©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Expression of a Functionally Active Human Renal Sodium-Calcium Exchanger Lacking a Signal Sequence (*)

(Received for publication, March 7, 1995; and in revised form, May 2, 1995)

Tip W. Loo Cheryl Ho (§) David M. Clarke (¶)

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

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The Na-Ca exchanger is an unusual membrane transport protein as it contains an NH(2)-terminal signal sequence which is co-translationally removed in the endoplasmic reticulum during synthesis. To determine if the signal sequence was essential for biosynthesis, mutations were introduced in the NH(2) terminus of the cDNA coding for the human renal Na-Ca exchanger in order to alter processing of the protein. To prevent cleavage of the signal sequence during biosynthesis, the last residue of the consensus signal sequence, Ala, was changed to Phe. Deletion mutants were also constructed to encode for exchangers which lacked the signal sequence, the signal sequence and the first extracellular loop, or all of the NH(2) terminus including the first transmembrane segment of the mature protein. These mutants were expressed in HEK 293 cells and assayed for Na-Ca exchange activity. Mutants lacking either a signal sequence or containing a noncleavable signal sequence were still targeted to the plasma membrane, where they exhibited Na-Ca exchange activity. By contrast, the mutants which had more than the signal sequence deleted did not demonstrate any exchange activity. These mutants were, however, still integrated into the membrane and were resistant to alkali extraction. These results show that the signal sequence is not essential for biogenesis of the Na-Ca exchanger and suggests that the molecule contains one or more internal signal sequences for insertion into the membrane during biosynthesis.


INTRODUCTION

The Na-Ca exchanger is an integral plasma membrane protein which functions primarily to extrude Ca from the cell (see reviews by Philipson and Nicoll (1992, 1993)). It is found in a wide variety of tissues including heart, brain, kidney, lung, large intestine, pancreas, and spleen (Kofuji et al., 1992; Lee et al., 1994). The physiological role of the Na-Ca exchanger has most clearly been established in cardiac muscle. Here, it acts as the dominant mechanism in extruding Ca from the cardiac myocyte after depolarization (Bers et al., 1990; Bridge et al., 1990).

The cardiac isoform of the Na-Ca exchanger, NCX1, was first cloned from canine heart (Nicoll et al., 1990). It soon became apparent that alternatively spliced variants of this gene were expressed in a tissue-specific manner (Kofuji et al., 1994; Lee et al., 1994; Loo and Clarke, 1994b). Recently, a second gene product, NCX2, has been identified in rat brain and has 65% amino acid identity to NCX1 (Li et al., 1994).

Hydropathy analysis of the NCX1 isoform of the exchanger predicts a molecule with 12 transmembrane segments (TM), (^1)with a large intracellular domain located between transmembrane segments 6 and 7 (Nicoll et al., 1990). This cytosolic domain contains the regulatory sites for calcium binding, inhibition by exchanger inhibitory peptide, and Na-dependent inactivation (Matsuoka et al., 1993). Mutant exchangers with deletions in the cytosolic domain retain the ability to carry out exchange activity (Matsuoka et al., 1993). These results indicate that ion binding and translocation sites are located within the membrane domains.

The first transmembrane segment of the exchanger was predicted to act as a signal sequence for insertion into the endoplasmic reticulum membrane during biosynthesis (Nicoll et al., 1990). It has been postulated that the signal sequence may be necessary to ensure correct orientation of the NH(2)-terminal end of the molecule (Durkin et al., 1991) and is removed during biosynthesis (Nicoll and Philipson, 1991; Durkin et al., 1991). This is in contrast to most polytopic membrane transport proteins, in which the first TM segment in the mature protein acts as an internal signal sequence that is not cleaved off during synthesis (Reithmeier, 1994). In addition, membrane transport proteins such as P-glycoprotein and the AE1 transporter contain multiple transmembrane segments which have topogenic properties of internal signal sequences (Zhang and Ling, 1991; Loo and Clarke, 1994a; Tam et al., 1994). It is possible that the Na-Ca exchanger may utilize a cleavable leader sequence at the NH(2)-terminal end because it lacks one or more internal signal sequences. To determine if the presence of a cleavable signal sequence is essential for biosynthesis of the exchanger, mutants were constructed to yield products which contained an uncleavable signal sequence or contained truncations at the NH(2)-terminal end of the molecule. The mutants were expressed in human kidney cells and assayed for their ability to carry out exchange activity. It was found that a mutant lacking a signal sequence or containing an uncleaved signal sequence were still targeted to the plasma membrane and were functional. By contrast, mutants with deletions greater than the signal sequence did not exhibit Na-Ca exchange activity.


EXPERIMENTAL PROCEDURES

Construction of Mutants

A 2,995-base pair EcoRI to XhoI fragment coding for the human renal isoform of the Na-Ca exchanger (Loo and Clarke, 1994b) was cloned into the EcoRI to XhoI sites of the mammalian expression vector, pMT21 (a gift from Dr. R. Kaufman, Genetics Institute, Boston), to create pMT21HEX.

An EcoRI to PstI fragment (nucleotides 1 to 532) of the exchanger was subcloned into the polylinker region of Bluescript vector (Stratagene) for site-directed mutagenesis by the method of Kunkel(1985). To create a mutant with an uncleavable signal sequence, the codon for Ala) of the consensus signal sequence was mutated to Phe with the oligonucleotide 5`-CCATGTAATTTTTGAGACAGAAA-3`.

In order to construct a mutant lacking the signal sequence, an NsiI site was created immediately following the initiating codon by changing the codon for Tyr^2 to His with oligonucleotide 5`-AGTGTCATGCATAACATGCGG-3`. Similarly, a second NsiI site was also created by changing the codons for Ala) to Met and Glu^1 to His by using oligonucleotide 5`-GACCATGTAATTATGCATACAGAAATGGAAGGA- 3`. The fragment coding for residues -1 to -35 was then removed by digestion with NsiI, followed by religation of the vector fragment.

Deletion mutants missing the first 49 or 64 amino acids of the mature protein were generated by first introducing an NcoI site at the initiating methionine, using oligonucleotide 5`-TTGGAAGTGCCATGGACAACATGC-3`. The fragment coding for residues 1 to 49 was removed as an NcoI fragment. The deletion mutant missing the first 64 amino acids was obtained by creating a second NcoI site at residue 65, with oligonucleotide 5`-CTGATCGGTCCATGGCCTCTATAGA-3`. Cleavage of this construct with NcoI, followed by religation, created an initiating methionine at residue 65 and also changed Ser to Gly.

Fragments containing the mutations were subcloned back into their original positions in pMT21HEX, for expression in HEK 293 cells. The integrity of the mutated segments of the cDNA were checked by sequencing the entire insert, including the cloning sites, by the dideoxy chain termination method (Sanger et al., 1977).

Construction of Chimeric Proteins

To monitor for size changes due to cleavage of the signal sequence or glycosylation, the fragments coding for the NH(2)-terminal end of the wild-type or mutant exchanger (residues -35 to 50) were ligated to the fragment coding for human calbindin D-28k (residues 47 to 261). The cDNA for human calbindin D-28k was cloned from the human kidney cortex library (Bell et al., 1986) and was found to be identical with that reported by Parmentier et al. (1987). (^2)

The EcoRI to PstI fragment (nucleotides 1 to 532) of the exchanger in Bluescript vector (Stratagene) was first digested with NcoI, filled-in with Klenow enzyme, and then digested with XhoI. The 1,100-base pair BstU1to XhoI fragment coding for residues 47 to 261 of human calbindin D-28k was then ligated into the NcoI-Klenow to XhoI sites in Bluescript vector containing the NH(2)-terminal end of the exchanger. The resulting construct in Bluescript vector contained the cDNA coding for residues -35 to 50 of the Na-Ca exchanger in-frame with that of human calbindin D-28k (residues 47 to 261). The chimeric cDNAs were then subcloned into the EcoRI to XhoI sites of vector pMT21 for expression in HEK 293 cells.

Cell Culture and Exchange Activity

Procedures for transfection of human HEK 293 cells have been described previously (Loo and Clarke, 1994c). Na-Ca exchange activity was measured on cells 48 h after transfection as [Na]-dependent [Ca] uptake as described by Li et al.(1992).

Immunological Procedures

Immunoblotting was performed using either 2 µg/ml anti-calbindin D-28k monoclonal antibody (Sigma) or a 1:500 dilution of rabbit polyclonal antibody against the large cytoplasmic domain of the exchanger (Loo and Clarke, 1994b) and the blots developed by enhanced chemiluminescence (Amersham).


RESULTS

Construction of Mutants and Expression in HEK 293 Cells

The first 35 amino acids of the human Na-Ca exchanger is a cleavable signal sequence which is removed during biosynthesis (Nicoll and Philipson, 1991; Durkin et al., 1991) (Fig. 1). The last residue of signal sequences (-1-position) has been observed to be one with a small uncharged side chain such as Ala, Gly, Ser, Cys, Thr, or Gln, which is postulated to fit into a pocket of the signal peptidase (von Heijne, 1983). It was suggested that the presence of a bulky side chain at the -1-position of the signal sequence would inhibit cleavage by the signal peptidase (von Heijne, 1983). Accordingly, in order to block the cleavage of the leader sequence of the exchanger, we mutated residue Ala Phe (Fig. 1, construct B). Deletion mutants were also constructed to test whether the NH(2)-terminal region encompassing the leader sequence and TM1 were essential for synthesis of a functional molecule that is targeted to the plasma membrane (Fig. 1). Mutants lacking the signal sequence (construct C), the signal sequence and the first extracellular loop (construct E), or lacking both the signal sequence and TM1 (construct F) were constructed. In addition, a glycosylation-deficient mutant (construct D) was also generated by mutating Asn^9 Ala. Hryshko et al.(1993) identified Asn^9 as the site of glycosylation using site-directed mutagenesis studies.


Figure 1: Amino acid sequences of the NH(2) termini of wild-type and mutants of the human renal Na-Ca exchanger. The linear representation of the Na-Ca exchanger showing hydrophobic segments (bold lines) predicted to form the leader sequence and 11 transmembrane segments is based on the model of Nicoll et al.(1990). The amino acid segment shown represents residues -35 to 66 of the unprocessed human Na-Ca exchanger. The sites where cleavage of the signal sequence () and glycosylation (Y) occur are indicated. The predicted amino acid sequences of the mature wild-type protein (A), mutant with a blocked signal sequence (Ala Phe) (B), mutant with deleted signal sequence (C), mutant lacking the glycosylation site (Asn^9 Ala) (D), and mutants with deletions past the signal sequence, Delta49 (E) and Delta64 (F) are shown.



A transient expression system utilizing human embryonic kidney cells (HEK 293) was used to monitor expression and function of the mutants. As shown in Fig. 2, HEK 293 cells transfected with the human kidney isoform of the Na-Ca exchanger (Loo and Clarke, 1994b) exhibited about 6-fold higher [Na]-dependent [Ca] exchange activity compared to cells transfected with vector alone. The exchange activity is rapid and is linear for the first 2 min. Recently, Gabellini et al. (1995) have reported that the level of exchange activity found in HEK 293 cells transfected with NCX1 is comparable with that found in isolated cardiac sarcolemmal membranes, when one considers that only a small percentage (10-20%) of the HEK cells express NCX1. These results suggest that the majority of the expressed NCX1 in HEK 293 cells resides in the plasma membrane. When the Na gradient across the cell membrane was abolished, no difference in exchange activity was observed between cells transfected with wild-type exchanger or with vector alone (data not shown). Low levels of Na-Ca exchange activity were, however, also present in cells transfected with vector alone. The low activity is due to the presence of endogenous Na-Ca exchanger in these cells. Indeed, the cDNA coding for Na-Ca exchanger used in this study was previously isolated from HEK 293 cells (Loo and Clarke, 1994b). The level of expression of the endogenous Na-Ca exchanger, however, was very low and did not appear to interfere with the expression studies (Fig. 3A).


Figure 2: Na-Ca exchange activity of HEK 293 cells transfected with wild-type Na-Ca exchanger. HEK 293 cells were transfected with cDNA coding for wild-type enzyme (bullet) or with vector alone (). 48 h after transfection, the cells were harvested by scraping, washed twice with phosphate-buffered saline, and loaded with Na by incubation in 10 mM MOPS, pH 7.4, 140 mM NaCl, 2 mM MgCl(2), 1 mM ouabain, and 25 µM nystatin as described by Li et al.(1992). The cells were washed in buffer containing no nystatin, and [Na]-dependent [Ca] activity was measured at room temperature in uptake medium containing either 140 mM KCl (uptake) or 140 mM NaCl (blank) and 10 mM MOPS, pH 7.4, 25 µM CaCl(2), 0.2 µCi/ml [Ca], and 1 mM ouabain. At various intervals, the reaction was stopped by addition of ice-cold stop solution containing 140 mM KCl, 1 mM EGTA, and 5 mM lanthanum chloride. The cells were collected by centrifugation, and the amount of radioactivity present was measured by liquid scintillation counting.




Figure 3: Immunoblot analysis of wild-type and mutant forms of the Na-Ca exchanger. A, HEK 293 cells were transfected with cDNAs of wild-type or mutant forms of the exchanger. After 48 h, the transfected cells were solubilized with SDS-PAGE sample buffer, heated for 2 min at 50 °C, and subjected to SDS-PAGE. The separated proteins were transferred onto nitrocellulose and analyzed with a rabbit polyclonal antibody against the cytoplasmic domain of the exchanger (Loo and Clarke, 1994b) and enhanced chemiluminescence (Amersham) as described under ``Experimental Procedures.'' B, N-glycanase F digestion. HEK 293 cells were transfected with wild-type exchanger, mutant with an uncleaved signal sequence (Ala Phe), glycosylation-deficient mutant Asn^9 Ala, or vector alone and solubilized with buffer containing 1% (w/v) SDS and 1% (v/v) 2-mercaptoethanol. The samples were then incubated in 50 mM sodium phosphate buffer, pH 7.5, containing 1% (v/v) Nonidet P-40 at 37 °C for 15 min in the presence (+) or absence(-) of PNGase F (1000 units, New England Biolabs). The reaction was stopped by addition of an equal volume of SDS-PAGE sample buffer, heated at 50 °C, and subjected to immunoblot analysis as described above.



Fig. 3A shows an immunoblot of wild-type renal Na-Ca exchanger and the NH(2)-terminal mutants expressed in HEK 293 cells. With the exception of the two largest deletions (Delta49 and Delta64 amino acids, respectively), the major immunoreactive product was a protein of an apparent mass of 115 kDa, which is in agreement with the predicted size of the protein. The 115-kDa products of wild-type exchanger, the mutant with an uncleavable signal sequence (Ala Phe), and the mutant with a deleted signal sequence (residues -1 to -35) were diffuse, suggesting that there was heterogeneity in glycosylation. Smaller immunoreactive products of approximately 80 kDa and 70 kDa were also observed in all cases and probably correspond to protease degradation products. A small increase in mobility on SDS-PAGE was noted for the glycosylation-deficient mutant Asn^9 Ala, consistent with the absence of oligosaccharide. We consistently observed that the level and pattern of expression were similar in cells expressing either wild-type or mutant exchangers. The full-length product of an apparent mass of 115 kDa was the most prominent product for the wild-type exchanger and all the mutants, except for mutants Delta49 and Delta64. In cells expressing these two mutants, the most prominent immunoreactive products had apparent masses of 70 kDa and 80 kDa, respectively. These observations suggest that the full-length products of mutants Delta49 and Delta64, were degraded more rapidly.

Expression of Chimeric Proteins

It was not possible to reliably ascertain by SDS-PAGE whether mutation of Ala Phe in the full-length exchanger did indeed prevent the cleavage of the signal sequence because of the small change in the size of the mutant product compared to wild-type enzyme. Similarly, removal of the oligosaccharide by N-glycanase F, resulted in only small changes in their molecular masses (Fig. 3B). In order to overcome these problems, small fusion proteins were made by joining the NH(2) termini (segment encompassing residues -35 to 50) of the Na-Ca exchanger to the soluble protein, calbindin D-28k (residues 47 to 261). Calbindin D-28k is a cytosolic vitamin D-dependent calcium-binding protein consisting of 261 amino acid residues (Fullmer and Wasserman, 1987). As shown in Fig. 4, expression of cDNA coding for calbindin D-28k in HEK 293 cells yields an immunoreactive product of an apparent mass of 26 kDa, which is not sensitive to treatment with endoglycosidase H. Endoglycosidase H removes core oligosaccharides which are added to glycosylated proteins in the lumen of the endoplasmic reticulum. Proteins whose carbohydrate moieties are further processed in the Golgi apparatus become resistant to treatment with endoglycosidase H. Expression of a chimeric protein consisting of the NH(2) terminus of the Na-Ca exchanger and calbindin D-28k (HEX-Calbindin) in HEK 293 cells yielded 3 immunoreactive products of apparent masses of 35 kDa, 32 kDa, and 28.5 kDa. The 32-kDa and 35-kDa products are likely to contain one and two core oligosaccharides, respectively, since only the 28.5-kDa product was observed after treatment with endoglycosidase H. The presence of two glycosylated sites was unexpected since the coding region for the NH(2)-terminal end of the exchanger does not contain any other consensus sites for N-linked glycosylation (Asn-X-Ser/Thr) other than at Asn^9. The calbindin passenger domain, however, contains two potential sites at residues Asn and Asn. Therefore, one of the glycosylated sites must occur within the calbindin passenger domain since the glycosylation-deficient mutant (Asn^9 Ala)-calbindin chimera was also glycosylated and sensitive to endoglycosidase H treatment (Fig. 4). These results indicate that at least part of the calbindin passenger domain in these chimeric proteins traversed the endoplasmic reticulum membrane and entered into the lumen. Mutation of Ala Phe successfully prevented cleavage of the signal sequence during biosynthesis since both glycosylated and endoglycosidase H-treated samples of HEX(A-1F)-Calbindin were about 3 kDa larger than the wild-type chimera (Fig. 4). The shift in molecular mass from 38.5 kDa to 31.5 kDa upon treatment with endoglycosidase H suggests that two sites had been glycosylated. It was also observed that the efficiency of glycosylation of mutant Ala Phe was much greater than that of the wild-type chimera (Fig. 4).


Figure 4: Expression and enzymatic deglycosylation of chimeric proteins constructed by fusion of the NH(2)-terminal domain of wild-type and mutant forms of the Na-Ca exchanger and human calbindin D-28k. The cDNAs coding for the chimeric proteins consisting of the fusion of residues 47 to 261 of human calbindin D-28k with the first 85 amino acids of wild-type exchanger (HEX-Calbindin), mutant exchanger with uncleavable (Ala Phe) signal sequence (HEX(A-1F)-Calbindin), or glycosylation-deficient exchanger (HEX(N9A)-Calbindin), were expressed in HEK 293 cells. The transfected cells were then solubilized with buffer containing 50 mM sodium citrate, pH 5.5, 0.5% (w/v) SDS, 10 mM EDTA, and 1% (v/v) 2-mercaptoethanol. The samples were divided into two equal portions, which were then incubated either with (+) or without(-) endoglycosidase H(f) (100 units, New England Biolabs) for 15 min at room temperature. The digestion was stopped by addition of an equal volume of buffer containing 0.25 M Tris-HCl, pH 6.8, 4% (w/v) SDS, 4% (v/v) 2-mercaptoethanol, and 20% (v/v) glycerol. The reaction mixtures were subjected to SDS-PAGE, transferred onto nitrocellulose, and analyzed with anti-calbindin D-28k monoclonal antibody (Sigma) and enhanced chemiluminescence (Amersham) as described under ``Experimental Procedures.''



Fusion of the NH(2) terminus of the exchanger to calbindin also resulted in translocation of at least a portion of the passenger domain into the lumen of the endoplasmic reticulum which was subsequently glycosylated. To determine whether the chimeric protein remained membrane-bound or was completely translocated into the lumen of the endoplasmic reticulum, the transfected cells were lysed and crude cytosolic and membrane fractions were prepared. Fig. 5shows that the majority of wild-type exchanger-calbindin chimeric protein was found in the cytosolic fraction, suggesting that the entire passenger molecule had entered the lumen of the endoplasmic reticulum following cleavage of the signal sequence. This chimeric protein was subsequently found to be exported from the cell (Fig. 6). By 48 h post-transfection, a greater amount of the wild-type exchanger-calbindin chimera was found in the culture medium compared to that found in the cellular extract. Similar results were obtained with the glycosylation-deficient mutant (Asn^9 Ala)-calbindin chimera (data not shown). By contrast, the mutant exchanger-calbindin chimera with an uncleaved signal remained associated with the membrane as an integral membrane protein since it was resistant to alkaline extraction (Fig. 5). Extraction of membranes with alkali removes all but integral membrane proteins from the membrane (Fujiki et al., 1982). The mutant exchanger-calbindin chimera with an uncleaved signal sequence is not exported since it was not detected in the culture medium (Fig. 6). These results indicate that the hydrophobic domain of the signal sequence (residues -7 to -30) was able to anchor the calbindin domain to the membrane. This would likely explain the increased efficiency of glycosylation of the (Ala Phe) mutant-calbindin chimeric protein relative to the wild-type chimera (Fig. 4). The wild-type chimeric protein may not be in the membrane of the endoplasmic reticulum long enough for glycosylation to be completed.


Figure 5: Alkali extraction of membranes from cells expressing wild-type or mutant chimeric proteins. HEK 293 cells were transfected with the cDNAs coding for the chimeric proteins generated by fusion of the NH(2)-terminal segments of wild-type exchanger (HEX-Cal) or mutant exchanger with an uncleavable signal sequence (HEX[A(-1)F]-Cal) with human calbindin D-28k. After 48 h, crude membranes were prepared from the the transfected cells as described by Clarke et al.(1989). The cells were harvested, a portion retained for immunoblot analysis (Whole Cells), and the remaining fraction was homogenized in the presence of buffer containing 10 mM Tris-HCl, pH 7.5, and 0.5 mM MgCl(2). Unbroken cells, nuclei, and mitochondria were removed by centrifugation at 4000 g for 10 min, The supernatant was then centrifuged at 180,000 g for 2 h, and the cytosolic material (Cytosolic Fraction) was retained for SDS-PAGE. The crude membrane fraction was suspended in buffer containing 10 mM Tris-HCl, pH 8.0, and 150 mM NaCl, and then an equal volume of 0.2 M sodium carbonate, pH 11.5 was added. After incubation on ice for 20 min, the samples were centrifuged at 180,000 g for 45 min. The pellet fraction (Carbonate-Extracted Membranes) was resuspended in a volume equal to the original cytosolic fraction, and equal volumes of samples were analyzed by SDS-PAGE and immunoblot analysis as described in the legend to Fig. 4.




Figure 6: Export of exchanger-calbindin chimeric proteins. HEK 293 cells were transfected with cDNAs coding for the wild-type exchanger-calbindin (HEX-Cal) or mutant exchanger with an uncleavable signal sequence-calbindin (HEX[A(-1)F]-Cal) chimeric constructs. After 48 h, the culture medium was harvested, and cells were removed by centrifugation at 4,000 g for 10 min (Extracellular fraction). The monolayer cells were harvested, washed with phosphate-buffered saline, and solubilized with SDS-PAGE sample buffer (Intracellular fraction). The samples were subjected to SDS-PAGE and immunoblot analysis with monoclonal antibody against calbindin D-28k as described in the legend to Fig. 4.



Na-CaExchange Activity of the Full-length Wild-type and Mutant Forms of the Na-CaExchanger

Cells transfected with the cDNAs coding for the full-length wild-type or mutant exchangers were assayed for Na-Ca exchange activity (Li et al., 1992). Fig. 7shows that the mutant with no signal sequence or with an uncleavable signal sequence exhibited exchange activity at levels comparable to that of the wild-type exchanger. These results indicate that the two mutants were correctly inserted into the endoplasmic reticulum and similar amounts of the exchanger must be present in the plasma membrane. Similarly, the glycosylation-deficient mutant, Asn^9 Ala, was also targeted to the plasma membrane and exhibited exchange activity, in agreement with the results obtained when the glycosylation-deficient mutant of rabbit cardiac exchanger was expressed in Xenopus oocytes (Hryshko et al., 1993). By contrast, no exchange activity was detected when mutants Delta49 or Delta64 were expressed in HEK 293 cells.


Figure 7: Comparison of the Na-Ca exchange activities of wild-type and mutant forms of the Na-Ca exchanger. HEK 293 cells were transfected with vector alone (A) or with the cDNAs coding for wild-type (B) or mutant forms of the Na-Ca exchanger lacking a signal sequence (residues -1 to -35) (C), lacking the glycosylation site (Asn^9 Ala) (D), containing an uncleaved signal sequence (Ala Phe) (E), or lacking residues 1 to 49 (F) or 1 to 64 (G). After 48 h, exchange activity was assayed for 1 min as described in the legend to Fig. 2. Each bar is the mean of 4 to 11 experiments and is expressed relative to wild-type activity (100%).



The deletion mutants Delta49 and Delta64 may have been inactive and subject to increased proteolytic digestion because they were not stably integrated into the membrane. To examine the ability of various deletion mutants to insert into the membranes, membranes were prepared from cells expressing these mutants and subjected to carbonate extraction. As shown in Fig. 8, all of the mutant proteins were recovered in the pellet fraction after alkali extraction of the membranes. These results indicate that the deletion mutants Delta49 and Delta64 were synthesized as integral membrane proteins.


Figure 8: Carbonate extraction of membranes containing wild-type and mutant forms of the Na-Ca exchanger. Membranes were prepared from HEK 293 cells transfected with the cDNAs coding for the wild-type exchanger or deletion mutants lacking the first 49 or 64 residues of the mature protein. The membranes were then extracted with sodium carbonate, pH 11.5. The resulting supernatant and pellet fractions along with a sample of untreated membranes (Total Membranes) were subjected to immunoblot analysis with a rabbit polyclonal antibody against the cytoplasmic domain of the exchanger, as described under ``Experimental Procedures.''




DISCUSSION

Cleavable signal sequences are usually present in eukaryotic proteins destined to be exported (Gierasch, 1989). They act to target the nascent polypeptide chain to the endoplasmic reticulum. In this study we found that the cleavable signal sequence was not essential for biosynthesis and targeting of the Na-Ca exchanger to the plasma membrane. An active exchanger targeted to the cell surface was obtained when the signal sequence was deleted or when cleavage of the signal sequence was blocked. In both instances, the protein product was glycosylated. By contrast, deletions that were greater than the signal sequence resulted in the synthesis of products that were rapidly degraded and whose exchange activity could not be detected. Deletions encompassing part of the TM1 segment of the mature protein may result in the inability of the protein to adopt its native conformation, since TM1 often acts as an internal signal-anchor sequence (Kolling and Hollenberg, 1994). For example, deletion of the first transmembrane sequence of SERCA1 Ca-ATPase resulted in an inactive enzyme and which was rapidly degraded (Skerjanc et al., 1993). In this case, the mutant was not stably inserted into the membrane, since most of the protein product was removed by alkali extraction. The deletion mutants Delta49 and Delta64, however, remained associated with the membrane, indicating that the exchanger contains other internal signal-anchor sequences. Internal signal-anchor sequences have recently been identified in a number of eukaryotic transport proteins such as AE1 (Tam et al., 1994) and P-glycoprotein (Loo and Clarke, 1994a, Zhang et al., 1995).

What is the role of the cleavable signal sequence in the Na-Ca exchanger? One possibility is that it may enhance membrane insertion. Such a situation has been observed for the human beta(2)-adrenergic receptor (Guan et al., 1992). It is a type IIIb membrane protein (NH(2) terminus on the extracellular side of the membrane but lacking a cleavable signal sequence). When a cleavable signal peptide was fused to this protein, translocation of the receptor protein into the endoplasmic reticulum was increased 2-fold. Such an increase would enhance expression of the exchanger in cell types which express low levels of the protein, but would not be detectable in the high levels of expression achieved by transient transfection in HEK 293 cells. Cleavage of the signal sequence may be required for the molecule to associate with itself or with other molecules. It has been noted that most membrane transport proteins are oligomeric (Klingenberg, 1981), but it has not been established whether oligomerization is required for transport. In the Na-H exchanger, it appears that the molecule forms stable dimers but individual monomers can function independently within the oligomeric state (Fafournoux et al., 1994). It is likely that the Na-Ca exchanger may also associate with other proteins in order to be localized to specific subcellular regions. It has been shown that the cardiac exchanger is preferentially localized to transverse tubules in heart sarcolemma (Frank et al., 1992), while renal exchanger is localized to the basolateral surfaces in the collecting ducts of the kidney (Reilly et al., 1993; Bourdeau et al., 1993). It is possible that the presence of a signal sequence would interfere with such associations if it was not removed.


FOOTNOTES

*
This research was supported by grants from the Medical Research Council of Canada and the Kidney Foundation of Canada (to D. M. C.). 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.

§
Supported by an Ontario Graduate Scholarship.

Scholar of the Medical Research Council of Canada. To whom correspondence and reprint requests should be addressed: Dept. of Medicine, University of Toronto, Rm. 7342, Medical Sciences Bldg., 1 King's College Circle, Toronto, Ontario M5S 1A8, Canada. Tel.: 416-978-1105; Fax: 416-978-8765.

(^1)
The abbreviations used are: TM, transmembrane segment; PAGE, polyacrylamide gel electrophoresis; MOPS, 3-(N-morpholino)propanesulfonic acid.

(^2)
T. W. Loo and D. M. Clarke, unpublished data.


ACKNOWLEDGEMENTS

We thank Dr. Randal Kaufman (Boston, MA) for pMT21.


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