Mapping the Ends of Transmembrane Segments in a Polytopic Membrane Protein
SCANNING N-GLYCOSYLATION MUTAGENESIS OF EXTRACYTOSOLIC LOOPS IN THE ANION EXCHANGER, BAND 3*

(Received for publication, January 15, 1997, and in revised form, May 14, 1997)

Milka Popov Dagger , Lisa Y. Tam Dagger , Jing Li and Reinhart A. F. Reithmeier §

From the Medical Research Council of Canada Group in Membrane Biology, Departments of Medicine and Biochemistry, University of Toronto, Toronto, Ontario, Canada M5S 1A8

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

Band 3, the anion exchanger of human erythrocytes, contains up to 14 transmembrane (TM) segments and has a single endogenous site of N-glycosylation at Asn642 in extracellular (EC) loop 4. The requirements for N-glycosylation of EC loops and the topology of this polytopic membrane protein were determined by scanning N-glycosylation mutagenesis and cell-free translation in a reticulocyte lysate supplemented with microsomal membranes. The endogenous and novel acceptor sites located near the middle of the 35 residue EC loop 4 were efficiently N-glycosylated; however, no N-glycosylation occurred at sites located within sharply defined regions close to the adjacent TM segments. Acceptor sites located in the center of EC loop 3, which contains 25 residues, were poorly N-glycosylated. Expansion of this loop with a 4-residue insert containing an acceptor site increased N-glycosylation. Acceptor sites located in short (<10 residues) loops (putative EC loops 1, 2, 6, and 7) were not N-glycosylated; however, insertion of EC loop 4 into EC loops 1, 2, or 7, but not 6, resulted in efficient N-glycosylation. Acceptor sites in putative intracellular (IC) loop 5 exhibited a similar pattern of N-glycosylation as EC loop 4, indicating a lumenal disposition during biosynthesis. To be efficiently N-glycosylated, EC loops in polytopic membrane proteins must be larger than 25 residues in size, with acceptor sites located greater than 12 residues away from the preceding TM segment and greater than 14 residues away from the following TM segment. Application of this requirement allowed a significant refinement of the topology of Band 3 including a more accurate mapping of the ends of TM segments. The strict distance dependence for N-glycosylation of loops suggests that TM segments in polytopic membrane proteins are held quite precisely within the translocation machinery during the N-glycosylation process.


INTRODUCTION

Band 3 (AE1) is the anion exchange (AE)1 protein of the erythrocyte membrane where it catalyzes the electro-neutral exchange of Cl- and HCO3- (1-6). Human Band 3 is a 911-amino acid polypeptide that consists of two domains, an amino-terminal domain located in the cytosol that is involved in cytoskeletal interactions and a carboxyl-terminal membrane domain responsible for anion exchange. Current models (see Fig. 1) indicate that the membrane domain may contain up to 14 TM segments, but the disposition of many of the hydrophilic loops connecting putative TM segments with respect to the membrane has not been established (4, 5, 7, 8).


Fig. 1. Initial folding model for the membrane domain of human Band 3 (TM 1/14 construct) containing 14 putative TM segments. EC loops are numbered EC 1-7 and IC loops are numbered IC 1-6. The single site of endogenous N-glycosylation in EC loop 4 is located at N642. This site is mutated to Asp (N642D) to create a non-glycosylated protein. The locations of the novel N-glycosylation acceptor sites created by site-directed mutagenesis are indicated by black diamonds. EC loop 4 insertions are indicated by the rectangular boxes. The 4-amino acid insert into EC loop 3 is indicated by the boxed VNSS. K539 and K851 are the residues cross-linked by extracellular H2DIDS. C843 is palmitoylated in native Band 3. Nt, amino terminus; Ct, carboxyl terminus; T, trypsin-sensitive site; C, chymotrypsin-sensitive site.
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Human Band 3 is N-glycosylated at Asn642 within EC loop 4 (9, 10). The mouse, rat, and chicken proteins are also N-glycosylated on the same loop (4). Interestingly, trout erythrocyte Band 3 is N-glycosylated on an expanded EC loop 3 (Z-loop) and does not contain an acceptor site in EC loop 4 (11). AE2 and AE3, like trout Band 3, contain N-glycosylation sites in an expanded EC loop 3 but none in EC loop 4 (4). The localization of N-glycosylated sites to single loops is a common feature of native polytopic membrane proteins (12), although N-glycosylation of multiple loops is possible (13).

The placement of novel N-glycosylation acceptor sites (Asn-Xaa-Ser/Thr) into putative extracellular or intracellular domains of polytopic membrane proteins has proven very useful in defining the folding pattern of the polypeptide in the membrane (14-20). Successful N-glycosylation requires that the acceptor site is exposed to the lumen of the endoplasmic reticulum during biosynthesis and is adequately spaced from the membrane surface (15, 20-23). In this paper, we have used this biosynthetic approach to refine the topology of Band 3 and to determine the requirements for N-glycosylation of loops in polytopic membrane proteins.


EXPERIMENTAL PROCEDURES

Materials

The following is a list of products and (in parentheses) their suppliers: MEGAscriptTM in vitro transcription kit, DNase I, and the cap analog m7GpppGp (Ambion); rabbit reticulocyte translation system, canine pancreatic microsomes, and RNasin (Promega); ModulisTM in vitro transcription/translation systems (MBI Fermentas); Single Tube Protein System 2, and T7 RNA polymerase (Novagen); [35S]methionine (Amersham Corp.); TransformerTM mutagenesis kit and Multi-PolTM DNA sequencing kit (CLONTECH); octaethylene glycol mono n-dodecyl ether (C12E8) (Nikkol); deoxyribonucleotides (Boehringer Mannheim); restriction enzymes, T4 DNA ligase, and Klenow DNA polymerase (New England Biolabs and Pharmacia Biotech Inc.); and [alpha -35S]ATP (ICN).

Oligonucleotide-directed Mutagenesis

The cDNA of human Band 3 in Bluescript vector BSSK+ (Stratagene) was a kind gift from Drs. A. M. Garcia and H. Lodish, Whitehead Institute. The endogenous N-glycosylation site in the membrane domain of Band 3 at Asn642 was mutated to Asp in the TM1/14 construct as described (24). A series of unique N-glycosylation acceptor sites (Table I) were created in the TM1/14 N642D construct using oligonucleotide-directed mutagenesis and the double-stranded mutagenesis system from CLONTECH.

Table I. N-glycosylation mutants in human Band 3 


N-glycosylation site Loop location and sizea Percent N-glycosylationb

Mutation
Endogenous site N642SS EC4 (35) 64  ± 6 (22)
  S644T N642ST EC4 (35) 59  ± 4 (6)
  E429N N429KT EC1 (10) 0 (4)
  L484S N482GS EC2 (10) 0 (4)
  Y555S, N556S N554SS EC3 (25) 18  ± 4 (5)
  Y555N, V557T N555NT EC3 (25) 24  ± 5 (6)
  L558T N556VT EC3 (25) 19  ± 4 (5)
  T627N N627YT EC4 (35) 0 (3)
  K631N, L632S N631ST EC4 (35) 0 (3)
  P635N, G637S N635DS EC4 (35) 0 (3)
  P635L, D636N, F638S N636GS EC4 (35) 0 (3)
  G637N, K639S N637FS EC4 (35) 35  ± 1 (3)
  F638N, V640S N638KS EC4 (35) 38  ± 3 (3)
  K639N N639VS EC4 (35) 68  ± 2 (3)
  S643N, A645S N643SS EC4 (35) 60  ± 2 (3)
  S644N, R646S N644AS EC4 (35) 66  ± 4 (3)
  A645N, G647S N645RS EC4 (35) 59  ± 3 (3)
  R647N, W648S N646GS EC4 (35) 67  ± 1 (2)
  G648N, V649S N647WS EC4 (35) 10  ± 9 (2)
  G648N, W648S, V649S N647SS EC4 (35) 58  ± 6 (3)
  W648N, I650S N648VS EC4 (35) 41  ± 4 (2)
  V649N, H650S N649IS EC4 (35) 0 (2)
  I650N, P652S N650HS EC4 (35) 0 (3)
  L655N N655RS EC4 (35) 0 (3)
  V729N N729RS IC5 (40) 0 (3)
  R730N, V732S N730SS IC5 (40) 0 (4)
  S731N N731VT IC5 (40) 4  ± 4 (9)
  V732N, H743S N732TS IC5 (40) 13  ± 2 (5)
  T733N, A735S N733HS IC5 (40) 0 (4)
  A735N, A737S N735NS IC5 (40) 26  ± 3 (2)
  L738S N736AS IC5 (40) 37  ± 3 (7)
  K743N N743AS IC5 (40) 38  ± 4 (7)
  P747N, A749S N747GS IC5 (40) 34  ± 4 (6)
  A749N, A751S N749AS IC5 (40) 32  ± 3 (6)
  A750N, Q752S N750AS IC5 (40) 11  ± 6 (5)
  A751N, I753S N751QS IC5 (40) 0 (4)
  Q752N, Q754S N752IS IC5 (40) 0 (4)
  I753N, E755S N753QS IC5 (40) 0 (3)
  E755N, K757S N755KS IC5 (40) 0 (3)
  R760N N760IS IC5 (40) 0 (6)
  P778A, I779N N779LS EC6 (5) 0 (4)
  P820Q, D821N, P823S N821VS IC6 (35) 0 (3)
  V828N N828KT IC6 (35) 0 (4)
  P854N N854AS EC7 (5) 0 (4)
  E882S N880VS Ct (40) 0 (3)
Insertion Mutants
  VNSSc N*557SS EC3 (29) 42  ± 6 (5)
  EC4 into EC1d N*642SS EC1 (45) 67  ± 8 (7)
  EC4 into EC2 N*642SS EC2 (45) 52  ± 9 (7)
  EC4 into EC6 N*642SS EC6 (40) 31  ± 6 (8)
  EC4 into EC7 N*642SS EC7 (40) 56  ± 13 (7)
  EC4 into IC3 N*642SS IC3 (50) 14  ± 2 (2)
  EC4 into IC5 N*642SS IC5 (75) 47  ± 1 (2)
  EC4 into IC6 N*642SS IC6 (70) 16  ± 1 (2)

a Loops are designated EC or IC according to the model shown in Fig. 1. Approximate loop sizes are estimated from the number of residues between the ends of consecutive hydrophobic TM segments as shown in Fig. 1.
b Quantitation of N-glycosylation was assayed by cell-free translation in the presence of microsomes. The percent N-glycosylation was calculated (± standard deviations) from scans of the autoradiographs. The number of determinations are in brackets.
c Four amino acid insertion into EC loop 3 between Y555 and N556.
d Insertion of EC loop 4 (endogenous N-glycosylation site) into EC loops 1, 2, 6, 7, or IC loops 3, 5, and 6 in the TM1/14 N642D construct.

Expansion of EC Loop 3

A "megaprimer" PCR method (25, 26) was used to expand the size and to insert a novel N-glycosylation acceptor site into EC loop 3 in the TM1/14 N642D construct. The resulting mutant had the additional sequence Val-Asn-Ser-Ser inserted between Tyr555 and Asn556. The flanking and mutagenic primers were: 5'-GCAGGGCATTCTCTTCGC-3' (forward primer), 5'-GTGGTGATCTGAGACTCCAGG-3' (reverse primer), 5'-ACTGCTGTTAACGTAGTTATAAGTCTTCTGTAGTG-3' (reverse mutagenic primer), and 5'-CTACGTTAACAGCAGTAACGTGTTGATGGTGCCCAAAC-3' (forward mutagenic primer). The bold lettering indicates the insertion that codes for the sequence Val-Asn-Ser-Ser. The underline indicates the position of a novel HpaI site that was used to screen for mutants.

Insertion of EC Loop 4 into EC Loops 1, 2, 6, and 7

Short EC loops were expanded by insertion of a 35-amino acid sequence derived from EC loop 4, containing the endogenous N-glycosylation acceptor site, into the TM1/14 N642D construct. Two oligonucleotide primers were used to synthesize a DNA segment corresponding to EC loop 4 with terminal XbaI sites by PCR (35 cycles at 94 °C, 1 min; 53 °C, 2 min; 72 °C, 3 min). Primer 1 was a sense oligonucleotide, 5'-GCTTCTAGATACCTACACCCAGAAACTCTC-3' and primer 2 was antisense oligonucleotide 5'-GAGTCTAGAAACTCGGAACGCAAGCCCAGT-3'. The underline indicates the XbaI cleavage sites. A unique XbaI cleavage site is located in Band 3 cDNA in a position corresponding to EC loop 2. The PCR product was ligated directly into this XbaI site in TM1/14 N642D construct. The endogenous XbaI site was mutated by a silent mutation CTA(Leu484)right-arrowCTG in TM1/14 N642D construct. Single novel XbaI restriction sites were then introduced at positions corresponding to EC loops 1, 6, and 7 and IC loops 3, 5, and 6. The same PCR product as described above was inserted into the novel XbaI sites. The resulting amino acid sequences at the insertion sites of the mutants are EC loop 1,  ... Leu427-Leu428-Asp626-Thr627 ... Glu658-Phe659-Leu428-Glu429-Lys430 ... ; EC loop 2,  ... Gly483-Leu484-Asp626-Thr627 ... Glu658-Phe659-Leu484-Glu485-Tyr486 ... ; EC loop 6,  ... Pro784-Leu785-Asp626-Thr627 ... Glu658-Phe659-Leu785-Asp786-Val787 ...; EC loop 7,  ... Thr853Leu854-Asp626-Thr627 ... Glu658-Phe659-Leu854-Asp855-Ser856 ... ; IC loop 3,  ... Pro598-Leu599-Asp626-Thr627 ... Glu658-Phe659-Leu599-Glu600-Leu601 ... ; IC loop 5,  ... Ile753-Leu754-Asp626-Thr627 ... Glu658-Phe659-Leu754-Glu755-Val756 ... ; and IC loop 6,  ... His819-Leu820-Asp626-Thr627 ... Glu658-Phe659-Leu820-Asp821-Val822... .

The amino acids in bold represent the inserted sequence corresponding to EC loop 4, and the underlined residues indicate amino acids that were altered or duplicated due to the creation of an XbaI restriction site. All mutations and insertions were confirmed by dideoxynucleotide chain termination DNA sequencing (27).

In Vitro Transcription and Translation

Plasmid DNA was linearized downstream of the stop codon with HindIII or with SacI; in the latter case, the ends were blunted with Klenow DNA polymerase. DNA (1 µg) was transcribed with T7 RNA polymerase in the presence of m7GpppGp for 4 h at 37 °C using the Ambion in vitro transcription kit according to the manufacturer instructions. The DNA template was removed by digestion with 2 units of DNase I for 15 min at 37 °C. The RNA was extracted with 1:1 phenol:chloroform, precipitated with 1/10 volume of 5 M ammonium acetate and 1 volume isopropyl alcohol, and then resuspended in diethylpyrocarbonate-treated water containing 2 units/µl RNasin.

Translation in a rabbit reticulocyte lysate was performed according to the suppliers. One-tenth (0.5-2 µg) of the RNA transcribed in vitro was added to 50 µl of translation mixture, and translation was allowed to continue for 2 h at 30 °C. The detergent C12E8 at 0.1% (w/v) was included in the translation mixture lacking microsomes to reduce aggregation of the translated protein. Translation was also carried out in the presence of microsomal membranes (7 units/50 µl, one unit of microsomes is the amount of membranes required to cleave the signal sequence of preprolactin by 50%). [35S]Methionine at 1 µCi/µl was included in all translation mixtures.

Alternatively, a coupled transcription/translation system supplied by MBI Fermentas was used. The transcription reaction was carried out with 0.3 µg of DNA using T7 RNA polymerase in a total volume of 3 µl. The sample was divided equally into three tubes, and the translation mix was added. Translation was carried out in the presence of 1) 0.1% C12E8, 2) 1 µl (2 units) of canine pancreatic microsomes, or 3) 1 µl microsomes and 32 µM Ac-Asn-Tyr-Thr-NH2 in a total volume of 10 µl. Each tube contained 10 µCi of [35S]Met. After incubation at 30 °C for 1 h, tRNA was digested with RNase A. Membrane integration of the translation products was determined by extracting samples (10 µl) with 100 µl of ice-cold 0.1 M Na2CO3, pH 11.5, followed by recovery of the stripped microsomes by centrifugation (16,000 × g for 15 min) (24). The translation products were examined by SDS gel electrophoresis (28) followed by autoradiography. The efficiency of N-glycosylation was determined by scanning autoradiographs of SDS gels of the cell-free translation products synthesized in the presence of microsomes using a Hoefer densitometer and MacIntegrator. The percentage of N-glycosylation was calculated from the integrated areas under the two peaks corresponding to the N-glycosylated and non-glycosylated full-length products. N-Glycosylation of the mutants was always compared with the wild-type TM1/14 construct synthesized in a parallel translation reaction.


RESULTS

Rationale

The requirements for N-glycosylation of EC loops and the topology of Band 3 were determined by scanning N-glycosylation mutagenesis. The endogenous site of N-glycosylation at Asn642 in EC loop 4 was mutated (N642D) to provide a non-glycosylated protein into which novel N-glycosylation acceptor sites (Asn-Xaa-Ser/Thr) could be introduced (Fig. 1). The loops connecting TM segments are poorly conserved (6), and therefore, single site mutations within them are not expected to have a profound effect on protein topology. A cell-free system consisting of a reticulocyte lysate supplemented with microsomal membranes was used to assay the N-glycosylation status of the mutants. N-glycosylation at a single site will produce a uniform increase of about 2,500 in the molecular weight of the protein corresponding to the cotranslational attachment of the high mannose oligosaccharide, which only occurs in the lumen of the microsomes. To confirm that the shift in mobility was due to N-glycosylation, one translation reaction in each set of translation mixes included a competitive inhibitor of N-glycosylation, the acceptor peptide Ac-Asn-Tyr-Thr-NH2. A truncated construct (TM1/14) (24) corresponding to the membrane domain of Band 3 (Fig. 1) was used in these experiments to improve the yield of full-length translation product and to better visualize the difference in molecular weight due to N-glycosylation.

EC Loops 3 and 4 Mutations

The endogenous site at Asn642 in EC loop 4 can be N-glycosylated by microsomal membranes in the reticulocyte translation system (Fig. 2, lanes 1 and 2). The non-glycosylated protein has a molecular mass of 48 kDa while the glycosylated protein is 3 kDa larger, consistent with the attachment of a single oligosaccharide chain. The average efficiency of N-glycosylation in the translation reaction was 65% (Table I). Increasing the amount of microsomes in the translation reaction did not result in a significant increase in the level of N-glycosylation. It has been reported (29, 30) that acceptor sites containing Thr can be more efficiently N-glycosylated than those containing Ser. To test this with Band 3, the endogenous site was mutated from Asn642-Ser-Ser to Asn642-Ser-Thr; however, no increase in the level of N-glycosylation was observed in the cell-free system (Table I). Both the N-glycosylated and non-glycosylated products were resistant to alkaline extraction showing that they were integrated into the lipid bilayer. The series of lower molecular mass bands around 35-40 kDa are due to internal initiation of translation at methionine residues located within TM 6 (24).


Fig. 2. Top panel, folding model showing the membrane domain of Band 3 and the position of the mutated endogenous N-glycosylation site at N642 (open circle) and the positions of the novel N-glycosylation sites and the VNSS insert in EC loop 3 (closed circles). Bottom panel, autoradiograph of the [35S]methionine-labeled cell-free translation products for the wild-type (lanes 1 and 2), EC loop 3 mutants: N554 (lanes 3 and 4), N555 (lanes 5 and 6), N556 (lanes 7 and 8), and the insert mutant VNSS (lanes 9 and 10). All translations were carried out in the presence of microsomes. The inhibitory peptide Ac-NYT-NH2 was present in the even numbered lanes. The open arrow indicates the position of the unglycosylated product, and the closed arrow indicates the N-glycosylated product. Numbers to the left indicate the migration positions of the standard proteins in kDa.
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EC loop 3 contains 25 residues and is sensitive to cleavage by extracellular chymotrypsin in intact red cells (31). A construct containing the mutated endogenous acceptor site and a novel site (Asn556) in the middle of EC loop 3 can be glycosylated as expected but only poorly (Fig. 2 and Table I). Other mutants containing acceptor sites one or two residues proximal (Asn554, or Asn555) were also N-glycosylated but again at low efficiency (Fig. 2 and Table I). This low efficiency may be due to the suboptimal size of EC loop 3 (12, 13), which may place the acceptor sites too close to the end of a TM segment to be properly N-glycosylated (23). To test this hypothesis, EC loop 3 was increased in size by insertional mutagenesis. This mutant contained the additional four residue sequence Val-Asn-Ser-Ser between Tyr555 and Asn556. The acceptor site within this insert was readily N-glycosylated (Fig. 2). Thus, this loop must be larger than 25 residues in size to be efficiently N-glycosylated, which places the acceptor sites 12 residues or more from the adjacent TM segments.


Fig. 3. Top panel, autoradiograph of the cell-free translation products for the scanning N-glycosylation mutagenesis of EC loop 4. Translation conditions were as in Fig. 2 except that all samples were extracted with alkali. Bottom panel (left), model of TM segments 7 and 8 and EC loop 4. N642 is the endogenous N-glycosylation site. Rectangles indicate limits of 21 residue TM segments as predicted by the 12 + 14 rule. Solid circles, efficient N-glycosylation; open circles, no N-glycosylation; hatched circle, low (less than 15%) N-glycosylation. The brackets indicate the 12 and 14 residues between sites of N-glycosylation and the hydrophobic ends of the bordering TM segments. Bottom panel (right), plot of average percent N-glycosylation (Table I) for separate translation experiments as a function of position of the acceptor site.
[View Larger Version of this Image (49K GIF file)]

Scanning N-glycosylation mutagenesis was performed on the endogenously N-glycosylated EC loop 4 to determine the spacing required for N-glycosylation of acceptor sites in loops. Acceptor sites located near the middle of this loop were N-glycosylated with an efficiency equal to the endogenous site (Table I and Fig. 3). One exception occurred with the sequon N647WS, which was poorly N-glycosylated relative to N647SS (Table I). This confirms the observation that sequons containing tryptophans are poor acceptor sites (32). Otherwise, the amino acid sequence context of acceptor sites in EC loop 4 had little effect on N-glycosylation in this cell-free assay. However, no N-glycosylation occurred at sites positioned close to either of the neighboring TM segments (Fig. 3). There was a sharp cut-off for N-glycosylation, which suggests that acceptor sites in loops must be positioned a minimum distance away from the lumenal ends of adjacent TM segments to reach the active site of the oligosaccharyl transferase. This requirement explains the poor N-glycosylation observed for the point mutations in EC loop 3. 

EC Loop 4 Insertions

The results of cell-free translation of mutants containing acceptor sites in other putative EC loops are shown in Fig. 4. No change in the mobility of the translation products occurred in the presence of microsomes for any of the mutants containing acceptor sites in EC loops 1, 2, 6, or 7. According to the folding model presented in Fig. 1, these loops are very short (5-10 residues), which may account for their inability to be N-glycosylated.


Fig. 4. Top panel, folding model showing the membrane domain of Band 3 and the positions of the EC loop mutations (closed circles) and the positions of the EC loop 4 insertion mutations. Bottom panel (left), autoradiograph of the cell-free translation products for point mutations in EC loop 1, N642WT (lanes 1 and 2), N429 (lanes 3 and 4); EC loop 2, N482 (lanes 5 and 6); EC loop 6, N779 (lanes 7 and 8), and EC loop 7, N854 (lanes 9 and 10). Bottom panel (right), autoradiograph of the cell-free translation products for constructs containing the EC loop 4 insertions into the regions corresponding to EC loop 1 (lane 1); EC loop 2 (lane 2); EC loop 6 (lane 3), and EC loop 7 (lane 4). Translation conditions as in Fig. 3.
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To determine the orientation of short loops with respect to the membrane, insertion of EC loop 4 into engineered XbaI sites was performed. A similar strategy was employed in investigations of the topology of 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase (15), the glucose transporter GLUT1 (18), and the sodium-dependent glucose transporter (20). The translation products containing the EC loop 4 insert are about 3 kDa larger than the corresponding TM1-14 proteins confirming that the expressed proteins contain the additional amino acid sequence. EC loop 4 inserted into EC loop 1 was N-glycosylated (Fig. 4), in contrast to the point mutation in EC loop 1. This confirms that acceptor sites in short loops facing the lumen of the endoplasmic reticulum cannot reach the active site of the oligosaccharyl transferase. The efficiency of N-glycosylation of this loop was close to the levels found for the wild type protein (Table I). This result shows that the inserted loop is accessible to the lumen of the endoplasmic reticulum and that this region of Band 3 is extracellular. Insertion of EC loop 4 into the endogenous XbaI site in EC loop 2 or EC loop 7 also resulted in optimal N-glycosylation (Fig. 4 and Table I). In contrast, the insert in EC loop 6 was N-glycosylated at a lower level (Fig. 4 and Table I). The low level of N-glycosylation observed for the insert in EC loop 6 suggests that only partial translocation of the loop into the lumen of the microsomes occurred in the cell-free system. A series of control EC loop 4 insertions into IC loops 3, 5, and 6 were also made. Insertions in IC loops 3 or 6 resulted in barely detectable N-glycosylation (Table I). The low level of N-glycosylation of these cytosolic loops suggests that some aberrant translocation had likely occurred during cell-free translation. Insertion into IC loop 5, however, resulted in a high level of N-glycosylation (Table I), inconsistent with a cytosolic disposition for this loop (see below). The insertion mutants suggest that EC loops 1, 2, and 7 and IC loop 6 in Band 3 are lumenal during biosynthesis while the disposition of putative EC loop 6 is ambiguous.

IC Loop Mutations

Figs. 5 and 6 show the results of a set of experiments with site-directed mutants containing acceptor sites in putative IC loops 5 and 6 and the carboxyl-terminal tail of Band 3. IC loop 5 contains a site at Lys743 that can be partially cleaved by trypsin contained within resealed ghosts (33). A point mutation (K743N) was made at this particular lysine to create a novel N-glycosylation acceptor site. Fig. 5 shows that this site, contrary to the 14 TM model, was N-glycosylated, suggesting that this hydrophilic loop is lumenal. This agrees with the results of the EC loop 4 insertion into IC loop 5. To confirm this finding, scanning N-glycosylation mutagenesis was performed on this loop. Acceptor sites located centrally between residues 735 and 748 were N-glycosylated; however, sites located close to the ends of the neighboring TM segments were not (Fig. 5 and Table I). This pattern of N-glycosylation was very similar to that found for the EC loop 4. These results show that this part of Band 3 is exposed to the lumen of the microsomes during biosynthesis and that acceptor sites in this loop must also be adequately spaced from the adjacent TM segments to be efficiently N-glycosylated. It is unlikely that all the individual point mutations in IC loop 5 resulted in a change in the orientation of this loop. Acceptor sites in IC loop 6 (Asn821,828) or in the carboxyl-terminal tail (Asn880) were not N-glycosylated, consistent with their intracellular disposition (Fig. 6). The lack of N-glycosylation of IC 6 and the carboxyl-terminal tail also confirms the specificity of the N-glycosylation assay.


Fig. 5. Top panel, autoradiograph of the cell-free translation products for the scanning N-glycosylation mutagenesis of IC loop 5. Translation conditions were as in Fig. 3. Bottom panel (left), model of new TM segments 9 and 10 and intervening loop. Rectangles indicate limits of 21-amino acid TM segments as predicted by the 12 + 14 rule. Solid circles, efficient N-glycosylation; hatched circles, low N-glycosylation; open circles, no N-glycosylation. The brackets labeled 12 and 14 indicate the number of residues between the optimal sites of N-glycosylation and the hydrophobic ends of the bordering TM segments. Bottom panel (right), plot of average percent N-glycosylation (Table I) for separate translation experiments as a function of position of the acceptor site.
[View Larger Version of this Image (43K GIF file)]


Fig. 6. Top panel, folding model showing the membrane domain of Band 3 and the positions of the IC loop 6 and carboxyl-terminal mutations. Bottom panel, autoradiograph of the cell-free translation products for IC loop 6 mutants: wild-type (lanes 1 and 2); N851 (lanes 3 and 4); N828 (lanes 5 and 6), and the C-terminal mutant, N880 (lanes 7 and 8). Translation conditions were as in Fig. 2.
[View Larger Version of this Image (36K GIF file)]


DISCUSSION

N-Glycosylation of Loops in Multispan Membrane Proteins

In this paper, we have defined the spacing requirements for the N-glycosylation of loops in the polytopic membrane protein Band 3. EC loop 3 in Band 3 contains about 25 residues, and three different point mutations near the center of this loop were barely N-glycosylated. Increasing the size of this loop by only four residues resulted in efficient N-glycosylation. The N-glycosylated EC loop 3 in trout Band 3 and in AE2 and AE3 contain an insert (Z-loop) doubling the size of the loop to about 50 residues. One reason for the larger size of EC loop 3 in these isoforms may be to allow efficient N-glycosylation. EC loop 4 in Band 3 contains about 35 residues, and sites located within a sharply defined threshold region near the middle of this loop were efficiently N-glycosylated. Point mutations in small EC loops in Band 3 were not N-glycosylated. However, insertion of EC loop 4 into these loops permitted efficient N-glycosylation. These findings are in agreement with observations made with other multispan membrane proteins in which short exofacial loops were also found to be poorly N-glycosylated (15, 18, 20-22).

A survey of polytopic membrane proteins revealed that EC loops containing N-linked oligosaccharides have a minimum size of about 30 residues, with the N-glycosylation site at least 10 residues away from a transmembrane segment (12). The distance between the oligosaccharyl transferase active site and the endoplasmic reticulum membrane has been determined using the Escherichia coli leader peptidase as a model membrane protein (23). This protein contains two TM segments with both the amino and carboxyl termini located in the periplasmic space. It was found that acceptor sites must be spaced greater than 12 residues COOH terminal from the second TM segment or greater than 14 residues NH2 terminal from the first TM segment to reach the active site of the lumenal oligosaccharyl transferase. The experiments described in this paper define similar limits for N-glycosylation of loops in the polytopic membrane protein Band 3. The distance requirements found for the leader peptidase can be applied to loops in polytopic membrane proteins. Thus, the acceptor site asparagine in loops must be spaced greater than 12 residues from the proximal and 14 residues from the distal TM segment to be efficiently N-glycosylated. For simplicity, this will be referred to as the "12 + 14 rule".

Mapping the Ends of TM Segments

The lumenal ends of TM segments can be mapped within a residue or two using the 12 + 14 rule. Acceptor sites at Asn554, Tyr555, or Asn556 in EC loop 3 are all poorly N-glycosylated, suggesting that they are closer than 12 or 14 residues from the ends of TM segments 5 and 6, respectively. The acceptor site in the four amino acid insertion (VNSS) between residues 555 and 556 in EC loop 3 can be N-glycosylated. This information maps the end of TM segment 5 to Phe544 and the beginning of TM segment 6 to Pro568, in agreement with the hydropathy analysis (Fig. 7).


Fig. 7. New folding model for Band 3 containing 12 TM segments. The lumenal ends of TM segments 5-10 have been refined using the 12 + 14 rule. The topology of the first eight TM segments is in good agreement with the starting 14 TM model. The original short TM segments 9 and 10 in Fig. 1 are now a single TM segment 9. The original IC loop 5 is on the lumenal side of the membrane during biosynthesis but is shown to loop into the protein to place K743 near the cytosolic side of the membrane. The original TM segments 11 and 12 in Fig. 1 are shown as a single TM segment but may span the membrane three times. The sequence between C843, which is palmitoylated, and the final EC loop is depicted as an extended structure. The final TM segment places the carboxyl terminus in the cytosol. Nt, amino terminus; Ct, carboxyl terminus; T, trypsin cleavage site. C, chymotrypsin cleavage site; Pa, papain cleavage site. Note that most of the trypsin and papain cleavage sites are only accessible after denaturation of the protein (45).
[View Larger Version of this Image (39K GIF file)]

TM segment 7 contains a well defined 21-residue hydrophobic sequence that extends from Val604 to Ile624 (Fig. 3). This segment is bordered by hydrophilic sequences including a pair of arginines on the cytosolic side of the membrane and a poorly-conserved Gln625 on the lumenal side (6). The closest acceptor site to TM segment 7 in EC loop 4 that can be N-glycosylated is located at Gly637. The 12 + 14 rule would place the end of TM7 at Ile624, 13 residues away from Gly637, in perfect agreement with the hydropathy profile prediction. Maximal N-glycosylation occurred at Lys639, two additional residues away from Gly637. Hydropathy profiles predict that a 21-residue segment corresponding to TM 8 begins at Pro660 and ends at Leu680. An acceptor site at Trp648 can be N-glycosylated, with maximal N-glycosylation one residue proximal at Gly647. The 12 + 14 rule defines the beginning of TM 8 at Met663, spaced 15 residues away from Trp648.

The N-glycosylation pattern for IC loop 5 is similar to EC loop 4, suggesting a lumenal disposition in the endoplasmic reticulum. The acceptor site at Ala735 is N-glycosylated, placing it greater than 12 residues away from the end of the preceding TM segment. This positions the end of TM segment 9 at Pro722. Similarly, the N-glycosylated acceptor site at Ala749 must be greater than 14 residues away from the beginning of TM 10. The first residue of TM 10 can be localized to Leu764, which defines the clear beginning of a very hydrophobic sequence (Fig. 7). This places the EC loop 4 insertion into EC loop 7 at Leu785 in the middle of the TM 10 (Fig. 7), which may account for its partial translocation.

Biosynthetic Considerations

The finding that acceptor sites in loops must be located a minimum distance away from adjacent TM segments to be N-glycosylated shows that TM segments are held quite precisely within the endoplasmic reticulum membrane during the N-glycosylation process (23). This precision is likely due to the interaction of TM segments with elements of the translocation machinery including the Sec61p complex and TRAM (34-37). These segments assume a helical conformation before entering the hydrophobic phase of the lipid bilayer (38, 39). The positioning of these helical TM segments within the translocon may be a prerequisite to their lateral movement into the membrane.

Polytopic membrane proteins usually contain a single N-glycosylated loop, and this loop is usually the largest EC loop in the protein (12). The results obtained in this study show that acceptor sites located in small loops or those in large loops located too close to a TM segment cannot reach the active site of the lumenal oligosaccharyl transferase. This would account for the lack of N-linked carbohydrate on acceptor sites found in short loops in some polytopic membrane proteins (12). The ability to N-glycosylate loops placed in various positions within the sequence of Band 3 and other polytopic membrane proteins (13, 15, 18, 20) shows that all lumenal loops of sufficient size are scanned by the active site of the oligosaccharyl transferase. This efficiency is due to intimate association of this lumenal enzyme with the translocation machinery in the endoplasmic reticulum (40-42).

Band 3 Topology

Fig. 7 presents a refined model for the folding of human erythrocyte Band 3 based on the results of scanning N-glycosylation mutagenesis. At the beginning of the membrane domain, there are five TM segments between the amino-terminal cytosolic domain and EC loop 3. Insertion of EC loop 4 into either EC loop 1 or 2 resulted in efficient N-glycosylation. The extracellular disposition of EC loop 1 is in agreement with labeling studies that show Lys430 is accessible to eosin maleimide (43) and reductive methylation (44) from the extracellular media. Also consistent with exposure of EC loop 1 is the finding that a peptide encompassing Leu427 to Gly436 can be released as a water-soluble fragment by pepsin digestion of ghost membranes (45). A recent study of the membrane insertion of truncated Band 3 molecules has shown that EC loop 2 is lumenal (46), in agreement with the results obtained in this paper. The conclusion that this loop is extracellular is not, however, supported by the examination of the orientation of Tyr486 (47). In that study, it was observed that Tyr486 could be radioiodinated by lactoperoxidase in inside-out vesicle but not in intact cells. Unfortunately, a labeled peptide corresponding to the predicted sequence Y486IVGR could not be unambiguously identified, and the yield of purified peptide was low, such that the total dpm in the final peptide fraction were only slightly above background. An expanded EC loop 3 can be N-glycosylated, and EC loop 4 contains the endogenous N-glycosylation site, consistent with the model in Fig. 1.

Contrary to the predictions of the model in Fig. 1, acceptor sites or the EC loop 4 insertion in the middle of IC loop 5 were N-glycosylated. These results suggest that during biosynthesis this loop is exposed to the lumen of the endoplasmic reticulum. This conclusion is consistent with the analysis of hydropathy profiles (6) that predicts that there is only one TM segment between residues Lys698 to Arg730 (Fig. 7). A previous study (33), however, showed that trypsin within resealed ghosts can partially cleave Band 3 at Lys743, producing a carboxyl terminal 20-kDa fragment at low yield. No such fragment could be produced by extensive treatment of intact cells with trypsin, suggesting that this site is exposed to the cytosolic side of the membrane. Soluble peptides derived from this loop can be produced by trypsin digestion of ghost membranes but only if Band 3 is first alkaline-denatured (45, 48). It appears that Lys743 is partially accessible from the cytosolic side of the membrane in the mature, folded protein but is lumenal during biosynthesis (Fig. 7). The lumenal exposure of this loop may be a feature of an intermediate in the folding pathway of Band 3. It was noted that the maximal level of N-glycosylation of sites within this loop was uniformly lower than that observed for the endogenous site (Table I). This hydrophilic loop is about 40 residues in length, which is large enough to be efficiently N-glycosylated. It is known that competition occurs between the processes of N-glycosylation and protein folding in the lumen of the ER (49). If this loop folds rapidly during biosynthesis, this may be responsible for limiting the extent of N-glycosylation. During the cell-free translation, this loop, in a fraction of the molecules, became trapped in the lumen by N-glycosylation at the novel acceptor site. Normally this loop folds into the protein to expose Lys743 to the cytosolic side of the membrane (Fig. 7). It is also possible that this part of Band 3 exhibits alternative topologies as has been reported for P-glycoprotein (50, 51) and has a dynamic aspect.

N-glycosylation acceptor sites located in the IC loop 6 were not N-glycosylated, consistent with their cytosolic location. This is in agreement with previous studies using monoclonal antibodies that localized the epitope encompassing Phe813 to Tyr824 on the cytosolic side of the membrane (52). The loop containing Lys743 and the cytosolic IC 6 are separated by a long hydrophobic sequence with a short hydrophilic and poorly conserved region (Glu777-Arg782), corresponding to putative EC loop 6 near the middle (Fig. 1). Insertion of EC loop 4 at the position of EC loop 6 resulted in poor N-glycosylation, inconsistent with a lumenal disposition for this region. We suspect that partial translocation of the EC loop 4 insertion occurred in the cell-free system due to disruption of the hydrophobic domain and its stop-transfer function. It is known that the amino-terminal halves of TM segments are not sufficient to act as a stop-transfer element in lactose permease-alkaline phosphatase fusions (53). Partial translocation and N-glycosylation of a large insert has also been observed with the glucose transporter (18).

Insertion of EC loop 4 into EC loop 7 resulted in efficient N-glycosylation of the mutant, placing EC loop 7 on the extracytosolic side of the membrane (Fig. 7). This agrees with the finding that the mutation P864L is associated with the Diego blood group antigen (54). In addition, Lys851, also located within EC loop 7, can be labeled with H2DIDS from the cell exterior, and a small peptide (Val849-Ala855) can be released from the membrane by extensive proteolysis (45). An N-glycosylation acceptor site within the carboxyl-terminal tail of Band 3 was not N-glycosylated. These results agree with previous studies (52, 55, 56) using antibodies to map the carboxyl terminus of Band 3 to the cytosolic side of the membrane. Cys843 is palmitoylated (57), which positions this residue on the cytosolic side of the membrane with the fatty acid extending into the inner leaflet of the lipid bilayer. The number of amino acids (~10) between the palmitoylated Cys843 and the last hydrophilic EC loop (K851STP) is not sufficient to cross a 30 Å bilayer as an alpha -helix. This short sequence is not likely to be exposed to the lipid bilayer but would be protected within a bundle of outer helices formed by other TM segments (58).


FOOTNOTES

*   This work was supported in part by a Group Grant from the Medical Research Council of Canada.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.
Dagger    Supported by University of Toronto Open Scholarship.
§   To whom correspondence should be addressed: MRC Group in Membrane Biology, Dept. of Medicine, Rm. 7344, Medical Sciences Bldg., University of Toronto, Toronto, Ontario, Canada M5S 1A8. Tel.: 416-978-7739; Fax: 416-978-8765; E-mail: r.reithmeier{at}utoronto.ca.
1   The abbreviations used are: AE, anion exchanger; C12E8, octaethylene glycol mono-n-dodecyl ether; EC, extracellular; IC, intracellular; TM, transmembrane; PCR, polymerase chain reaction.

ACKNOWLEDGEMENTS

We thank Dr. David Andrews (McMaster University) and MBI Fermentas (Flamborough, Canada) for the generous donation of high quality microsomal membranes and a coupled transcription/translation system.


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