem in either the presence or absence of GPA cRNA. The amounts of each cRNA injected were (per oocyte) 1.5 ng, band 3, b3(1:12), or GPA; 1.25 ng, b3(1:8); 1.0 ng, b3 mem or mem(1:12); 0.75 ng, mem(1:8); 0.5 ng, (13:14) or (9:14). Chloride influx (over 60 min) was measured 24 h after injection as described in Ref. 11. Each value shows the mean DNDS-sensitive chloride influx estimated from the difference between the means of groups of 13-14 oocytes, which were assayed in the presence and absence of 400 µ M DNDS. The bar shows the standard error of the mean DNDS-sensitive chloride influx in each case. Cross-hatched bars, with GPA absent; shaded bars, with GPA present.



Time-dependent Accumulation of Band 3 Fragments at the Cell Surface

We have shown previously that functional intact band 3 accumulates steadily in the plasma membrane of oocytes with time after injection of the cRNA (13) . Groups of oocytes were co-injected with equimolar quantities of the cRNAs encoding either b3(1:12) and (13:14) or b3(1:8) and (9:14) or intact band 3. GPA cRNA was also co-expressed in each case. The injected oocytes were incubated at 20 °C, and the the DNDS-sensitive chloride uptake into the oocytes was measured at various times over a 3-day period (Fig. 7). The results show that the expressed pairs of complementary fragments mediate increasing levels of DNDS-sensitive anion transport with the time of expression in a similar manner to the intact protein. In contrast, oocytes injected with a cRNA encoding b3(1:14*), which lacks 30 amino acids at the carboxyl-terminal cytoplasmic tail of band 3 (containing amino acid residues 1-881) (Fig. 1), induced no significant transport activity even after 3 days of expression in the oocytes.


Figure 1: Structure of fragments of band 3. Various fragments of band 3 were prepared as described under ``Materials and Methods.'' The amino acid ( aa) residues and the nomenclature used throughout this paper are indicated. Putative transmembrane segments are represented by analogy with those of the 14 span model for intact band 3. The arrows indicate the extracellular chymotrypsin cleavage site in each case.



Cell Surface Expression of Band 3 Fragments

Chymotrypsin cleavage has been used as a probe for the band 3 expressed at the surface of oocytes (11) . Band 3 in red cells is cleaved by chymotrypsin at a single extracellular site in the loop between putative transmembrane helices 5 and 6 to yield an amino-terminal 60-kDa fragment and a carboxyl-terminal 35-kDa fragment (18) . We used this assay to investigate the factors affecting the cell surface expression of each of the recombinant fragments that contains this chymotrypsin cleavage site. Since this method provides an assay for plasma membrane-expressed protein that is independent of the anion transport function, it also enabled us to investigate whether the individual fragments of band 3 (which do not mediate chloride transport) were translocated to the plasma membrane, rather than being retained in internal membranes or targeted for degradation in the cells.

Groups of oocytes were injected with an equimolar amount of the cRNA encoding either b3 (1:12) or b3(1:8) in either the presence or absence of their respective complementary partners (13:14) and (9:14) and the presence or absence of GPA. After pulse-chase labeling with S-labeled amino acids, the intact oocytes were treated with chymotrypsin. Subsequently, the radiolabeled protein was immunoprecipitated using the monoclonal antibody BRIC 170. The immunoprecipitated proteins were separated by SDS-PAGE to distinguish the intact fragments from the 60-kDa chymotryptic cleavage product, which originates from the fraction of the protein that is present at the oocyte cell surface. The percentage of the band 3 construct that had been cleaved by chymotrypsin was used to estimate the proportion of the protein located at the cell surface.

The results of the immunoprecipitations are shown in Fig. 8. As expected (11, 13) , the expression of intact band 3 at the cell surface was markedly GPA dependent and attained a high level in the presence of GPA (Fig. 8, lanes 9 and 10). Similarly high proportions of b3(1:12) and b3(1:8) were expressed at the cell surface when expressed as complementary pairs with (13:14) and (9:14), respectively, in the presence of GPA (Fig. 8, lanes 4 and 8). Co-expression of b3(1:12) with GPA in the absence of (13:14) also resulted in the expression of b3(1:12) at the cell surface (Fig. 8, lane 2), although the level was reduced to about 60% of that in the presence of both (13:14) and GPA (Fig. 8, lane 4). In contrast, co-expression of b3(1:8) and GPA in the absence of (9:14) did not result in a significant amount of b3(1:8) being expressed at the cell surface (Fig. 8, lane 6). This result was in marked contrast with the high level of cell surface expression of b3(1:8) in the presence of both (9:14) and GPA (Fig. 8, lane 8).

The co-expression of GPA substantially increased the proportion of b3(1:12) (Fig. 8, lanes 3 and 4) or b3(1:8) (Fig. 8, lanes 7 and 8) expressed at the cell surface when their respective complementary partners (13:14) and (9:14) were also present. The extent of the increase in cell surface expression induced by GPA was similar to that observed with intact band 3 (Fig. 8, lanes 9 and 10). In the absence of (13:14), the proportion of b3(1:12) expressed at the cell surface was markedly enhanced by the co-expression of GPA (Fig. 8, lanes 1 and 2). This result shows that GPA is able to facilitate the translocation of b3(1:12) to the plasma membrane in a manner similar to that with the intact protein and that (13:14) is not essential for the surface expression of b3(1:12). In contrast, very little b3(1:8) was translocated to the plasma membrane when expressed without (9:14) in either the presence of absence of GPA (Fig. 8, lanes 5 and 6).

Analysis by the chymotrypsin digestion assay showed that the carboxyl-terminally truncated fragment b3(1:14*) is not translocated to the plasma membrane of oocytes. The amount of b3(1:14*) recovered in these experiments suggests this construct is degraded in the oocytes, even though the more extensively truncated fragments b3(1:8) and b3(1:12) are expressed stably in oocytes. This low yield of b3(1:14*) was observed when the protein was expressed in either the presence (Fig. 8, lane 12) or absence (Fig. 8, lane 11) of GPA. Absence of the carboxyl terminus may cause the inappropriate exposure of a site in the protein (presumably in the region of transmembrane spans 13 and 14, between amino acid residues 825 and 881), which results in the intracellular retention and probable degradation of the protein by the oocyte. The cytoplasmic carboxyl-terminal tail of band 3 may be required for the expression at the cell surface of a band 3 construct containing all 14 transmembrane spans.


DISCUSSION

In this paper, we demonstrate that fragments of band 3 containing the first 8 and last 6 transmembrane spans co-expressed from separate cRNAs in Xenopus oocytes generate chloride transport activity in the oocyte plasma membrane with the stilbene disulfonate sensitivity characteristic of intact band 3. Similarly, reconstitution of anion transport activity can be achieved by co-expression of fragments of band 3 containing the first 12 and last 2 transmembrane spans from separate cRNAs. None of the individual fragments induced anion transport in oocytes without their complementary partner, and co-expression of GPA was required for high levels of cell surface expression to be observed, as is found with the intact protein (11, 13) . These results show that anion transport activity is not sensitive either to the introduction of a breakage in the polypeptide chain within the loops between transmembrane spans 8 and 9 or between spans 12 and 13 or to the consequent generation of -COOor -NH charged groups at the new termini of the fragment proteins within these loops. When designing the positions of our truncated band 3 constructs, we were careful to avoid introducing significant alterations to the charge distribution between cytoplasmic and extracellular sides of the fragment, since these could lead to an altered topology in the membrane (19, 20) . Furthermore, all of the fragments we have studied show a distribution of protease cleavage sites that is similar to that of intact band 3, suggesting that the topology of the truncated mutants (when expressed without their complementary partner) is similar to that of the same regions of the intact protein. The results suggest that there is no single signal sequence for inserting the different transmembrane spans of band 3 into the endoplasmic reticulum. It is clear that the band 3 fragments examined in this study can insert independently into this membrane so that each fragment contains sufficient information in its protein sequence to carry out this process.

The properties of the fragment (13:14) are of some interest since it contains two rather short hydrophobic segments. In the intact protein, the two ends of this fragment are located in the cytoplasm (reviewed in Refs. 4 and 7), while the region around Lysis accessible from the extracellular surface. Lysis a site of intramolecular cross-linking of band 3 by extracellular 4,4`-diisothiocyanato-2,2`-dihydrostilbene disulfonate at high pH, and small soluble peptides can be released from this region by proteolysis (21) . In addition, the mutation Pro Leu results in the formation of a new extracellular antigen on band 3 (22) . In the intact protein, the ends of the two short hydrophobic segments are accessible at both surfaces of the membrane. This could be achieved by these segments adopting either an extended structure or rather short helices at a location within the structure of the protein that is shielded from contact with the lipid by the remainder of the polypeptide (22) . Our results with fragment (13:14) indicate that this fragment inserts into microsomal membranes by itself and shows a resistance to proteolysis that is similar to that of the other fragments and to intact band 3. This suggests that the fragment is tightly folded in the absence of the remainder of the protein. If the membrane-penetrating portion of (13:14) takes up a predominantly helical conformation when expressed by itself, it seems unlikely that it would be able to span the bilayer completely. However, (13:14) efficiently forms a transport-active structure when co-expressed with fragment b3(1:12), and it is likely that (13:14) adopts the same membrane-spanning structure in the associated complex as in the intact protein. This suggests that the integral membrane portion of (13:14) may undergo substantial rearrangement of its structure when it associates with the remainder of the polypeptide chain.

The translocation of b3(1:12) to the cell surface is faster when (13:14) is co-expressed than when b3(1:12) is expressed without (13:14). Similarly, b3(1:8) moves faster to the cell surface in the presence of (9:14) than in its absence. These results suggest that the complementary pairs of fragments first associate in internal membranes rather than at the plasma membrane and that the complex is better able to translocate to the plasma membrane than either b3(1:12) or b3(1:8) individually.

In the absence of (9:14), fragment b3(1:8) was poorly expressed at the cell surface whether or not GPA was co-expressed. However, the movement of b3(1:12) to the plasma membrane in the absence of (13:14) was markedly accelerated by the presence of GPA. These results, which were also obtained in a duplicate set of experiments, suggest that the first 12 transmembrane segments of band 3 are sufficient for GPA-dependent translocation to the cell surface but that the first 8 spans alone will not suffice. Since it was possible to restore GPA-dependent translocation of b3(1:8) to the cell surface by complementation with (9:14), we conclude that a portion of the region containing transmembrane segments 9-12 is involved in the interaction between band 3 and GPA, which results in the enhanced movement of band 3 to the oocyte surface.

GPA has been shown to facilitate the translocation to the oocyte cell surface of the mutant form of band 3 (band 3 SAO) present in Southeast Asian ovalocytosis (23) . Band 3 SAO (reviewed in Ref. 5) contains a deletion of amino acid residues 400-408 (located at the cytoplasmic boundary of the first transmembrane segment of normal band 3), which probably results in the mis-assembly of the membrane domain. The deletion causes the loss of anion transport function and the inability to bind stilbene disulfonates (24) . However, extracellular cleavage of band 3 SAO by chymotrypsin appears to occur at the same site as normal band 3 (25, 26) , suggesting that the loop between transmembrane spans 5 and 6 has the normal extracellular location. It is likely that some or all of the amino-terminal four transmembrane segments are mis-folded in this variant form of band 3. The observation that the translocation of band 3 SAO to the plasma membrane is facilitated by GPA like the native protein is consistent with a portion of the region around membrane spans 9-12 being required for the GPA-dependent translocation of band 3. However, it is possible that some part of the band 3 membrane domain within transmembrane spans 5-8 is also involved in the interaction with GPA.

A ``two-stage model'' has been proposed to explain the process by which polytopic integral membrane proteins attain their folded structure (27) . This postulates that transmembrane -helices fold independently on insertion into the membrane, stabilized by main chain hydrogen bonds and interaction with the lipid bilayer, and that subsequently these autonomous -helical domains associate to yield the final structure without significant rearrangement of their secondary structure (reviewed in Refs. 28-30). In support of this hypothesis, several workers have generated functional bacterial membrane proteins by co-expression or reconstitution of two or more complementary protein fragments (reviewed in Ref. 31). Bacteriorhodopsin was refolded from three peptides comprising the first transmembrane span, the second span, and the remaining five spans, and after addition of retinal, a functional pump was reconstituted (32) . Similarly, lactose transport was restored by co-expression of fragments of the lac permease of E. coli containing 2 and 10 spans (33) or two 6-span fragments (34) . Iron (III) hydroxamate transport was restored by co-expression of two complementary fragments of the the FhuB protein (35) . There are a few examples where this complementation approach has been extended to the functional expression of eukaryotic membrane proteins. The adrenergic receptors have been co-expressed in oocytes from fragments containing 5 and 2 spans (36) , and human and rat muscarinic acetylcholine receptors have been expressed in COS cells, also from a 5- and a 2-span fragment (37) . Recently, the glucose transporter GLUT1 was expressed in the baculovirus system from fragments corresponding to the two homologous 6-span fragments that are a feature of this family of transporters (38) , and human P-glycoprotein activity has been reconstituted by co-expression of two polypeptides each containing six transmembrane spans (39) . In many of these cases, the individual fragments were expressed as stable proteins but were not functional unless both fragments were co-expressed.

The complementary fragments we have prepared are able to generate stilbene disulfonate-sensitive chloride transport and show several of the properties of the intact protein (GPA-dependent translocation to the surface, time-dependent accumulation at the cell surface, and inhibitor-sensitivity of anion transport). The individual fragments appear to take up tightly folded native-like structures, which can combine with their complementary partners to give a structure similar to that of intact band 3. While the association of the fragments may involve some minor structural rearrangements (for example, movements of the transmembrane helices), it seems unlikely that major unfolding and refolding events occur because of the high energy barriers involved. Our results are consistent with the ``two-stage model'' for membrane protein biosynthesis (27) , in which individual transmembrane helices fold independently on insertion into the membrane and retain this fold in the fully assembled protein.


FOOTNOTES

*
This work was supported by the Medical Research Council. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 44-117-9288271; Fax: 44-117-9288274.

The abbreviations used are: PCR, polymerase chain reaction; NLT, N-benzoyl-Asn-Leu-Thr- N-methylamide; DNDS, 4,4`-dinitro-2,2`-stilbene disulfonate; GPA, glycophorin A; PAGE, polyacrylamide gel electrophoresis.


ACKNOWLEDGEMENTS

We thank Dr. David Anstee for antibodies, Peter Martin and Julian Ng for assistance with preparation of the mutants, and Rhiannan Jowers for assistance with automated DNA sequencing.


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