(Received for publication, December 22, 1994; and in revised form, June 6, 1995)
From the
The rat brain Na-Ca
exchanger
(RBE) gene, as well as other isoforms of this protein family, can be
organized into 12 transmembrane
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.
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
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
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.
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
, 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
, 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) .
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%
-mercaptoethanol and addition of Nonidet P-40 to a final
concentration of 1%.
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.
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.
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
[
-
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.
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).
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).
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 -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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank®/EMBL Data Bank with accession number(s) X68812[GenBank].