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INTRODUCTION |
Mammalian Na+/H+ exchangers
(NHE)1 are a ubiquitous
family of transmembrane proteins that catalyze the antiport of
Na+ and H+ at the plasma membrane. Under
physiological conditions, NHE drive H+ out of the cell by
coupling to the Na+ gradient, which is generated and
maintained by the activity of the
Na+/K+-ATPase. NHE are implicated in a variety
of important physiological functions, including intracellular pH
regulation, cell volume control, and Na+ homeostasis.
Hydropathy analysis of the NHE1 isoform predicts that 10 to 12 transmembrane helices comprise the N terminus and that the C terminus
constitutes a large cytoplasmic domain (1). This structure delineates
function; the N-terminal domain, which is highly conserved among the
various NHE isoforms, confers transport activity of the exchanger,
whereas the considerably divergent C-terminal tail corresponds to a
regulatory domain (2, 3). Numerous studies have shown that NHE isoforms
can be post-translationally modified in a variety of ways. The
cytosolic C-terminal tail domain modulates exchanger activity by
becoming phosphorylated in response to extracellular signals like
growth hormones (4) and through association with a variety of
regulatory molecules, such as Ca2+/calmodulin (5). Some NHE
isoforms are glycosylated in extracellular loops between transmembrane
segments, although the functional significance of such modifications is
unclear (6, 7).
Over the last several years, phenomenological evidence of intracellular
Na+/H+ exchange has been documented in a
variety of mammalian tissues (8-10), and in each case this activity
colocalized with V-type H+-ATPase activity, suggesting an
endosomal residence for Na+/H+ exchange. Our
laboratory recently identified and cloned a Saccharomyces cerevisiae homologue of mammalian NHE, called Nhx1 (11), which represents the founding member of a unique and growing subfamily of
intracellular Na+/H+ exchangers, including
proteins identified in Schizosaccharomyces pombe,
Caenorhabditis elegans, Arabidopsis thaliana, and
humans. Nhx1 localizes to a late endosomal/prevacuolar compartment
where it mediates intracellular sequestration of Na+ in a
pH-dependent manner (11, 12), coupling Na+
movement to the proton gradient established by the vacuolar
H+-ATPase (13, 34). Hydropathy analysis of Nhx1
reveals a domain structure similar to NHE isoforms, suggesting that the
structure/function relationship for intracellular exchangers may be
homologous to the plasma membrane-type antiporters. Here, we take a
first step toward understanding how Nhx1 is regulated, specifically by
investigating post-translational modification of the protein. We have
determined that Nhx1 is an N-linked glycosylated protein and
have shown that these glycosylations uniquely map to residues in the
C-terminal hydrophilic tail portion of the exchanger. The presence of
glycosylated asparagines in the C terminus predicts that at least some
portion of the Nhx1 tail is exposed to the lumen of the late endosomal compartment. An earlier report demonstrated that epitopes within the
NHE3 C-terminal tail were exposed to the exoplasmic side of the
membrane (31), although there is no evidence for glycosylation of the
tail region of NHE3 or any other NHE isoform. Taken together with the
results of our study, these findings support an unpredicted topological
arrangement for the C-terminal region of Na+/H+
exchangers that warrants further study.
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EXPERIMENTAL PROCEDURES |
Yeast Strains, Media, and Growth Assays--
R100, the Nhx1 null
strain (
nhx1), is isogenic to W303 and has
been described previously (11). R100 was the host strain unless
otherwise indicated. sec7 and sec18 are
temperature-sensitive strains isogenic to NY13 (14). The
temperature-sensitive sec53 mutant was a gift of
Peter Orlean. The wild-type strain W303 1A was a gift of
Susan Michaelis. The synthetic minimal medium APG was used
throughout and contained 10 mM arginine, 8 mM
phosphoric acid, 2% glucose, 2 mM MgSO4, 1 mM KCl, 0.2 mM CaCl2, trace
vitamins and minerals, pH ~6.7 (15). In some cases, NaCl was added,
and the pH value was adjusted to 4.0 with acetic acid. For growth assays, 0.2-1-ml cultures, supplemented with NaCl as specified, were
inoculated at an A600 value of 0.05 in a
multi-well plate and incubated at 30 °C for 96 h, at which time
A600 was measured.
Plasmids and Mutagenesis--
pRin73, a 2 µ plasmid
harboring NHX1 tagged with a C-terminal triple hemagglutinin
epitope (NHX1::HA), under control of its endogenous promoter, was created previously (12).
512 is a C-terminal truncation of Nhx1 at residue 512, followed by the triple HA
tag, and was created by a PCR-based method as follows (16). A
0.4-kilobase piece at the 3' end of NHX1 was
amplified from pRin73 using a forward primer at the SpeI
site (5'-CTGAAGTAGAACTAGTCTATAAGCCAC-3') and a reverse
primer
(5'-AACATCGTATGGGTAAAAGATGCGGCCGCCATTTATCGCCCTTGGAGCCTCTATATC-3'), which specified the junction of the truncated NHX1 gene with
the HA tag sequence. The amplified product was used as a
"megaprimer" in a second round of PCR using a reverse primer at the
3' untranslated region (5'-GGCACGAGCTCGTCTTCATCCATGACGGAAG-3') to give
a 1.9-kilobase pair product. The latter was digested with
SpeI and SacI and inserted into pRin72, a plasmid
harboring the 5' end of the NHX1 gene (12), to give the
final plasmid pKW403. Asparagine to aspartate point mutants of
NHX1::HA were generated by a one-step PCR method
(17) using pRin73 subclones as templates. For each PCR reaction, the point mutation (underlined) was introduced in one of the two
oligonucleotides (forward and reverse) as follows: N121A/D,
5'-CTACTTTTTTAATGTTCTATTG-3' and
5'-GATGAAGC(T/C)AAAAGTAACCG-3';
N334D, 5'-CCTATTATGACATGTCAAGAAG-3' and
5'-CGTAATGTTTTAAAGTAATTCC-3'; N420D,
5'-GGAGAAGATATTTCTGTTCCC-3' and
5'-GGTTATGCCGCTCATAGATCTG-3'; N515D,
5'-TTATTGGACGGTAGTTCTATTCAG-3' and
5'-ATTTATCGCCCTTGGAGCCTCTATATC-3'; N550D,
5'-CCCAATGACATATCCACAAC-3' and 5'-GAGATTCTTGTTACTGCTG-3';
N563D, 5'-GAGGCCTTGATGAAACTGAGAATAC-3' and
5'-CAAAAGTATTACCACCAGTTG-3'. Double and triple point mutants were
generated by consecutive rounds of the above PCR method, and all
mutations were confirmed by sequencing.
Biochemical Methods--
Endo H and tunicamycin were from Roche
Molecular Biochemicals and Sigma, respectively. For Endo H treatments,
samples were pooled from sucrose density gradient fractions (30-46%
w/w sucrose) enriched for Nhx1 (18). Briefly, cells from 600-ml
overnight cultures (A600 = 0.5-1.0) were
harvested and converted to spheroplasts using yeast lytic enzyme (10 mg/1600 OD units; ICN) for 45 min at 37 °C. Spheroplasts were
suspended in 3-6 ml of lysis buffer (0.3 M sorbitol, 20 mM triethanolamine acetate, pH 7.2, 1 mM EDTA, and protease inhibitors) and homogenized with ~60 strokes of a Wheaton A Dounce homogenizer. The cleared lysate (~3 ml) was layered on top of and centrifuged through (2 h, 27,000 rpm, Beckman SW28 rotor,
4 °C) a 10-step sucrose gradient (3 ml each of 18-54% (w/w) in 4%
increments). Fractions were collected from the top, pooled, and
centrifuged for 1 h at 27,000 rpm; the final pellet was
resuspended in ~50 µl of buffer (0.5 M sucrose, 10 mM MES/KOH, pH 6, 150 mM KCl) plus protease
inhibitors. 30-50 µg of total protein was denatured for 10 min at
60 °C in 0.5% sodium dodecyl sulfate and 1%
-mercaptoethanol, then diluted into 50 mM sodium citrate buffer, pH 5.5, and
incubated either with or without Endo H at 37 °C for 18 h, as
specified in legends to figures. Following treatment, samples were
precipitated in 10% (v/v) trichloroacetic acid and centrifuged for 30 min at 4 °C, and pellets were resuspended in sample buffer prior to
loading on gels.
For treatments with tunicamycin, 20-200-ml cultures were grown
overnight at 30 °C to an A600 value of ~1,
divided in two, and grown for two more hours following addition of
tunicamycin (10 µg/ml final concentration) or an equal volume of
carrier (5 µM NaOH). Aliquots (10-40 ml) were removed at
indicated times, and total membranes were prepared by a glass bead
method (18). Briefly, cells from each aliquot were harvested and
resuspended in ~200 µl of bead buffer (10 mM Tris/HCl,
pH 7.4, 0.3 M sorbitol, 0.1 M NaCl, 5 mM MgCl2) plus protease inhibitors. Acid-washed glass beads were added to the meniscus, and samples were vortexed 3 times for 1 min. Lysates were washed 3 times with 1 ml of bead buffer
and, using a drawn-out pasteur pipette, removed to new tubes. Cleared
lysates were centrifuged for 1 h at 45,000 rpm (Beckman Ti50
rotor, 4 °C), and final pellets were resuspended in ~30 µl of 20 mM K-Hepes, pH 7.4, plus protease inhibitors. Samples
(30-100 µg) were diluted in sample buffer prior to loading on gels.
Protein concentrations were determined by the method of Lowry et
al. (19) for total membrane preparations and by a modified Lowry
method (20) for fractionation and pooled preparations. SDS-PAGE
and Western blotting were as described previously (12). Antibodies used
were mouse anti-HA monoclonal antibody, 12CA5 (Roche Molecular
Biochemicals) at 1:5,000, and horseradish peroxidase-coupled sheep
anti-mouse antibody (Amersham Pharmacia Biotech) at 1:10,000.
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RESULTS |
Nhx1 Is an N-Linked Glycosylated Protein--
Nhx1 exhibits a
consistently multi-banded appearance upon gel electrophoresis, strongly
suggestive of post-translational modification of the protein (12). To
determine whether Nhx1 was N-glycosylated, a combination of
in vivo and in vitro biochemical and genetic approaches were employed. Membrane fractions enriched for Nhx1 (see
"Experimental Procedures") were treated with endoglycosidase H, an
enzyme that cleaves high-mannose complex-type N-linked
glycosylations (21, 22). As shown in Fig.
1A, Endo H treatment resulted
in the disappearance of the upper collection of Nhx1 bands, indicative of a loss of N-linked glycosylation. To independently
corroborate this observation, growing yeast cultures were treated with
tunicamycin, an antibiotic that blocks all N-linked
glycosylation of proteins by inhibiting the synthesis of dolichol
diphosphate N-acetylglucosamine (23). As indicated in Fig.
1B, tunicamycin treatment directed the following
time-dependent effects: (i) a gradual decline in the
abundance of the Nhx1 protein; and (ii) a loss of the higher molecular
weight bands, similar to the loss observed following Endo H
treatment.

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Fig. 1.
Nhx1 is an N-linked
glycosylated protein. Western blots of HA-tagged Nhx1 are shown.
A, Endo H treatment. Membranes (50 µg) pooled from sucrose
gradients, as described under "Experimental Procedures," were from
left to right, untreated, incubated in reaction
buffer alone (Mock), or incubated in reaction buffer plus 1 milliunit of Endo H/µg of protein (Endo H). The positions
of molecular weight markers are shown at the extreme left;
the arrow to the right indicates disappearance of
the upper set of bands. B, tunicamycin treatment.
10 µg/ml tunicamycin (Tu) was added to yeast cultures
(W303/pRin73), and aliquots were removed at the indicated times.
Total membranes were prepared, and 150 µg of protein was loaded per
lane. The arrow to the right indicates
the upper set of bands.
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Biosynthesis of a glycosylated membrane protein begins in the
endoplasmic reticulum, where cotranslational attachment of core glycosylation occurs, followed by movement to the Golgi, where there is
further modification and maturation of glycosylation (24). In S. cerevisiae, a well defined set of temperature-sensitive sec mutants interrupt the biosynthetic pathway at discrete
stages (14). We examined the expression of Nhx1 in the
sec18, sec7, and sec6 mutants, which
show temperature-dependent retention of protein in the
endoplasmic reticulum, Golgi, and secretory vesicles, respectively. In
the sec18 mutant, shift to the nonpermissive temperature
resulted in a striking redistribution of Nhx1 bands from the higher to
lower molecular weight (Fig.
2A), indicating that exit from
the endoplasmic reticulum is required for the appearance of the higher
molecular weight bands. Conversely, the upper bands became more intense
following incubation at 37 °C in the sec7 mutant,
indicative of increased modifications (Fig. 2B). There were
no significant changes in Nhx1 mobility upon temperature shift in the
sec6 mutant, suggesting that the modifications are completed
in the Golgi or that Nhx1 does not traffic through secretory vesicles
(not shown). To directly examine glycosylation status in
vivo, Nhx1 was expressed in the sec53 mutant, which
harbors a temperature-sensitive mutation in phosphomannomutase, an
enzyme essential for the biosynthesis of both N- and
O-linked glycosylations (25). Consistent with both Endo H
and tunicamycin sensitivities, shifting Nhx1 from permissive (25 °C)
to nonpermissive (37 °C) temperatures in sec53 resulted
in loss of the upper bands on SDS-PAGE (Fig. 2C).

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Fig. 2.
Expression of Nhx1 in temperature-sensitive
sec mutant strains. Western blots of HA-tagged
Nhx1 are shown; arrows on left indicate upper
sets of bands. NHX1::HA
(CEN, heat shock promoter) was transformed into
sec18 (A) and sec7 (B)
mutant strains. Cultures were grown to an A600
value of ~1, then divided, and allowed to grow for an additional
2 h at either permissive (25 °C) or nonpermissive (37 °C)
temperatures. Total membranes were prepared, and 50 µg of protein was
loaded in each lane. C,
NHX1::HA (pRin73) was transformed into a
sec53 mutant strain. The procedure was as above, except 70 µg of total protein was loaded in each lane.
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Nhx1 Contains Six Consensus Sites for N-Linked
Glycosylation--
Hydropathy analysis of Nhx1 (Fig.
3A) by the Kyte-Doolittle
method (26) predicts multiple hydrophobic regions capable of spanning
the lipid bilayer 10 to 13 times. Although less well defined, the
hydropathic peaks numbered 6 and 7 share a high degree of identity with
other NHE isoforms and are predicted to be involved in transport (27).
Based on hydropathy analysis, transmembrane predictions, and models of
plasma membrane NHE isoforms (3), we developed a working model for the
membrane topology of Nhx1 (Fig. 3B). As drawn, the six
consensus sites for N-linked glycosylation, -NX(S/T)- (28), are polarized into two groups;
Asn-121, Asn-334, and Asn-420 are in loop regions of the N-terminal
transmembrane domain and are predicted to face the lumen of the
prevacuolar compartment, and Asn-515, Asn-550, and Asn-563 are in the
C-terminal tail domain and are predicted to reside in the cytosol.

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Fig. 3.
Predicted structure of Nhx1.
A, hydropathy analysis of the primary sequence of Nhx1 using
the Kyte-Doolittle algorithm (26). The asterisk indicates a
possible transmembrane segment not included in the model shown in
B. B, topological model of Nhx1, indicating
strong consensus sites (-NX(S/T)-) for N-linked
glycosylation (28) and sites of truncation and point mutation; numbers
of transmembrane segments correspond to the numbered peaks
on the hydropathy plot (A).
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Nhx1 Is Glycosylated in the C-terminal Tail Domain--
Based on
the preliminary topological model and the known sites of glycosylation
in other NHE isoforms, we hypothesized that the likely site(s) of
N-linked glycosylation were on the loops between
transmembrane segments, namely at one or more of the residues Asn-121,
Asn-334, and Asn-420. Each of these asparagines was individually replaced with aspartate, as described under "Experimental
Procedures." Mutants N334D and N420D were as abundant as wild-type
Nhx1; however mutant N121D was expressed at significantly lower levels.
Regardless of expression levels, the aspartate point mutants conferred
salt-tolerant growth that was indistinguishable from wild-type (Fig.
4A) and was clearly above the
nhx1 null strain (11). An alternate
substitution, N121A, was generated; this mutant exhibited normal
abundance levels, although salt tolerance was intermediate to wild-type
and null mutant (not shown). The individual point mutants (N121A,
N334D, and N420D) each exhibited a sensitivity to tunicamycin similar to that of wild-type (Fig. 4B), indicating a retention of
N-linked glycosylation in vivo.

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Fig. 4.
Effect of mutations in the N-terminal
transporter domain of Nhx1. A, NaCl sensitivity
of wild-type or mutant strains was assayed as described under
"Experimental Procedures." Relative growth is the
A600 value at a given NaCl concentration divided
by A600 in the absence of NaCl, expressed as a
percentage. Data points represent averages of two to four independent
experiments. Western blots of HA-tagged Nhx1 are shown in B
and C. B, tunicamycin-sensitivity. Lanes
correspond to 2-h treatments with (+) or without (-) 10 µg/ml
tunicamycin. Total membranes were loaded in the following amounts:
upper panel, Tu (50 µg) and +Tu
(100 µg); lower panel, Tu (100 µg) and
+Tu (200 µg). C, Endo H treatment of the
N-terminal triple loop mutant N121A/N334D/N420D. Endosomal membranes
(30 µg) from sucrose density gradients were treated as described in
the legend to Fig. 1, using 3.33 milliunits of Endo H/µg of
protein.
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It was possible that replacement of a glycosylated asparagine residue
could lead to improper glycosylation at another, normally unmodified,
consensus site for N-linked glycosylation. To address this
concern, we created the triple loop mutant, N121A/N334D/N420D, and
determined its glycosylation state. This triple mutant exhibited normal
abundance and subcellular distribution but had decreased activity, as
inferred from a reduction of salt tolerance to levels similar to the
null strain (not shown). Most importantly, treatment with tunicamycin
(Fig. 4B) and Endo H (Fig. 4C) resulted in a collapse of the upper collection of bands to a lower molecular weight,
as in the single point mutants. Taken together, these results prove
that mutation of all N-linked glycosylation consensus sites
in the N-terminal domain does not prevent the normal glycosylation of Nhx1.
Next, we investigated whether the C-terminal tail harbored site(s) of
N-linked glycosylation. There are three strong consensus sites for N-linked glycosylation at Asn-515, Asn-550, and
Asn-563, as well as several weak sites. To test the hypothesis that
some or all of these sites are normally glycosylated, we engineered two
mutant Nhx1 constructs (see Fig. 3B), a C-terminal
truncation at asparagine 512 (
512), which deleted the three strong
consensus sites for N-linked glycosylation in the tail
region, and a triple mutant (N515D/N550D/N563D) in which aspartates
replaced each of the three consensus asparagines. Fig.
5A clearly shows that unlike the wild-type control, the
512 mutant was insensitive to Endo H
treatment. As indicated in Fig. 5B, both
512 and the
triple mutant also appeared insensitive to tunicamycin treatment,
strongly suggesting a loss of the site(s) of in vivo
glycosylation. In contrast, the N334D mutant, used as a positive
control because of its strong upper banding pattern (see Fig.
4B), showed a clear sensitivity to tunicamycin.
Additionally, expression of either
512 or the triple mutant in
sec7 and sec18 strains resulted in no change in
banding pattern upon shift to the nonpermissive temperature of 37 °C
(Fig. 5C), an observation in stark contrast to that of the
wild-type protein (see Fig. 2). It was possible that deletion of the
C-terminal tail or mutation of the three asparagine residues resulted
in polypeptides that were misfolded or abnormally localized and
therefore not properly glycosylated. In earlier work, we have described
the subcellular fractionation of yeast lysates on sucrose density
gradients and shown colocalization of Nhx1 with endosomal markers (12).
Here we show that the triple mutant was identical to wild-type Nhx1 in
its distribution on sucrose density gradients (Fig.
6A), and in its tolerance to
high salt (Fig. 6B), indicating that this protein was
properly localized and fully functional. Distribution of the
512
mutant showed some overlap with Golgi-containing fractions (12),
suggesting a delay in trafficking out of the Golgi (Fig.
6A). Interestingly,
512 had a sodium-sensitive phenotype intermediate to that of wild-type Nhx1 and
nhx1 (Fig. 6B), suggesting that the
N-terminal transmembrane domain retains some transport function.

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Fig. 5.
Effect of C-terminal tail mutations on the
glycosylation of Nhx1. Western blots of HA-tagged Nhx1 are shown.
A, Endo H treatment. Endosomal membranes, pooled from
sucrose gradient fractions, were incubated with (+) or without (-) 0.5 milliunits of Endo H/µg of protein; protein loaded was 30 µg for
NHX1 and 20 µg for 512. The
arrow on the right indicates the disappearance of
the upper set of wild-type bands. B, tunicamycin
treatment was as in Fig. 1 legend; all lanes were loaded
with 50 µg of protein. The arrow on the right
indicates the disappearance of the upper set of bands.
C, both 512 and the C-terminal triple mutant
(N515D/N550D/N563D) were transformed into sec7 and
sec18 mutant strains as described in the legend to Fig. 2.
Cultures were grown overnight, divided, and allowed to grow for an
additional 2 h at either 25 or 37 °C. Total membranes were
prepared, and 50 µg of protein was loaded in each
lane.
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Fig. 6.
Effect of C-terminal tail mutations on the
subcellular localization and salt tolerance of Nhx1. A,
Western blots of HA-tagged Nhx1. Equal protein was loaded in each
lane as follows: 50 µg for 512,
30 µg for Triple (N515D/N550D/N563D), and 50 µg for
NHX1, from each of ten sucrose gradient fractions (18 to
54% sucrose; L = load). B, NaCl sensitivity
of wild-type or mutant strains was assayed as described in the legend
to Fig. 4.
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Asn-515 and Asn-550 Are the Sites of N-Linked Glycosylation of
Nhx1--
To map the precise site(s) of N-linked
glycosylation, we created the single point mutants N515D, N550D, and
N563D. Each mutant appeared fully functional, as evidenced by
salt-tolerant growth (Fig.
7A). The abundance of each
mutant was approximately equal to that of wild-type, but the banding
pattern was upwardly shifted for the N515D and N550D mutants (Fig.
7B). Based on this observation, we constructed the double
mutant N515D/N550D. Fig. 7C shows that although each of the
three single point mutants appeared to retain tunicamycin sensitivity,
the double mutant N515D/N550D, like the C-terminal triple mutant, was
insensitive to tunicamycin treatment. The double aspartate mutant also
showed an upward shift compared with wild-type, perhaps because of the
substitution with charged residues. To test this hypothesis, we created
a double mutant substituted with alanines rather than aspartates. As
illustrated in Fig. 7D, mutant N515A/N550A does not show the
mobility shift of the aspartate mutant and also exhibits an
insensitivity to tunicamycin. Thus, the data indicate that Nhx1 is
normally glycosylated at residues Asn-515 and Asn-550. We also verified
a normal subcellular localization of the double mutant on sucrose
density gradients (data not shown). These results confirmed that (i)
both Asn-515 and Asn-550 were the sites of N-linked
glycosylation; and (ii) none of the remaining consensus sites were
glycosylated, as evidenced by the insensitivity of the double mutant to
tunicamycin.

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Fig. 7.
Nhx1 is glycosylated at residues Asn-515 and
Asn-550. A, NaCl sensitivity of point mutants, as
described in the legend to Fig. 4. Western blots of HA-tagged Nhx1 are
shown in B-D. B, abundance of point mutants.
Total membranes were prepared, and 50 µg of protein was loaded in
each lane; duplicate lanes represent separate
yeast transformants. C, tunicamycin treatment.
Lanes correspond to 2-h treatments with (+) or without (-)
10 µg/ml tunicamycin; protein loaded was 50 µg ( Tu)
and 100 µg (+Tu). Double is the double point
mutant N515D/N550D. D, tunicamycin treatment, as in
C; protein loaded was 100 µg ( Tu) and 150 µg (+Tu). DblA is the N515A/N550A mutant, and
DblD is the N515D/N550D mutant.
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DISCUSSION |
N-Linked Glycosylation of the Na+/H+
Exchanger Nhx1--
The present study shows that Nhx1, the yeast
endosomal Na+/H+ exchanger, is
post-translationally modified with N-linked glycosylations. Two independent experimental approaches, employing tunicamycin (in vivo) and Endo H (in vitro), resulted in
similar effects; the uppermost collection of bands on Western blots of
Nhx1 disappeared following treatment. The sensitivity of Nhx1 to Endo H
indicates that the N-linked glycosylations are of the high
mannose complex-type, created first by attachment of a core
glycosylation in the endoplasmic reticulum and followed by maturation
of that modification by enzymes in the Golgi. Temperature-sensitive
blocks in vesicular traffic out of the endoplasmic reticulum
(sec18) or Golgi (sec7) resulted in the expected
loss or accrual of the higher molecular weight bands of Nhx1,
respectively. These observations are consistent with those documented
for other N-linked glycosylated membrane proteins in yeast
(24, 29).
Using site-directed mutagenesis of the consensus sites for
N-linked glycosylation, we have mapped Nhx1 glycosylation to
the C-terminal hydrophilic tail domain, specifically to residues
Asn-515 and Asn-550. Importantly, loss of N-linked
glycosylation had no apparent effect on in vivo exchanger
function, as evidenced by normal profiles of sodium-tolerant growth.
Polypeptide expression levels and subcellular localization of
nonglycosylated Nhx1 appeared similar to that of wild-type. Thus, the
specific role of N-linked glycosylation of Nhx1 remains to
be determined. Although the triple loop mutant was glycosylated
normally, its sodium-sensitive growth was identical to the null strain,
suggesting that this combination of point mutations (N121A/N334D/N420D)
is deleterious for exchanger function. This observation may not be
surprising, as each residue lies within the N-terminal transporter
domain of the exchanger, and the single point mutant N121A exhibited a
salt tolerance intermediate to wild-type and the null strain (data not shown).
Expression levels were drastically reduced only for the N121D mutant,
which was not unexpected, given the predicted location of Asn-121 one
to four amino acid residues inside the
-helical M4 transmembrane
segment. Introduction of the negatively charged aspartate within the
membrane may have led to destabilization and possibly increased
degradation of the mutant polypeptide. It was noteworthy that the
relative intensities and mobilities of the Nhx1 bands varied with the
position(s) of substitution, as well as with the host strain and growth
medium. For example, the mobilities of the bands in N334D was
markedly changed, whereas N420D appeared very similar to wild-type. One
explanation is that introduction of a negative charge at certain sites
in the polypeptide affects migration of the protein on SDS-PAGE,
perhaps in a way similar to that observed for other negatively charged
groups such as phosphate. It is for this reason that we believe the
double mutant N515D/N550D is shifted upward relative to wild-type Nhx1, and indeed this shift is ablated by mutation of each asparagine residue
to alanine rather than aspartate (see Fig. 7D).
Topological Implications--
The mutational constructs of Nhx1
thus far created, and the analysis of these mutants in terms of
glycosylation status, exchanger function, and localization, begins to
elucidate the domain structure and topology of Nhx1, particularly as a
model protein for the new subfamily of intracellular
Na+/H+ exchangers. The demonstration of
N-linked glycosylation in the C-terminal tail region of Nhx1
is novel for the following reasons: (i) to date, there is no evidence
for glycosylation of any NHE isoform on its C-terminal tail domain; and
(ii) the location of glycosylations implies an unusual topological
disposition of the C-terminal tail of Nhx1. Thus far, only NHE1 has
been shown to be N-linked glycosylated, and this site has
been mapped to an extracellular loop between transmembrane domains M1
and M2 (6). Both NHE1 and NHE2 are O-linked glycosylated,
each at sites between M1 and M2 (6, 7). There is no evidence, for any
of the known NHE isoforms, that N-linked glycosylations
exist on the C-terminal tail domain. Numerous functional studies
suggest that the large hydrophilic C-terminal tail of NHE assumes a
cytosolic orientation and constitutes a regulatory region of the
exchanger. It is in fact this cytosolic domain that is implicated in pH
"set point," calmodulin binding, protein kinase
C-dependent phosphorylation, and volume sensitivity (27,
30). However, and of particular significance with regard to the present
study, recent results indicate that some regions of the C-terminal
domain of NHE3 assume an exoplasmic orientation. Two monoclonal
antibodies directed against the C-terminal tail of NHE3 reacted with
epitopes exposed at the extracellular surface (one epitope lies between
residues 702 and 756, and the other epitope lies C-terminal to residue 756) (31). In contrast, during the preparation of this manuscript, a
substituted cysteine-based topological analysis of the NHE1 isoform
demonstrated that three residues (538, 561, and 794) in the C-terminal
tail assumed the predicted cytosolic orientation (33). Our results,
combined with these recent topological studies of NHE1 and NHE3,
suggest either that there is considerable topological divergence in the
C-terminal tail topology of various exchanger isoforms or that the
organization of the tail domain is considerably complex.
The mapping of glycosylation sites in Nhx1 to the C-terminal domain
strongly suggests that the hydrophilic tail, or some portion thereof,
resides within the lumen of the prevacuolar compartment, rather than in
the cytosol. Although this observation was unexpected in terms of
homology to NHE isoforms, the physiological function(s) of
intracellular Na+/H+ exchangers remains to be
elucidated and thus may be consistent with some structural divergence
from plasma membrane-type antiporters. A hallmark of the NHE family is
the strong sequence conservation within the N-terminal transmembrane
domain, which mediates transport function, whereas the hydrophilic
C-terminal tail has diverged considerably, allowing for tissue- and
cell-specific regulation (2, 3). A lumenal disposition of at least some
portion of the putative regulatory domain of Nhx1 may be relevant for
its proposed roles in endosomal volume control, Na+
sequestration, and pH homeostasis (12, 32).
A revised topological model for Nhx1 is essential, based on our new
evidence that this protein is N-linked glycosylated on its
hydrophilic C-terminal tail. Because we know very little about the
physiological function or the orientation of the C terminus for this
emerging subfamily of intracellular Na+/H+
exchangers, a variety of topology models are possible; one commonality must be that at least some portion of the C terminus (including residues Asn-515 and Asn-550) is lumenally accessible. The simplest model would assume a lumenal disposition for the entire C-terminal tail; the loss or gain of one transmembrane segment in Nhx1 would result in a lumenally oriented tail. Wakabayashi et al. (33) discovered in their recent study on NHE1 topology that a hydrophobic region, located between M11 and M12 of the original model, in fact
constitutes an additional transmembrane segment. The homologous hydrophobic peak in Nhx1, indicated by an asterisk in Fig.
3A, could provide an unpredicted membrane-spanning segment
that would dispose the C terminus to the lumen. An alternative model is
that only certain portions of the Nhx1 C-terminal tail are accessible to the lumen; this would imply that part of the tail would have to
traverse the lipid bilayer. Hydropathy analysis of Nhx1 and NHE
isoforms indicates no putative membrane-spanning
-helices in the
C-tail regions; hence, for NHE3, Biemesderfer et al. (31) suggest that region(s) of the C terminus may span the membrane as a
-sheet. Such a scenario represents another plausible model that
could explain our observations about Nhx1. To address these and other
possibilities, we are currently pursuing a rigorous experimental
dissection of the membrane topology of Nhx1.
The present study supports the idea that the modular domain structure
of Nhx1 parallels exchanger function in a way similar to that of NHE
isoforms. In particular, the large N-terminal transmembrane domain
appears sufficient for Na+/H+ exchange
activity; the truncation mutant
512, in which 120 amino acids were
deleted from the C-terminal end, can still confer salt tolerance,
albeit at levels somewhat lower than wild-type. However, the C-terminal
deletion also resulted in significantly increased abundance of the
polypeptide, as well as a partial shift in subcellular localization
into Golgi-containing fractions of the sucrose density gradient. It is
possible that slower trafficking of the truncated protein out of the
Golgi also delays its proteolytic turnover, accounting for increased abundance.
It is interesting that even in the absence of N-linked
glycosylation, Nhx1 retains some multi-banded appearance upon gel
electrophoresis. Expression of Nhx1 in the sec53 mutant,
which blocks all O-linked and N-linked
glycosylations, retains some measure of multi-banded character, as
well. These data suggest the existence of additional post-translational
modifications of Nhx1. In particular, the sequence of the first
predicted transmembrane helix reveals features reflective of a signal
peptide, and we have preliminary evidence for an N-terminal cleavage
event. Additionally, there are numerous consensus sites for protein
kinase C- and casein kinase II-dependent phosphorylation distributed throughout the polypeptide.
In conclusion, our finding that Nhx1 is N-linked
glycosylated at two residues in its hydrophilic C-terminal tail domain
makes an experimental dissection of membrane topology imperative; this should contribute greatly to future studies on molecular structure and
function. The observation that a C-terminally truncated mutant and the
N515D/N550D double mutant retain heterogeneity on SDS-PAGE Western
blots paves the way for further characterization of post-translational modification(s) and of how this intracellular exchanger might be regulated.