INTRODUCTION

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Membrane proteins on the secretory pathway in eucaryotic cells are integrated into the membrane at the endoplasmic reticulum (ER)¹ and acquire their final membrane topology. Almost all the membrane proteins are integrated into the membrane during protein synthesis. Several sequences of their nascent polypeptide chains (the so-called "topogenic sequences") regulate the cotranslational insertion to define the final membrane topology (1-3). Signal sequences are responsible for ER targeting and for initiating the translocation. The signal sequences have been classified into three classes consisting of a signal peptide and two signal-anchor sequences (type I and type II signal-anchor sequences (SA-I and SA-II), respectively; Refs. 2 and 4). Signal peptide and SA-II mediate the translocation of their following portion showing N_cyt/C_exo orientation. Signal peptide is processed by signal peptidase, whereas SA-II is not cleaved to become a membrane-anchoring segment. In contrast, SA-I mediates the translocation of its amino-terminal portion and leaves the following portion on the cytoplasmic side of the membrane. All three signal sequences emerging from the ribosomes are recognized by the signal recognition particle and target the ribosomes to the ER membrane. The signal sequences are released from the signal recognition particle by signal recognition particle receptor and then enter into the protein translocation channel in the ER membrane forming loop structure. The stop-transfer sequence (St) is a hydrophobic segment that interrupts the ongoing protein translocation initiated by signal peptide and SA-II (5, 6). The cotranslational integration of the membrane proteins is mediated by the protein translocation channel (or the so-called translocon) consisting of the Sec61p complex and translocating chain-associated membrane protein (TRAM) (7, 8).

Although the topogenic functions of these sequences in simple single spanning membrane proteins have been established, their contribution to the membrane topogenesis of multispanning proteins remains to be clarified. According to a hypothesis that has been widely accepted (9, 10), nascent polypeptide on the ribosomes is targeted by the amino-terminal signal sequence in a signal recognition particle-mediated fashion, and then the following hydrophobic segments are sequentially integrated into the membrane from the N terminus while showing the alternative functions for either translocation initiation or stop-translocation (11); after the synthesis of cytoplasmic domain, the following transmembrane segment (TM) initiates the translocation of the following portion (internal SA-II), and the next transmembrane segment is supposed to stop the ongoing translocation (St; Ref. 9). This model, however, is not always applicable; in some contexts, certain transmembrane segments are left outside the membrane (12, 13), and some TM segments in the translocation initiation context cannot mediate the translocation of the following portion (14, 15). In this study, we aim to clarify the topogenic functions of individual TMs of a multispanning membrane protein, band 3.

Band 3 is a major multispanning membrane protein in erythrocytes, and its topology has been extensively studied from various approaches (16, 17). It consists of two domains: (a) an amino-terminal cytoplasmic domain of about 400 amino acid residues that bind to the cytoskeleton to regulate the cell shape of the erythrocytes, and (b) the carboxyl-terminal half, which is a transmembrane domain responsible for the anion-exchanging activity. A current model of the membrane topology of human band 3 deduces 14 TMs (Fig. 1; Ref. 18). We herein elucidate the topogenic functions of each TM and demonstrate that those of individual TMs are not always high enough to explain the integration process. We also suggest that several TMs in the stop-transfer context possess the potential of internal SA-I, which mediates the insertion of the preceding segment into the membrane.

View larger version (38K): [in this window] [in a new window]   Fig. 1.   The deduced transmembrane segments assessed in this study and topological model of human band 3. A, the amino acid sequence of the transmembrane domain of the human band 3. Acidic amino acid residues are underlined twice, and the basic residues are indicated by a dot. The transmembrane segments deduced in the model of Wood et al. (18) are indicated by the numbered boxes. The cytoplasmic (C) and extracellular (E) loops are indicated, which were included in the assay of the topogenic functions. B, the topological model of the transmembrane domain of band 3 on which this study is based.

	EXPERIMENTAL PROCEDURES

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Construction of Model Proteins

Systematic Constructs for Stop-Transfer and Translocation Initiation Assay-- Using each set of the primers, each DNA fragment encoding TM was amplified and digested with EcoRI and XhoI, whose sites were included in the PCR primers. In cases in which the TM is adjacent to cytoplasmic (Fig. 1, C) and extracellular (Fig. 1, E) loops of more than 5 amino acid residues, either of them was included in the amplified sequence. These TMs are designated according to their numerical order and the flanking loops included (Fig. 1; e.g. E8 corresponds to TM8 (M664-T685) including the preceding extracellular loop, Q625-M663).

Prolactin Fusions-- TM1 (Thr³⁷⁵-Arg⁴³², NcoI/XhoI), TM1-2 (Thr³⁷⁵-Gln⁴⁵⁷, NcoI/XhoI), and TM1-3 (Thr³⁷⁵-Ile⁴⁸⁷, NcoI/XhoI) were ligated with mature prolactin fragment (XhoI/XbaI) on pCITE2b (NcoI/XbaI) to yield pTM1-P, pTM1-2-P, and pTM1-3-P, respectively. TM1 (Thr³⁷⁵-Leu⁴²⁷, NcoI/XhoI), mature prolactin fragment SalI/MunI, TM2 (Gly⁴²⁸-Gln⁴⁵⁷, EcoRI/XhoI), and mature prolactin fragment XhoI/XbaI were sequentially ligated to yield pTM1-200-2-P. In all of these constructs, Thr³⁷⁵ was mutated to create the NcoI site. To monitor the translocation, the reporter of prolactin was mutated to include the N-glycosylation site by point mutagenesis (T90N).

Constructs for the Orientation Assay-- The glycosylated loop of band 3 (Asp⁶²⁶-Trp⁶⁶², NcoI/EcoRI), inserted fragment (EcoRI/XhoI), and mature prolactin (XhoI/XbaI) were sequentially ligated into pCITE2b (NcoI/XbaI). The Asp⁶²⁶ residue was changed to create an initiation codon and the NcoI site. In these cases, both the flanking cytoplasmic and extracellular loops were included in the inserted TMs (TM1 (Ala⁴⁰⁰-Gly⁴³⁶), TM4 (Phe⁴⁷⁸-Arg⁵¹⁸), TM6 (Leu⁵⁴⁰-Arg⁶⁰³), and TM8 (Gln⁶²⁵-Gly⁷⁰¹)).

In Vitro Transcription Translation and Protease Treatment

Both in vitro transcription and a topology assay were carried out as described previously (19). Plasmids were linearized by ScaI and then transcribed using T7 RNA polymerase. The RNAs were translated in reticulocyte lysate in either the absence or presence of rough microsomal membranes (RM). After translation, the aliquots were treated with proteinase K (200 µg/ml) and/or endoglycosidase H.

	RESULTS

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The major objective of this study was to examine whether or not the anticipated TMs of band 3 function individually during the cotranslational integration process. To this end, each TM with either an extracellular or a cytoplasmic loop was positioned in either the St or the internal SA-II context as described below. Four loops (L2, L9, L11, and L13) consisting of less than 5 residues were not examined.

Assessment of the Stop-Transfer Function-- The assessment of a stop-transfer function was carried out essentially as described by Kuroiwa et al. (5, 6) using systematically constructed model proteins (Fig. 2A). Translocation was initiated by the amino-terminal signal peptide. If the inserted TM shows the St function, then the carboxyl-terminal half is exposed on the cytoplasmic side of the membrane, where the carboxyl-terminal PL domain is sensitive to externally added proteinase K (Fig. 2B). If the inserted TM does not show the St function, the nascent polypeptide should be translocated into the lumenal space, where it becomes fully resistant to the externally added protease (Fig. 2B).

View larger version (25K): [in this window] [in a new window]   Fig. 2.   Assay for the stop-transfer function. A, the model protein for the systematic assessment of stop-transfer function. The signal peptide (S) of prolactin, mature prolactin (PL), the inserted TMs, and the second prolactin were sequentially arranged. Each prolactin domain is 200 amino acid residues long. B, the expected membrane topology of the model proteins. When the inserted TM functions as St, the carboxyl-terminal portion is located on the cytoplasmic side, where the second PL domain is sensitive to externally added proteinase K (Pro K). When it possesses no St function, the molecule is fully translocated into the lumen, where it is fully resistant to the protease. C, typical results using TM2 (E2; Gly428-Gln458), TM3 (3E; Leu459-Val488), and TM2-3 (Gly428-Val488). The model proteins were expressed in vitro in the absence () or presence (+) of RM. The aliquots were treated with proteinase K (PK+). The proteins were analyzed by SDS-PAGE and by a subsequent image analysis. The molecular weight markers are indicated on the left. The stop-transferred form and the fully translocated form are indicated by a and b, respectively. Both forms were quantitated by an image analysis in which the intensities were normalized according to the number of methionines in each protected band, and the stop-transfer efficiency was calculated by the formula [a] × 100/[(a) + (b)]. D, the results of the stop-transfer assay. The extracellular (E) and cytoplasmic (C) loops included in the assessed segment were indicated (e.g. C5 and 5E correspond to segments Gly509-Lys539 and Tyr519-Leu567, respectively).

Assessment of the Translocation Initiation Function-- To assess the internal SA-II function of the anticipated TMs, we constructed another series of model proteins (Fig. 3A). The H1 segment translocated the amino-terminal domain, which is glycosylated in the ER lumen (13, 20). If the inserted TM initiates the translocation of the following domain, then the carboxyl-terminal PL domain becomes proteinase K resistant (Fig. 3B).

View larger version (23K): [in this window] [in a new window]   Fig. 3.   Assay for the translocation initiation function. A, the model protein for the systematic assessment of the translocation initiation function. The Nexo/Ccyt SA-I of Escherichia coli leader peptidase (H1) preceding the domain with the N-glycosylation site, TMs, and the prolactin (PL) were sequentially arranged. B, the expected membrane topology of the model proteins. Membrane-targeted protein was glycosylated at the amino-terminal domain. When the inserted TM shows an internal SA-II function, the PL domain is translocated into the lumenal side, where it is resistant to externally added proteinase K (ProK). When it does not function, the molecule remains outside of the membrane and is degraded by the protease. C, typical results using TM9 (C9; Thr685-Pro722), TM10 (10C; Trp723-Val756), and TM9-10 (Thr685-Val756). The model proteins were expressed in vitro in the absence () or presence (+) of RM. Aliquots were treated with proteinase K (PK+). The glycosylated form (d, indicated by dots) indicates the ER targeting of the molecule. The translocated domain gave a truncated form (c) after proteinase K digestion. Both forms were quantitated by image analysis, and the translocation initiation efficiency was calculated by the formula [(c) × k1/(d)] × 100. k is the proteinase K protection efficiency of the translocated mature prolactin by the membrane that had been estimated by our experimental conditions using the following formula k = (mature PL after proteinase K treatment)/(mature PL before proteinase K treatment). The number of methionines in each protected form was compensated. D, the results of quantitation from the data from the translocation initiation assay. The numbers and symbols (E and C) are the same as those described in the Fig. 2 legend.

Efficient Membrane Integration of TM2 Requires the Existence of Flanking TM1-- TM2 and even the TM2-3 segment seemed to be insufficient for the stop-transfer sequence when they were assessed in the model constructs in which those were separated 200 residues from the amino-terminal signal peptide (Fig. 2). We then examined the topogenic properties of the amino-terminal TMs in the original context using three fusion constructs (Fig. 4A). When synthesized in the absence of RM, they gave single bands (Fig. 4B, lanes 1, 6, and 11). The lower molecular weight (M_r 25,000) bands in lanes 1 and 2 are most likely the results of an irregular translation initiation. When translated in the presence of RM, TM1 fusion gave a higher molecular weight form (lane 2). This band disappeared upon endoglycosidase H treatment (lane 5). The lower molecular weight band in lane 5 is the deglycosylated form of processed mature prolactin. Upon proteinase K treatment, a substantial quantity of the prolactin domains was resistant (lane 3), whereas these bands were completely degraded by treatment in the presence of detergent (lane 4). In contrast, the other two constructs with TM1-2 and TM1-3 were hardly glycosylated, if at all (lanes 7 and 12), and were almost completely degraded by proteinase K treatment (lanes 8 and 13). These data indicate that TM1 efficiently translocated the following prolactin domain, whereas TM2 interrupted the translocation under the original context. Furthermore, it was suggested that TM3 possesses no internal SA-II function.

View larger version (14K): [in this window] [in a new window]   Fig. 4.   Length between TM1 and TM2 affects the St function of TM2. A, constructs for examining the topogenic properties of the amino-terminal segments. Mature prolactin was fused to the ends of TM1 (Arg432), TM2 (Gln457), and TM3 (Ile487) of band 3 membrane domain. In TM1-200-2, the distance between TM1 and TM2 of the TM1-2 construct was elongated by inserting mature prolactin (200 residues). , the loop-connecting TMs. The reporter domains of prolactin were point mutated (T90N) to create a N-glycosylation consensus site (). B, a topology assay of the constructs. mRNAs were translated in the absence () or presence (+) of RM. The aliquots were treated with proteinase K (PK+) or endoglycosidase H (H). The proteinase K treatment was also carried out in the presence of detergent, Tx-100 (T). The efficiency of N-glycosylation is indicated (as a percentage), except for that of the TM1 construct, because a substantial amount of the TM1 construct was accessible to the signal peptidase.

TM4, TM6, and TM8 Show N_exo/C_cyt Topology-- The observations described above suggest that TM3, TM5, and TM7 are integrated into the membrane via a novel mode that differs from the conventional model. We hypothesized that the following TMs (TM4, TM6, and TM8) might possess an internal SA-I function by which their preceding segments could be integrated into the membrane. To test this possibility, we examined which orientation (N_exo/C_cyt or N_cyt/C_exo) is favored by these segments using model proteins (Fig. 5A). If the segment shows the SA-I function, the amino-terminal portion is translocated and glycosylated (Fig. 5B). If it shows the SA-II function, then the prolactin domain is translocated and becomes proteinase K resistant (Fig. 5B). When synthesized in the presence of RM, the constructs with TM4, TM6, and TM8 were glycosylated (Fig. 5C, dots). In contrast, the construct with TM1 was only slightly glycosylated, if at all. When treated by proteinase K after the translation, the TM1 construct gave a substantial amount of the proteinase K-resistant band (Fig. 5C, lane 12), whereas the others were degraded by proteinase K (lanes 3, 6, and 9). As shown in Fig. 5D, the three segments, TM4, TM6, and TM8, showed a SA-I tendency rather than a SA-II one. In contrast, TM1 showed an efficient SA-II function.

View larger version (22K): [in this window] [in a new window]   Fig. 5.   Nexo/Ccyt-topogenic function of even-numbered segments. A, the constructs for examining the preferred orientation of each TM. The glycosylated loop of band 3 (Asp626-Trp662), each segment including both flanking loops, and mature prolactin were fused sequentially. B, the expected membrane topology of the constructed proteins. If the segment shows Nexo/Ccyt SA-I function, the protein is glycosylated and is not resistant to proteinase. If it shows Ncyt/Cexo SA-II function, the PL domain becomes resistant to proteinase K, but the N terminus is not glycosylated. C, an in vitro assay of the topologies of the constructs. The constructs were expressed in vitro in the absence () or presence (+) of RM. Aliquots were treated by proteinase K (PK+). TM8 gave a diglycosylated form, because the segment included another glycosylated loop of band 3. The mono- and diglycosylated forms were indicated by single and double dots, respectively. The proteinase K-resistant forms were indicated by arrowheads. D, quantitation of the orientation of the segments. The glycosylated (g) and nonglycosylated (ng) forms and the proteinase K-resistant form (r form) were quantitated by image analysis, and the topogenic functions were calculated from the formulas (SA-I efficiency) = (g form) × 100/[(g form) + (ng form)] and (SA-II efficiency) = (r form) × k1 × 100/[(g form) + (ng form)], where k is the proteinase K protection efficiency of the translocated PL domain (see Fig. 3). The number of methionines in each protected form was compensated. Note that the ng form includes polypeptides that had not been targeted to the membrane as well as those with SA-II topology.

	DISCUSSION

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Each anticipated TM of human band 3 is assessed for the topogenic functions of internal SA-II and St, which are postulated to be responsible for the topogenesis of the multispanning membrane proteins. We found TMs to possess unequal topogenic functions. Several segments showed either insufficient or no supposed topogenic functions. The stop-transfer function of TM2 is affected by the distance from the preceding TM1. Some segments could not show the supposed internal SA-II function, but those were followed by highly hydrophobic segments with a de novo N_exo/C_cyt topogenic function. These facts could not be explained by the conventional model.

TM2 partially interrupts the ongoing translocation (only 40%) when it is placed 200 residues away from the preceding signal peptide (Fig. 2). In contrast to this fact, the stop-transfer action of TM2 improved when it was located near the TM1 segment, as in the case of the original band 3 molecule (Fig. 4). This result demonstrated that the stop-transfer action of the segment depends on its location. When the connecting loop is long, the preceding segment should be allowed to exit from the protein translocation channel (22). If the loop becomes shorter, the signal-anchor sequence should still be in the translocation channel. The existence of the preceding hydrophobic segment within the translocon would affect the translocation of the following portion; the presence of the preceding segment may affect the character of the translocon, or the two segments may directly interact with each other, so that translocation of the latter segment would easily stop within the translocon. Under such a context, the segment with a lower hydrophobicity can thus be integrated into the membrane.

A protein chemical analysis has already demonstrated that segments TM1 to TM3 of band 3 are stabilized in the membrane via an interaction between the peptide segments but not via protein-lipid interaction, because these segments were readily extracted from the erythrocyte membrane by alkali denaturation and subsequent protease treatment (16). These alkali-extractable transmembrane segments should be surrounded by other transmembrane segments that interact directly with the membrane lipids. This fact supports the idea that a direct interaction between TM1 and TM2 exists during the membrane insertion step. These segments are likely to be assembled within the translocon and are released into the hydrophobic environment as pointed out by Borel and Simon (23). The structure requirements that define such interaction are now under investigation.

Although the odd-numbered TMs of band 3 molecule have been supposed to possess an internal SA-II function that mediates the translocation of their following portion, TM3 and TM5 showed an unexpectedly weak internal SA-II function (Fig. 3). TM3 did not mediate the translocation of the reporter, even in the original context (Fig. 4). Despite these observations, the third loop connecting TM3 and TM4 was demonstrated to be in the lumenal space of the ER, because the glycosylated loop inserted between TM3 and TM4 was efficiently glycosylated (21). The loop connecting TM5 and TM6 has also been found in the lumen (21). To explain these discrepancies, we hypothesized that TM4 and TM6 should be an internal SA-I that possesses a N_exo/C_cyt orientation and promotes the integration of TM3 and TM5, respectively. Our results indicated this to be the case. Thus, it is strongly suggested that the odd-numbered segments (TM3, TM5, and TM7), which possess either no or low topogenic function, are integrated by the SA-I function of their following segments.

Consistent with the consideration above, Tam et al. (24) have reported that TM7 possesses partial SA-II activity, whereas the translocation of the loop between TM7 and TM8 was improved by the presence of TM8 (24). This observation supports our proposal that SA-I activity of TM8 contributes correct integration of its preceding region. Furthermore, it was demonstrated that fragments of band 3 membrane domain (e.g. TM9-14, TM8-14, and TM13-14) were correctly integrated into the membrane, indicating that there are unique topogenic sequences in these fragments (25, 26). In the case of the TM8-14 construct, the amino-terminal TM8 should be correctly inserted in a SA-I orientation. In the case of triple-spanning coronavirus M protein, each transmembrane segment was demonstrated to possess its intrinsic preferred orientation (27). The amino-terminal TM1 of cystic fibrosis transmembrane conductance regulator was inserted into the membrane depending on the following TM2 (28). Pairing of two transmembrane segments in the H⁺-ATPase was suggested to be essential for integration into the microsomal membrane (29). These findings also suggested that the transmembrane segment can contribute membrane insertion of their preceding segment.

We assessed the translocation initiation function of TM4, TM6, and TM8 in two different contexts (Figs. 3A and 5A). In the former construct, the loop connecting the H1 and the segment to be assessed was only 40 residues long, which is too short to allow the segments to form SA-I topology. In such a context, the presence of H1 itself dictates that the following TM cannot be inserted in a SA-I orientation. On the other hand, when they were placed near the N terminus, as in the latter case (Fig. 5A), they showed a strong SA-I function. In the original context of band 3 molecule, their preceding sequences including the TMs (TM3 and TM5) were sufficiently long and should be integration competent (as the glycosylated loop connecting TM7 and TM8). Thus, it is highly likely that in the original context of band 3, the even-numbered TMs mediate the integration of their preceding TMs.

The segment including both TM9 and TM10 was suggested to form one topogenic unit as SA-II. This conclusion is consistent with the report that the loop connecting these segments is on the lumenal side of the membrane at least during the process of biosynthesis (Fig. 6; Ref. 21). Based on these findings, TM11 is most likely a stop-transfer sequence. TM12 was demonstrated to be left outside of the cytoplasmic side of the membrane (21), whereas our observations suggested TM12 to be a highly functional internal SA-II sequence. This discrepancy remains to be clarified.

View larger version (22K): [in this window] [in a new window]   Fig. 6.   Model of topogenic functions of the TMs of band 3. An efficient St function of TM2 was observed only when it was close to the preceding TM1. Integration of TM3 and TM5 should be mediated by the function of internal SA-I of TM4 and TM6, respectively. TM 9 and TM10 functioned as a single topogenic unit, and the loop between TM10 and TM11 was located on the lumenal side of the membrane. Some parts of TM11 and/or TM12 segments were left out of the membrane. The segments classified by category 2 are indicated by the shadowed boxes (16).

Based on the abovementioned findings, we propose the following model of the topogenic process for band 3 (Fig. 6). TM1 is a SA-II that is responsible for ER targeting and for initiating the translocation of the following portion. TM2 interrupts the translocation by interacting with the closely positioned TM1. TM3, which has a weak topogenic function and low hydrophobicity, is integrated by the internal SA-I function of the following TM4. TM5 is also likely to be integrated by the SA-I function of TM6. TM7 has a moderate (but not sufficiently high) internal SA-II action to translocate the long hydrophilic loop with the N-glycosylation site. TM8 stops the translocation initiated by TM7 and also mediates the translocation of the glycosylated loop that had not been inserted by TM7. In this connection, the membrane topology in the TM7-loop-TM8 region is established by both the function of internal SA-I of TM8 and that of internal SA-II of TM7. TM9-10 functions as a single unit of internal SA-II as a whole. TM11 and/or TM12 is a stop-transfer segment. Some part of this segment may be left out of the hydrophobic core of the membrane.