©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Putative Amino-terminal Signal Peptide of the Cloned Rat Brain Na-Ca Exchanger Gene (RBE-1) Is Not Mandatory for Functional Expression (*)

(Received for publication, December 22, 1994; and in revised form, June 6, 1995)

Ian Furman Orna Cook Judith Kasir Walter Low Hannah Rahamimoff (§)

From theDepartment of Biochemistry, Hebrew University-Hadassah Medical School, Jerusalem 91120, Israel

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

The rat brain Na-Ca exchanger (RBE) gene, as well as other isoforms of this protein family, can be organized into 12 transmembrane alpha helices, the first of which was proposed by Durkin et al.(14) to constitute a cleavable signal peptide. We have prepared three amino-terminal mutants, in which 21, 26, and 31 amino acids beyond the initiating methionine were deleted. The deletions include the hydrophobic core of the putative signal peptide(N21), the entire putative signal peptide and parts of the putative signal peptidase cleavage site(N26), and the entire putative signal peptide and putative signal peptidase cleavage site(N31). All three mutant clones were transiently expressed in HeLa cells. The average Na gradient-dependent Ca transport activity of the mutant exchangers was 108%(N21), 37.2% (N26), and 60.06%(N31) of the wild-type clone. Mutation of the putative cleavage site by an exchange of Ala-32 Asp, resulted in a decrease in Na-Ca exchange activity to 7.7%, relative to the wild-type exchanger. Functional reconstitution of the proteins that were expressed in the transfected cells, resulted in transport activities of: 60.1%(N21), 26.75%(N26), 85.36%(N31), and 31% (Ala-32 Asp) relative to the wild-type exchanger. Western blot analysis of the protein profile of RBE-1, N21, N26, N31 and Ala-32 Asp-transfected HeLa cells was carried out by using an antipeptide antibody directed against a pentadecapeptide segment derived from the large putative cytoplasmic loop of the cloned rat exchanger gene. In the total cell extract and in the plasma membrane-enriched fraction, in addition to a major protein band of about 125 kDa, which corresponds to the molecular mass of the mature fully processed Na-Ca exchanger, an additional protein of about 135 kDa is revealed in the profile of N21- and N26-transfected cells. This band is not detected in the protein profile of RBE-1, N31, or Ala-32 Asp. The amino-terminal truncated mutants of the cloned Na-Ca exchanger could be expressed and processed also in a reticulocyte lysate supplemented with dog microsomes. Our results suggest that the putative signal peptide of the cloned Na-Ca exchanger gene does not play a mandatory role in functional expression of the protein in HeLa cells.


INTRODUCTION

The presence of an amino-terminal signal peptide earmarks the protein for insertion into the endoplasmic reticulum from where it is targeted to the plasma membrane via the Golgi apparatus. Many polytopic membrane proteins, however, do not contain cleavable amino termini that can be identified as a signal peptide, yet they are correctly targeted to the plasma membrane(1, 2, 3, 4) . It is thought that transmembrane segments in these proteins have targeting information.

The Na-Ca exchanger is a major Ca-regulating protein present in all excitable and many nonexcitable cells(5, 6) . The protein has been cloned(7, 8, 9, 10, 11, 12) and functionally expressed, and the presence of multiple isoforms, which are the product of two different genes(12, 13) , was established. Hydropathy analysis using a window of 20 amino acids suggested (7, 8, 13) that the cloned Na-Ca exchanger proteins can be organized into 12-transmembrane alpha helices. Partial sequencing, however, of the amino terminus of the purified bovine cardiac Na-Ca exchanger indicated that the first amino acid of this protein corresponds to amino acid number 33 of the cloned gene(14) . Hence it was suggested that the first putative transmembrane alpha helix (amino acids 1-24) and the next eight amino acids (25-32) that precede the amino terminus (amino acid 33), constitute a signal peptide that is presumably cleaved and hence not detectable in the ``mature'' protein.

In this work, we have examined the importance of the putative signal peptide of the cloned Na-Ca exchanger gene in functional expression of the transporter. Our studies indicate that neither the hydrophobic core of the putative signal peptide nor the following eight amino acids, which were suggested to be part of the putative signal peptidase cleavage site, are mandatory for functional expression of Na-Ca exchange activity.


EXPERIMENTAL PROCEDURES

Preparation of RBE-1^1 Mutants

Truncation of clone RBE-1 (9) has been carried out by the method of Kunkel(15) . Appropriate phosphorylated antisense primers were annealed to the uracil-containing single-stranded DNA derived from RBE-1. The primers were typically 30-40-mer long and flanking (about half of the nucleotides upstream and the other half downstream) the desired deletion. Synthesis of second strand was carried out in vitro using T4 DNA polymerase and T4 DNA ligase. Selection of mutant plasmids was based on sequencing of the segment containing the mutation. To ensure that no other mutations beyond the planned occurred, either a 0.4-kilobase pair HindIII-NcoI fragment (N21, N26, or N31) or a 0.4-kilobase pair SalI-NcoI fragment (Ala-32 Asp) containing the desired mutation was subcloned into a corresponding HindIII-NcoI- or SalI-NcoI-digested wild-type RBE-1. The entire subcloned fragment including the ligation sites were fully sequenced. The following antisense primers (5`to3`) were used: CAACATGGGTAAACAACATGTTGTACAATGAG(N21); CTGCAGTTATATGGTCCATGTTGTACAATGAG(N26); TTTCTGCCTCTGTATCCATGTTGTACAATGAG(N31); TTCTGCCTCTGTATCATCAGTTATATGGTCAAC (Ala-32 Asp). Plasmid preparations were carried out by standard procedures (16) or with the Wizard® Promega plasmid preparation kit. Sequencing was carried out by the dideoxy method (17) using the Sequenase II kit (U. S. Biochemical Corp.).

Expression of Na-CaExchange Activity

Transient expression of the wild-type cloned (in pBluescript) rat brain Na-Ca exchanger gene RBE-1 and its mutants in HeLa or L-cells was done essentially as described in (8) and (9) . 2.5 10^5 cells/well in a 24-well culture plate or 5 10^6 cells/90-mm Petri dish or 12.5 10^6 cells/135-mm Petri dish were transfected with 1.5, 15, or 37.5 µg of plasmid DNA, respectively. Dotap (Boehringer Mannheim, Germany) was used for transfection. Prior to transfection, the cells were infected with the recombinant vaccinia virus VTF-7(18) . Expression of transport activity was determined 16-17 h following transfection as described previously (8, 9) . Culture media were obtained from Biological Industries, Beit Haemek, Israel.

For determination of transport activity in whole cells, these were preincubated for 10 min with 0.14 M NaCl, 0.01 M Tris-HCl, pH 7.4, after which they were exposed to Ca in either 0.01 M Tris-HCl-buffered 0.14 M NaCl or 0.14 M KCl. The reactions were stopped by aspiration of the uptake media and two washes with 0.14 M KCl at 4 °C, after which the cells were solubilized with 0.3 N NaOH, neutralized with 0.2 M NaP(i), pH 4.5, and counted in a liquid scintillation counter. Net Na gradient-dependent Ca uptake was calculated by subtraction of the amount of Ca associated with the cells in the presence of external NaCl (no gradient) from the amount of Ca associated with the cells in the presence of external KCl. Each measurement was done in triplicate. For reconstitution experiments, cells were harvested and dissolved in a solution containing 0.2 M NaP(i), pH 7.4, 2% cholate, and 15-20 mg/ml brain phospholipids as described previously(8, 9) . Reconstitution and determination of Na gradient-dependent Ca uptake was done as in (19) . Protein was determined by the method of Lowry et al.(20) .

Expression of Cloned Na-CaExchangers in Reticulocyte Lysate

The TNT® mRNA-free combined transcription/translation reticulocyte lysate system (Promega) was used. To detect protein synthesis, [S]methionine (Amersham Corp., SJ1515) was added to the lysate. The manufacturer's instructions were followed with fidelity, except that the K concentration was increased by 40 mM. When stated, dog microsomes (Promega) were added to the lysate (2 µl of microsomes/25 µl of final volume of lysate reaction mixture). The proteins synthesized were analyzed on SDS-containing polyacrylamide gels(21) . The acrylamide and N,N`-methylenebisacrylamide concentrations were reduced to 5 and 0.125%, respectively. 8 M urea was always added to the sample buffer, otherwise the proteins synthesized in the lysate aggregated and did not enter the gel.

To evaluate the extent of the glycosylation by the microsomes, translation products were treated with peptide N-glycosidase F (New England BioLabs), and the size of the proteins synthesized before and after the treatment was determined by SDS-polyacrylamide gel electrophoresis (see above). Peptide N-glycosidase treatment was carried out as specified by the manufacturer, and it involved boiling for 10 min in 0.5% SDS, 1% beta-mercaptoethanol and addition of Nonidet P-40 to a final concentration of 1%.

Northern Blots

Total RNA was extracted from 17 h postinfected/transfected HeLa cells, using the TRI® reagent (Molecular Research Center, Inc. Cincinnati OH). RNA separation using formaldehyde-containing 1% agarose gels was carried out by standard procedures(16) . Following electrophoresis, the gels were soaked in 0.5 N NaOH and then in 20 SSC, after which the denatured RNA was transferred to nitrocellulose by capillary elution. Hybridization was carried out in 50% formamide buffer overnight at 53 °C. As probes, we have used either a [alpha-P]dCTP-labeled polymerase chain reaction-amplified DNA fragment of 863 base pairs corresponding to nucleotides 1936-2799 of the cloned exchanger gene or a [-P]ATP-labeled 53-mer synthetic antisense oligonucleotide corresponding to nucleotides 14-67 (the numbering refers to the open reading frame of the cloned exchanger RBE-1) of the following sequence: 5`-AGAGAGCCACCAGAGTTACCAGACGAAATCCCATTGAAACATTGGGTGGGAGAC-3`. Labeling of the 863-base pair probe was done by random priming using the Klenow fragment of DNA polymerase, and that of the oligonucleotide with T4 polynucleotide kinase. Quantitative densitometric analysis of the Northern blots was done with the Fuji thermal imager FTI 500 using the Macintosh program Image.

Western Blot Analysis

Transfected HeLa cells were rinsed twice in phosphate-buffered saline, scraped from the 135-mm culture dish with a rubber policeman, and divided into three parts. One part was used for determination of expression of Na gradient-dependent Ca transport activity. The second part was pelleted, suspended in a minimal volume of phosphate-buffered saline, clarified by a 10-s sonication, and used without further treatment. This total cell extract contained the entire repertoire of proteins produced in the transfected HeLa cells. The third part was suspended in 5 mM Tris-HCl, pH 7.4, 1 mM EDTA, 0.02 mM phenylmethylsulfonyl fluoride and subfractionated by differential centrifugation into a plasma membrane-enriched fraction P30, sedimenting at 30,000 g and an endoplasmic reticulum-enriched fraction P100 sedimenting at 100,000 g. From each fraction an aliquot was kept for the determination of protein. The different fractions were separated by SDS-containing gel electrophoresis (21) and transfered electrophoretically by standard procedures to nitrocellulose(22) . As primary antibody, we have used antiserum (AbO-8) obtained from a rabbit immunized with a synthetic pentadecapeptide derived from amino acids 645-659 (9) of the cloned rat brain exchanger RBE-1. The peptide was coupled to large nonimmunogenic carriers keyhole limpets hemocyanin, bovine serum albumin, and thyroglobulin using glutaraldehyde(22) . Each subsequent injection of the peptide was done with a different carrier. Antibody production was tested by enzyme-linked immunosorbent assay first against the peptide and then against transfected HeLa cells. Mock transfected cells, with pBluescript were used as controls. For Western blots, the antiserum was purified by preadsorption on HeLa cells. This was done by incubating the antiserum with the cells at a dilution of 1:20 (in 3% bovine serum albumin, 0.02% NaN(3) in phosphate-buffered saline) for 60 min at 25 °C, after which the cells were sedimented and discarded. Usually, confluent cells harvested from a 25-ml culture flask were used for each ml of diluted antiserum. Before use, the purified antiserum was further diluted in 3% bovine serum albumin, 0.02% NaN(3) in phosphate-buffered saline to a final dilution of 1:500. I-Protein A (DuPont NEN) was used as a secondary antibody.

Quantitative analysis of the protein profile was carried out with the Fujix Bas 1000 PhosphorImager using the Tina 2.06 analysis program.

The plasma membrane fraction was characterized by measuring the specific activity of 5`-AMP nucleotidase. In the P30 fraction, the specific activity of the enzyme increased between 3- and 6-fold in different preparations over the total cell extract and about 15-fold over the P100 fraction.


RESULTS

The Role of the Amino-terminal Segment of the Cloned Na-Ca Exchanger Gene in Functional Expression

In order to investigate the role of the amino-terminal segment of the cloned exchanger gene RBE-1 in functional expression of Na-Ca exchange activity, we have prepared three amino-terminal deletion mutants of the protein: N21, N26, and N31 (see Table1). All of the deletions conserved the initiating methionine. Mutant N21, in which we have deleted the initial 21 amino acids beyond the initiating methionine, was designed to test the role of the hydrophobic core of the putative signal peptide in functional expression. The deletion of amino acids 2-21, removed most of the hydrophobic core of the amino-terminal segment and the two positive charges within it that are thought to be important in the interaction with the negatively charged surface of the inner membrane of the endoplasmic reticulum(24) . In mutant N26, we had deleted amino acids 2-27. This deletion included the entire hydrophobic segment of the putative signal peptide (amino acids 2-24), and three of the amino acids that presumably form part of the putative signal peptidase recognition and cleavage site(24, 25) . The longest truncation was done in mutant N31, in which all of the amino acids between the initiating methionine and the putative amino-terminal aspartic acid were deleted. This mutant lacks the entire putative signal peptide and the putative signal peptidase cleavage site.



All of the amino-terminal mutants were expressed transiently in HeLa cells using the recombinant vaccinia virus VTF-7 expression system (18) . The wild-type exchanger (clone RBE-1) was always expressed in parallel to the mutant exchangers. Na-Ca exchange activity was determined in ``whole cells'' exactly as described previously(8, 9) .

Fig.1summarizes the results obtained. In Fig.1A, Na gradient dependent Ca uptake of the three amino-terminal mutants N21, N26, and N31 in whole cells is shown. Since the expression of transport activity varied in different transfection experiments, the numerical value of the average steady state rate of Na gradient-dependent Ca uptake (nmol of Ca transported/mg of HeLa cell protein) of the wild-type exchanger RBE-1 in each experiment has been defined as 100%, and the transport activity of the mutant clones is presented in relative values. Fig.1B, shows the numerical value of the average Ca transport activity in these experiments (n = 12), of the wild-type exchanger RBE-1 in the presence (dottedbar) and in the absence (clearbar) of a driving Na gradient (for details, see ``Experimental Procedures''). Net Na gradient-dependent Ca uptake is calculated by subtracting the amount of Ca transported in the absence of a driving Na gradient from that obtained in its presence. It should be also noted, that no endogenous Na-Ca exchange activity could be detected in infected/nontransfected HeLa cells (8, 9) or infected/control plasmid (pBluescript SK) (mock) transfected cells. From the data presented in Fig.1A, it can be seen that the expression of transport activity of N21 is similar to that of the wild-type clone RBE-1, suggesting that the hydrophobic core of the putative signal peptide does not seem to play a significant role in functional expression of the Na-Ca exchanger. Determination of the transport activity of whole HeLa cells transfected with N26 and N31 revealed that these mutants are functional as well. Na gradient-dependent Ca transport activity of these mutants was 35.4% (S.D. = 5.3) and 60.06% (S.D. = 8.48) of that of the wild-type exchanger. Although the expression of transport activity of these amino-terminally truncated mutants is somewhat lower than that of the wild-type clone, the results suggest that mutant protein is synthesized and is inserted in functional form into the plasma membrane.


Figure 1: Expression of Na gradient-dependent Ca uptake in transfected HeLa cells. Infected HeLa cells (for details see ``Experimental Procedures'') were transfected with wild-type or mutant plasmid DNA. 17 h posttransfection, Ca uptake in the presence and in the absence of a driving Na gradient was measured for 10 min. The amount of Ca taken up by the cells in a Na gradient dependent manner was calculated. A, expression of the relative Na gradient-dependent Ca uptake in HeLa cells transfected with the wild-type and amino-terminal truncated Na-Ca exchanger mutants N21, N26, and N31. The average transport activity of the wild-type clone RBE-1 was defined as 100% in each separate experiment, and the transport activity of the mutant exchangers that were expressed in parallel is presented in relative values. B, the numerical value of the average (n = 12) Ca uptake of the wild-type Na-Ca exchanger gene RBE-1 determined in the presence (dottedbar) and in the absence (clearbar) of a driving Na gradient. n = number of experiments; each separate experiment was done in triplicate.



Functional expression of amino-terminal truncated mutants of the cloned Na-Ca exchanger gene is not restricted to HeLa cells, since transfection of L-cells with the same mutant exchangers resulted in functional expression similar to that obtained in HeLa cells. Relative to the wild-type exchanger RBE-1, 65, 43, and 38.3% of the Na-Ca exchange activity was obtained when L-cells were transfected with the truncated mutants N21, N26, and N31, respectively (data not shown).

There are several ways to explain the somewhat lower transport activity of the truncated mutants N26 and N31 relative to the wild-type exchanger. One explanation could be that these mutants have an impaired trafficking machinery to the plasma membrane. To test this hypothesis, we reconstituted into brain phospholipids the proteins expressed in wild-type and mutant Na-Ca exchanger-transfected HeLa cells and examined their transport activity. Our rationale was that by solubilization of the transfected cells and reconstitution of the proteins into proteoliposomes, the presence of intracellular Na-Ca exchangers that were completed but not targeted to the plasma membrane would be revealed. If the proportion of these nontargeted Na-Ca exchangers within HeLa cells transfected with the mutant exchangers, N26 and N31 is significant, transport activity following reconstitution, should increase and result in values similar to those detected in cells transfected with the wild-type exchanger or mutant N21.

The results of these experiments are shown in Fig.2A. It can be seen, that the transport activity of the reconstituted amino-terminal truncated Na-Ca exchangers relative to the transport activity of the wild-type exchanger, is in principle similar to the transport activity obtained in the whole cell experiments. Fig.2B shows the average Ca transport activity (n = 9) of the reconstituted wild-type exchanger RBE-1 in the presence (dottedbar) and in the absence (clearbar) of a driving Na gradient.


Figure 2: Expression of Na gradient-dependent Ca uptake in transfected HeLa cells, measured following reconstitution of the proteins synthesized into proteoliposomes. The experimental conditions are identical to those described in Fig.1, except that 17 h posttransfection the proteins produced in the HeLa cells were solubilized and reconstituted into proteoliposomes. Reconstitution and determination of transport activity is described under ``Experimental Procedures.'' A, expression of the relative Na gradient-dependent Ca uptake activity determined in the wild-type and amino-terminal-truncated mutant-transfected HeLa cells. The transport activity of the wild-type clone, RBE-1 transfected HeLa cell preparation was defined as 100% in each separate experiment, and the transport activity of the mutant exchangers, which was determined in parallel, is presented in relative values. B, the numerical value of the average (n = 9) Ca uptake of the wild-type Na-Ca exchanger gene RBE-1 measured in the presence (dottedbar) and in the absence (clearbar) of a driving Na gradient. n = number of experiments; each experiment involved triplicate determinations.



These experiments suggest that truncation of the entire putative signal peptide of the cloned Na-Ca exchanger gene RBE-1 including the signal peptidase cleavage site (mutant N31) do not result in an accumulation of significant amounts of intracellular Na-Ca exchangers.

To try and elucidate the possible role of the signal peptide and that of the signal peptidase cleavage site in functional expression of the Na-Ca exchanger, a mutant exchanger was prepared in which the putative cleavage site Ala-32 was changed to Asp. We chose this exchange since analysis of the patterns of amino acids near the cleavage site (23) indicated, that Asp was not found in position -1.

Transfection of HeLa cells with this mutant clone indicated that its Na-Ca exchange activity was only 7.7% (S.D. = 4.4; n = 5) when compared with the wild-type exchanger RBE-1 (see Fig.3), which was tested in parallel, suggesting that the amino acids in the vicinity of cleavage of the signal peptide might be of importance in functional expression.


Figure 3: Expression of Na gradient-dependent Ca uptake in HeLa cells transfected with the exchange mutant Ala-32 Asp. Infected HeLa cells were transfected with the cloned wild-type Na-Ca exchanger gene RBE-1 and with the exchange mutant Ala-32 Asp in parallel. 17 h posttransfection Na gradient-dependent Ca uptake activity has been determined in whole cells (barA) and following reconstitution (barB) of the proteins present in the transfected cells exactly as described in Fig.1and Fig. 2. The average transport activity of the wild-type exchanger was defined as 100% in each separate experiment and the transport activity of the mutant clone is presented in relative values. (n = 5)



It was interesting to note that reconstitution of the proteins synthesized in the Ala-32 Asp-transfected HeLa cells led to an increase in transport activity. Compared with the 7.7% relative to the wild-type exchanger in whole cells, following reconstitution, the transport activity increased to 31% (S.D. = 12.07; n = 5). This would suggest that either some protein was not targeted to the plasma membrane or it acquired functional conformation only following reconstitution.

Can the Lower Transport Activity of the Amino-terminal Truncated Mutants be Related to Lower Transcription?

To test this possibility, we have isolated RNA from HeLa cells infected with the recombinant vaccinia virus VTF-7 and mock transfected with the plasmid pBluescript KS and from HeLa cells transfected with the wild-type and the three mutant cloned Na-Ca exchanger genes. Northern blot analysis (Fig.4A) using a 863-base pair probe (spanning nucleotides 1936-2799 of the cloned rat Na-Ca exchanger gene) revealed that 1) the probe did not hybridize to RNA isolated from infected HeLa cells (laneA) or infected and mock-transfected (with pBluescript KS) HeLa cells (laneB); 2) the probe hybridized to RNA isolated from HeLa cells transfected with either wild-type (laneC) or the mutant exchangers (lanesD-F); 3) in most experiments (as also in that shown in Fig.4A), we did not detect significant differences between the amount of the wild-type and mutant mRNAs, although their transport activity was somewhat lower ( Fig.1and 2). Hence, there is no clear correlation in most experiments between the expression of Na-Ca exchange activity, which usually was somewhat lower when the cells were transfected with N26 and N31 and the amount of mutant-derived mRNA.


Figure 4: Expression of wild-type and mutant mRNAs in transfected HeLa cells. Total RNA was isolated from HeLa cells transfected with the wild-type Na-Ca exchanger gene RBE-1 and its truncated mutants N21, N26, and N31 (for details see ``Experimental Procedures''). 10 µg of RNA was layered in each lane and separated on denaturing formaldehyde containing agarose gels. A, a polymerase chain reaction-amplified 863-base pair-long DNA segment labeled with [alpha-P]dCTP derived from the cloned wild-type Na-Ca exchanger gene (nucleotides 1936-2799) was used as a probe. RNA isolated from infected HeLa cells (A), RNA isolated from infected and pBluescript KS transfected HeLa cells (B), and RNA isolated from HeLa cells infected and transfected with the wild-type Na-Ca exchanger gene RBE-1 (C) and its truncated mutants N21, N26, and N31 (D-F), respectively. B, a 53-mer antisense oligonucleotide (nucleotides 14-67) labeled with [-P]dATP was used. RNA isolated from infected and pBluescript KS-transfected HeLa cells (A) or infected and transfected with the wild-type exchanger RBE-1 or its truncated mutants N21, N26, and N31 (B-E), respectively.



We have also hybridized to these RNA preparations an antisense 53-mer-long oligonucleotide derived from the truncated amino-terminal region of the cloned Na-Ca exchanger genes (see ``Experimental Procedures''). Fig.4B shows that the oligonucleotide hybridized only to RNA isolated from HeLa cells transfected with the wild-type Na-Ca exchanger (laneB). This rules out any possibility that a contaminant signal peptide containing plasmid, either in the transfecting DNAs or in the cells, was responsible for the transport activity of the truncated mutants.

Distribution of the Wild-type and Mutant Na-Ca Exchanger Protein in Transfected HeLa Cells

We have also tested directly the correlation between the functional expression of Na-Ca exchange activity, the length of the putative signal peptide, and the nature of the signal peptidase cleavage site with the amount of Na-Ca exchanger protein synthesized and targeted to the plasma membrane. Western blot analysis was carried out to detect Na-Ca exchanger-derived protein in a total extract obtained from transfected HeLa cells, in a plasma membrane-enriched fraction P30 (see ``Experimental Procedures'') obtained from the transfected cells and in the microsomal fraction, sedimenting at 100,000 g (P100). Equal amounts of protein from the different cellular fractions to be tested were layered on each lane of a SDS-containing polyacrylamide gel and separated by electrophoresis. The amount of protein derived from the Na-Ca exchanger was determined by selective binding of an antipeptide antibody (AbO-8) directed against amino acids 645-659 of clone RBE-1. Fig.5shows a typical Western blot of transfected HeLa cell proteins. In this figure, the proteins obtained from the different HeLa cell extracts (lanesA) and the corresponding plasma membrane enriched fractions (lanesB) are shown. It can be seen, that protein species of different molecular mass are highlighted in the different lanes. In the total cell extract obtained from HeLa cells transfected with the wild-type clone RBE-1, two protein species are visible: the major one of about 125 kDa, and a minor band of 108 kDa. The major band of 125 kDa, corresponds presumably to the fully processed mature protein. We do not know whether the low molecular mass protein represents incomplete polypeptide chains or degradation products. Quantitative analysis (see ``Experimental Procedures''), indicates that the amount of the 108-kDa protein is about 15% of that of the 125-kDa one. In the extract prepared from HeLa cells transfected with clones N21 and N26, two bands are highlighted as well. The lower band of about 125 kDa corresponds presumably to the fully processed exchanger protein derived from the mutant clones. The molecular mass of the upper band is about 135 kDa. This is a much higher molecular mass than one would expect, even if part of N21 and N26-derived Na-Ca exchanger proteins would retain their residual signal peptides and undergo glycosylation. Similar protein pattern is revealed in the plasma membrane-enriched fractions (lanesB). Quantitative analysis indicates that there is about 2-fold more 125-kDa protein than 135-kDa protein both in the N21- and N26-transfected cells. The amount of all the different protein species derived from the Na-Ca exchanger (except that which is derived from N31) is enriched about 2.5-fold in the plasma membrane-enriched fraction relative to the corresponding cell extracts.


Figure 5: Western blot analysis of the proteins synthesized in RBE-1, N21, N26, N31, and Ala-32 Asp transfected HeLa cells. 17 h posttransfection HeLa cells were divided into three fractions: 1) a total cell extract (see ``Experimental Procedures''); 2) A plasma membrane-enriched fraction (P30); and 3) a 100,000 g sedimenting (P100) fraction. The proteins present in each fraction were separated by SDS-polyacrylamide gel electrophoresis, transfered to nitrocellulose, and analyzed by incubation with a polyclonal antibody AbO-8, produced against a pentadecapeptide derived from the large cytoploasmic loop of the exchanger protein. I-Protein A was used as secondary antibody. 7% acrylamide and 0.18% N,N`-methylenebisacrylamide were used. 100 µg of protein was layered in each well. The total cell extract (A) and the plasma membrane-enriched fraction (B) are shown.



In the N31-transfected HeLa cells, both in the total cell extract and in the plasma membrane-enriched fraction, only a single protein species of about 125 kDa is visible. The amount of this protein is only about 36% in the total cell extract and 26% in the plasma membrane-enriched fraction when compared with the wild-type exchanger RBE-1. But based on 60% and 85%, Na-Ca exchange activity obtained in whole cells (Fig.1) and following reconstitution (Fig.2), respectively, it seems to be a fully functional protein of similar specific transport activity (or even higher) than that of the wild-type exchanger.

Analysis of the protein expression pattern of the exchange mutant Ala-32 Asp reveals that in a similar manner to the wild-type exchanger, this mutant also is expressed in two molecular forms: one corresponds to about 125 kDa and the other to about 110 kDa. Both bands are revealed by the antibody at about equal intensity.

Analysis of the Na-Ca exchanger-derived proteins in the P100 fraction of the transfected HeLa cell preparations indicates that this fraction contains only small amounts of the exchanger protein. When identical amounts of P100 proteins to those that were layered on the gel shown in Fig.5were analyzed, detection required an excessive overexposure of the immunoblots. Compared with 1-2 h that were required to detect the proteins highlighted in Fig.5, between 24-36 h were required to detect the proteins in the P100 fraction. It should be noted that in the immunoblots of the P100 fraction, proteins of 125 and 135 kDa were detected in all of the different P100 fractions (results not shown).

Processing of the Signal Peptide-truncated Mutants by Microsomes

In order to investigate whether the wild-type Na-Ca exchanger and its amino-terminal truncated mutants follow similar pathways of post-translational modification, we have decided to use the mRNA-free reticulocyte lysate system supplemented with dog microsomes. In this model system, we could potentially examine whether the amino-terminal truncated mutants of the Na-Ca exchanger gene can be both cleaved and glycosylated.

The cloned Na-Ca exchanger gene RBE-1 and its truncated cloned mutant genes N21, N26, and N31 were added to an mRNA-free combined transcription/translation reticulocyte lysate system (see ``Experimental Procedures''). Protein synthesis was monitored by labeling with [S]methionine and separation of the products by gel electrophoresis. Posttranslational modification was tested following the addition of dog microsomes to the lysate. Glycosylation was monitored by the addition of the deglycosylating enzyme peptide N-glycosidase to control assays. Fig.6shows a typical profile of the proteins synthesized after separation by SDS containing polyacrylamide gel electrophoresis. It should be noted that in order to prevent aggreggation of the translation products and to facilitate entry into the gel, 8 M urea was added to the sample buffer, and the acrylamide and N,N`-methylenebisacrylamide concentrations were reduced to 5 and 0.125%, respectively.


Figure 6: Expression of the wild-type Na-Ca exchanger gene RBE-1 and its truncated mutants N21, N26, and N31 in reticulocyte lysate. RBE-1 (W.T.) and its truncated mutants N21, N26, and N31 were added to a combined transciption/translation mRNA free reticulocyte lysate system alone (lanesA) or with dog microsomes (lanesB) or to a combined reticulocyte lysate system with dog microsomes as in B, except that the translation products were treated with peptide N-glycosidase as well (lanesC). A control experiment in which the pBluescript KS plasmid was added to the lysate is shown as well (D). 3 µg of plasmid DNA were added to 25 µl of lysate. 10 µl of lysate were layered in each lane. Electrophoresis conditions are described in detail under ``Experimental Procedures.''



It can be seen that in the combined transcription/translation mRNA-free reticulocyte lysate (lanesA), large amounts of protein are synthesized, and they migrate as a wide band on this gel. The molecular mass of these proteins is between 112 and 120 kDa, which fits well the calculated molecular mass of 120.8, 118.2, 117.5, and 116.9 kDa of the completed nonprocessed unglycosylated wild-type exchanger RBE-1 and its truncated mutants N21, N26, and N31, respectively. The lower molecular mass migrating proteins (below 116 kDa) represent probably some uncompleted translation products, and the high molecular mass proteins (on the upper part of the separating gel), represent probably aggregates that were not separated by the SDS-urea treatment. Addition of microsomes to the reticulocyte lysate (lanesB) leads to a reduction in protein synthesis(26) . Cleavage of the signal peptide and glycosylation is expected to result only in small changes in molecular mass(27) , especially when the wild-type exchanger is processed. This fits the observed molecular mass distribution of 120-125 kDa (W.T. laneB). When, however, microsomes are added to mutant exchanger containing reticulocyte lysate, proteins of molecular mass between 115 and 130 kDa, are obtained (see lanesB, N21, N26, and N31). Since mutant exchangers have only short (N21 and N26) or no(N31) signal peptides, processing should in principle reflect the extent of glycosylation. And indeed, the addition of the deglycosylating enzyme peptide N-glycosidase (lanesC) leads to reduction in molecular mass, formation of sharper bands, and uniform migration on the gel. The total amount of protein is somewhat reduced, which could be a result of either some degradation or aggregation following the prolonged treatment with the deglycosylating enzyme (see ``Experimental Procedures''). The molecular mass of the peptide N-glycosidase-treated proteins is about 115 kDa, which is in good agreement with the calculated molecular mass of the unglycosylated signal peptide-free protein of 116 kDa.

We have also tested the sensitivity of the processed translation products to the deglycosylating enzyme endoglycosidase H. Our results indicate that there is a partial sensitivity to the enzyme as observed by the small decrease in molecular mass of the proteins (data not shown).


DISCUSSION

In this work, we have shown, that the initial segment of 32 amino acids of the cloned Na-Ca exchanger gene RBE-1 (9) are not mandatory for functional expression of the transporter in HeLa cells. We have shown that amino-terminal truncated mutants (for their list, see Table1) of the cloned gene code for functional exchangers that catalyze Na gradient-dependent Ca influx into transfected HeLa cells (Fig.1). Na-Ca exchanger protein, derived from the cloned wild-type and amino-terminal truncated mutant genes, can be detected by Western blot analysis in the transfected cells (see Fig.5). Determination of the transport activity following reconstitution of the proteins expressed in HeLa cells suggested, that nontargeted amino-terminal truncated mutant Na-Ca exchangers did not accumulate in the transfected cells since the transport activity was similar to that determined in whole cells (Fig.2). This is also supported by Western blot analysis of the protein profile derived from the microsomal fraction P100. The amount of Na-Ca exchanger protein in this fraction was much lower than that which was detected in comparable amount of either crude cell extract or in a plasma membrane-enriched fraction. We have also shown that the amino-terminal truncated mutants of the cloned Na-Ca exchanger undergo glycosylation when exposed in a reticulocyte lysate to dog microsomes (Fig.6).

Our findings would presumably be less surprising if the initial 32 amino acids of cloned Na-Ca exchanger gene would not have been identified previously as a cleavable signal peptide and signal peptidase cleavage site(14) . Such an identification would imply that this segment of amino acids should be instrumental in targeting the transporter to the ER from where it should subsequently be targeted via the Golgi apparatus to the plasma membrane. Our results indicate that the Na-Ca exchanger can insert into the ER in the absence of the endogeneous amino-terminal signal peptide. In addition, the cell-free translation results show that translocation and glycosylation of the first extracellular domain does not require amino-terminal signal peptide.

One could argue of course, that the absence of the initial segment of 32 amino acids in the mature protein, was shown directly only in the case of the bovine heart exchanger and do not apply to the rat brain exchanger. But since there is an overall 88% sequence identity between the bovine heart exchanger and the rat brain exchanger RBE-1 (excluding the two gaps of the deleted 35 amino acids in the cytoplasmic loop of the brain clone(9) , this is probably unlikely. Even when the amino-terminal segments of the cloned rat brain exchanger RBE-1 and the bovine heart exchanger are compared (amino acids 1-32, which are the least similar segments of the protein), 20 amino acids are identical (28) , and out of the remaining 12 amino acids, six exchanges are conservative. Moreover, all of the consensus topological signals that identify signal peptides and signal peptidase cleavage sites that are present in the bovine heart exchanger are present also in the brain clone: the initial hydrophobic segment of 24 amino acids, the positive charges within that segment, and the putative signal peptidase cleavage site(14, 23, 25) .

Many polytopic membrane proteins exhibiting the 12-helix motif (29) do not contain an amino-terminally localized cleavable signal peptide. The Na-Ca exchanger is an unusual protein since it does seem to have a signal peptide, but this putative signal peptide is redundant for the very function it is supposed to carry out.

One can only speculate what is the role of this putative signal peptide. The addition of a cleavable amino-terminal signal peptide to the beta(2)-adrenergic receptor (2) enhanced its translocation to the ER. If translocation into the ER and protein synthesis are coupled processes, and if the ``naturally'' occurring signal peptide of the Na-Ca exchanger is indeed instrumental in enhancing the entry of the protein into the ER, it could provide an explanation for the somewhat lower transport activity and for the lower amount of N31-derived protein as detected on the blots. Since the transport activity of N21 is not significantly different than that of RBE-1, it would suggest, that the residual stretch of 11 amino acids of the original 32 is sufficient to provide the enhancement in translocation to the ER. Coupled protein synthesis and enhancement of translocation into the ER is probably not the entire explanation for the lower transport activity of N26 since immunoblots reveal that the amount of protein that is detected is not significantly different than that of N21.

The exchange mutant Ala-32 Asp, had much lower transport activity in transfected HeLa cells than either of the three amino-terminal truncated mutants. The transport activity, which was only 7% relative to the wild-type exchanger, increased somewhat following reconstitution and it reached 31%. Western blot analysis (see Fig.5) indicates that the amount of protein synthesized was not substantially different than that of the wild-type exchanger in the total cell extract. Moreover, the amount of mutant protein that was detected in the plasma membrane-enriched fraction was also similar to that of the wild-type clone. Hence, the lower transport activity could result from retention of the signal peptide, an additional negative charge in the vicinity of the signal peptidase cleavage site, misfolding of the native protein, or a combination of any of these.

It is also possible that the proximity of the signal peptide to a potential glycosylation site regulates the extent of glycosylation, which in turn can affect functional expression. The first putative glycosylation site of the rat brain Na-Ca exchanger is at asparagine 41. This site is removed only by eight amino acids from the putative signal peptidase cleavage site. There are two lines of evidence that support this possibility. Western blot analysis suggests that mutants N21 and N26 are expressed as two protein species; one of these is of 125 kDa, and the other is of 135 kDa. The 125-kDa protein fits well with the calculated molecular mass of the mature fully processed Na-Ca exchanger. The 135-kDa protein, however, has a much higher molecular mass than would be expected for a normally glycosylated Na-Ca exchanger, even if it would have retained its partially truncated residual signal peptide that would be 11 or 4 amino acids for N21 and N26, respectively. It should be noted, that high molecular mass proteins are produced also when amino-terminal truncated mutants serve as templates for protein synthesis in the reticulocyte lysate-dog microsomal cell-free system (see Fig.6). It is not known at present whether a higher level of glycosylation reduces transport activity. Expression of dog heart Na-Ca exchanger gene in insect cells(30) , where no glycosylation takes place, resulted in lower specific transport activity. Similar observation was obtained also with the deglycosylated glycine transporter(31) . Further experiments, however, have to be carried out to see which of these speculations applies in the case of the cloned Na-Ca exchanger gene.


FOOTNOTES

*
This work was supported in part by the Basic Research Fund of the Israel Academy of Sciences, the Israel Ministry of Science and Technology, the U. S.-Israel Binational Science foundation and the Bernard Katz Minerva Center. 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.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank®/EMBL Data Bank with accession number(s) X68812[GenBank].

§
To whom correspondence should be addressed. Tel.: 972-2-758511; Fax: 972-2-757379; Hannah{at}HUJIMD.

^1
The abbreviations used are: RBE, rat brain exchanger; ER, endoplasmic reticulum.


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