(Received for publication, March 7, 1995; and in revised form, May 2, 1995)
From the
The Na-Ca
exchanger is an
unusual membrane transport protein as it contains an
NH
-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
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
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.
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), ()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-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
-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
-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.
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 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
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).
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-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.
Figure 1:
Amino acid sequences of the NH 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
Ala) (D), and mutants with
deletions past the signal sequence,
49 (E) and
64 (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 (
) 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
, 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
, 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
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
-terminal mutants expressed in
HEK 293 cells. With the exception of the two largest deletions (
49
and
64 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
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
49 and
64.
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
49 and
64, were degraded more rapidly.
Figure 4:
Expression and enzymatic deglycosylation
of chimeric proteins constructed by fusion of the
NH-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
(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 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
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-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
. 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.
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
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 49 and
64 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
49 and
64 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.''
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
49 and
64,
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
-adrenergic receptor (Guan et al., 1992). It is a type IIIb membrane protein (NH
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.