Molecular Cloning and Characterization of a Novel
(Na+,K+)/H+ Exchanger Localized to
the trans-Golgi Network*
Masayuki
Numata and
John
Orlowski
From the Department of Physiology, McGill University,
Montréal, Québec H3G 1Y6, Canada
Received for publication, February 12, 2001
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ABSTRACT |
The luminal pH of organelles along the secretory
and endocytic pathways of mammalian cells is acidic and tightly
regulated, with the [H+] varying up to 100-fold
between compartments. Steady-state organellar pH is thought to reflect
a balance between the rates of H+ pumping by the
vacuolar-type H+-ATPase and H+ efflux through
ill-defined pathways. Here, we describe the cloning of a novel gene
(NHE7) in humans that is homologous to
Na+/H+ exchangers, is ubiquitously expressed,
and localizes predominantly to the trans-Golgi network.
Significantly, NHE7 mediates the influx of Na+ or
K+ in exchange for H+. The activity of NHE7 was
also found to be relatively insensitive to inhibition by amiloride but
could be antagonized by the analogue benzamil and the unrelated
compound quinine. Thus, NHE7 displays unique functional and
pharmacological properties and may play an important role in
maintaining cation homeostasis of this important organelle.
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INTRODUCTION |
The luminal ionic composition of many, if not all, intracellular
compartments differs from the surrounding cytoplasm and is an important
determinant of their function. The establishment of this differential
composition is achieved through the concerted actions of distinct
integral membrane ion carriers, including pumps, channels, and
transporters. For example, alkalinization of the mitochondrial matrix,
driven by the respiratory chain, contributes to the inner membrane
H+ gradient used to drive ATP synthesis (1) and,
indirectly, to extrude matrix Ca2+ through the functional
coupling of Na+/H+ and
Na+/Ca2+ antiport pathways (2-4).
By contrast, organelles of the secretory and endocytic pathways
are distinguished by their luminal acidity, which is generated by the
activity of an electrogenic vacuolar-type H+-ATPase
(V-ATPase)1 (5, 6).
Progressive acidification of vesicles in the endocytic pathway (early
and late endosomes, pH ~6.5
lysosomes, pH ~4.5) is essential
for the redistribution and degradation of internalized membrane
proteins, such as ligand-receptor complexes and fluid-phase solutes (5,
7). Likewise, increasing luminal acidification of compartments of the
exocytic pathway (endoplasmic reticulum, pH ~7.0
Golgi complex,
pH ~6.5
trans-Golgi network (TGN), pH ~6.0
secretory vesicles, pH ~5.0) is important for proper post-translational processing and sorting of newly synthesized proteins (5, 8, 9).
At present, little is known about the mechanisms controlling the
steady-state [H+] within the lumen of different
endomembrane compartments. Although distinct isoforms of some of the
V-ATPase subunits have been reported in different tissues (10, 11) or
specialized cell types (12), there is no clear evidence that the
V-ATPase functions differently in particular organelles within a single
mammalian cell (although this may not be the case in yeast (13)). It
has been suggested that because the pump is electrogenic, its activity
could be influenced by the membrane potential and by the
availability of permeant counterions such as chloride and potassium
(14). However, in the case of the Golgi complex, the endogenous
counterion conductances were found to exceed the rate of H+
pumping at the steady state, implying that the electrical potential across the membrane is negligible and therefore not a defining factor
in setting organellar pH (15-17). In addition, despite extensive work,
differential control of V-ATPase activity by hormones or other factors
has not been found along the endo- or exocytic pathways. Rather, the
luminal [H+] is thought to be regulated by a complex
interplay between the V-ATPase and unidentified leak pathways for
protons, based on the rapid dissipation of the transmembrane proton
chemical gradient (
pH) observed after inhibiting the V-ATPase with
macrolide antibiotics (15, 16, 18). A component of this H+
leak in the Golgi complex was recently identified as a
Zn2+-inhibitable H+ conductance (17) but could
not fully account for H+ turnover. Nevertheless, it
highlights the H+ leak as a key determinant of organellar
pH and emphasizes the need to identify the molecular components of this
pathway which, in addition to putative H+ channels, could
conceivably involve H+ proton-coupled cotransporters or exchangers.
In this study, we describe the cloning and functional characterization
of a unique monovalent cation/proton exchanger that localizes
predominantly to the trans-Golgi network and suggests a
novel molecular mechanism for controlling the luminal cation composition of this important organelle.
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EXPERIMENTAL PROCEDURES |
Molecular Cloning--
Two overlapping human ESTs with homology
to known mammalian Na+/H+ exchangers were
identified in GenBankTM (accession number AA279477 and
AA648924) and obtained from Genome Systems Inc. (St. Louis, MO).
Examination of the cDNA sequences (designated as NHE7) indicated
that they were missing coding information at the 5' and 3' regions
(i.e. start and stop codons, respectively). To obtain the
complete nucleotide sequence, we isolated cDNA fragments corresponding to the missing 3'-end (amino acids 624-725) from a human
bone marrow cDNA library using rapid amplification of cDNA ends
methodology (19). However, we were unable to clone the 5'-end of the
cDNA using this approach. While this work was in progress, the
sequences covering the 3'-end of the cDNA, but not the 5'-end, were
found to match nucleotide sequences present in a human genomic fragment
(GenBankTM accession number AL022165) that mapped to
chromosome Xp11. To determine the missing 5'-sequence, we screened a
human X chromosome library (20) available from the American Tissue
Culture Collection (ATCC) using a 123-base pair polymerase chain
reaction (PCR)-generated fragment corresponding to the most 5'-end of
one of the EST clones (GenBankTM accession number
AA648924). Four overlapping positive clones were isolated by screening
4 × 104 independent clones from the library. The
largest insert was subcloned into a plasmid vector, and the missing
sequence (the first 53 amino acids), in addition to downstream sequence
that precisely overlapped the 5'-end of the EST clone, was found to
reside within a single predicted exon. The presence of this sequence in
the NHE7 transcript was verified by reverse transcriptase-polymerase chain reaction (PCR) using human bone marrow and skeletal muscle poly(A+) RNA. The predicted translation initiation codon is
preceded by a purine nucleotide in position
3 and downstream contains a purine at position +4, placing it in a good context for initiation by
eukaryotic ribosomes, as defined by Kozak (21). In addition, the
apparent translation initiation site is also preceded by an in-frame
stop codon at nucleotide position
336. The 5'-end of the NHE7
cDNA sequence was subsequently found to match uncharacterized genomic sequences that map to chromosome Xp11.1-11.4
(GenBankTM accession number AL050307). The full-length
cDNA was reconstituted by PCR and the integrity of the construct
was verified by DNA sequencing. The complete cDNA sequence was
deposited in GenBankTM (accession number AF298591).
RNA Blotting--
Human poly(A+) mRNA Northern
and Master dot blots (CLONTECH) were hybridized
with a 0.2-kilobase pair PCR fragment generated from the 3'-end of NHE7
that shares minimal sequence identity with other NHE isoforms. The PCR
probe was agarose gel-purified and radiolabeled with
[
-32P]dCTP by the random primer method. Hybridization
was done at 65 °C in Church buffer containing 7% SDS, 0.5 M sodium phosphate, pH 7.2, 1% bovine serum albumin and 2 mM EDTA overnight. The blots were washed twice in 2× SSC,
0.05% SDS at room temperature for 30 min each, followed by three
higher stringency washes in 0.1× SSC containing 0.1% SDS at 68 °C
for 30 min each. The radioactive signals were analyzed by a
PhosphorImager (Molecular Dynamics, Sunnyvale, CA).
Stable Transfection and Expression of NHE7--
The full-length
NHE7 cDNA was engineered to include the influenza virus
hemagglutinin (HA) epitope, YPYDVPDYAS (preceded by a single G amino
acid linker inserted to create peptide flexibility), at the very
C-terminal end (called NHE7HA) using PCR mutagenesis to
allow for immunological detection of the protein. In a separate construct, an HA epitope was also inserted at an internal site, Leu488 (NHE7488HA). The NHE7HA
construct was subcloned into the ecdysone-inducible expression vector
pIND (Invitrogen) and transfected into Chinese hamster ovary cells that
constitutively express an ecdysone-activated receptor (EcR-CHO cells;
Invitrogen). Cells stably expressing both NHE7HA and EcR
were selected in
-minimum Eagle's medium supplemented with 10%
fetal bovine serum, 100 units/ml penicillin, 100 µg/ml streptomycin,
25 mM NaHCO3, pH 7.4, and containing 600 µg/ml G418 and 250 µg/ml zeocin. The cells were maintained in an
humidified atmosphere of 95% air and 5% CO2 at 37 °C.
Single colonies were isolated, and the regulated expression of
NHE7HA was verified by Western blotting and
immunofluorescence microscopy in the presence of increasing
concentrations (0-10 µM) of the ecdysone analogue,
ponasterone A, for 24 h.
Western Blotting--
Cells were washed three times with
ice-cold PBS and then lysed with triple detergent buffer (150 mM NaCl, 0.1% SDS, 1% IGEPAL CA-630, 0.5% sodium
deoxycholate and 50 mM Tris-HCl, pH 8.0) supplemented with
proteinase inhibitor mixture (Roche Molecular Biochemicals) for 5-10
min on ice. Cell lysates were spun at 12,000 × g for 5 min to remove insoluble cell debris, separated in a 7.5% SDS-PAGE, and
then transferred to a polyvinylidene difluoride membrane (Amersham Pharmacia Biotech). The blot was briefly rinsed with PBS, blocked with
5% non-fat skim milk in PBS for 1 h, and then incubated with mouse monoclonal antibodies against either the HA epitope (Babco, Berkley, CA) at a 1/5,000 dilution or cytochrome oxidase subunit IV
(COX IV) (Molecular Probes, Eugene, OR) at a 1/200 dilution. After
extensive washes with PBS containing 0.1% Tween 20, the blot was
incubated with goat anti-mouse IgG second antibody conjugated with
horseradish peroxidase (Jackson Laboratory, Bar Harbor, ME) at a
dilution of 1/20,000. Green fluorescent protein (GFP) was detected with
a rabbit polyclonal anti-GFP antibody (1/100 dilution) (CLONTECH) followed by incubation with a mouse
anti-rabbit IgG secondary antibody conjugated with horseradish
peroxidase (New England Biolabs) at a dilution of 1/3000.
Immunoreactive bands were detected by enhanced chemiluminescence
(Amersham Pharmacia Biotech) and exposed to an x-ray film.
Immunofluorescence Confocal Microscopy--
For
immunofluorescence confocal microscopy studies,
NHE7HA-transfected EcR-CHO cells were grown on glass
coverslips and incubated in the presence of 5 µM
ponasterone A for 24 h. The cells were subsequently fixed with 2%
paraformaldehyde/PBS for 20 min, permeabilized in 0.1% Triton X-100,
blocked with 5% non-fat skim milk in PBS for another 20 min, and then
incubated with monoclonal anti-HA antibody at a dilution of 1/1000 for
1 h. After extensive washing, cells were incubated with
Cy3-conjugated goat anti-mouse IgG secondary antibody (1/800 dilution)
for 1 h. For double labeling experiments with polyclonal
antibodies to organelle-specific markers, the signals were visualized
using Oregon Green or FITC-conjugated donkey anti-rabbit IgG (Molecular
Probes and Jackson Laboratory, respectively). In the case of the TGN
marker, a mammalian expression vector containing the CD25-TGN38
chimeric gene (22) was transiently transfected into the cells, and the
protein was visualized by FITC-conjugated anti-CD25 antibody (Serotec,
Raleigh, NC). The coverslips were washed, mounted onto glass slides,
and analyzed by confocal laser scanning microscopy using a Zeiss
inverted microscope. Images were processed using Adobe®
PhotoshopTM version 5.5 and CorelDrawTM version
8.0.
Measurements of Organellar
22Na+ and 86Rb+
Influx--
Rates of 22Na+ and
86Rb+ influx into endomembrane structures were
measured in control and ponasterone A-induced (10 µM;
24-h treatment) NHE7-transfected EcR-CHO cells that were permeabilized
with saponin (50 µg/ml) in K+-rich buffer (in
mM: 140 KCl, 2 CaCl2, 2 EGTA, 1 MgCl2, 2 Mg2+-ATP, 20 HEPES, pH 7.2) at
20 °C for 4.5 min, followed by multiple washes. Influx measurements
were conducted in choline chloride-rich buffer (in mM: 140 choline chloride, 2 CaCl2, 1 MgCl2, 2 Mg2+-ATP, 1 EGTA, 10 HEPES-Tris, pH 7.8). Following a 5-min
uptake period, the cells were quickly washed three times with ice-cold stop buffer (in mM: 140 NaCl (for
22Na+ influx) or 140 KCl (for
86Rb+ influx), 2 CaCl2, 1 MgCl2, 10 HEPES, pH 5.5). To extract the radiolabel, the
monolayers were solubilized with 0.5 N NaOH and neutralized
with 0.5 N HCl, and the pooled extracts were assayed by
liquid scintillation spectroscopy. Protein content was determined using
the Bio-Rad DC protein assay kit according to the manufacturer's protocol. Each experiment was repeated at least three times.
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RESULTS AND DISCUSSION |
A search of the GenBankTM data base for candidate
genes homologous to known mammalian Na+/H+
exchangers (i.e. NHE1-NHE6) (23-25) identified two novel
overlapping expressed sequence tags from human (GenBankTM
accession numbers AA279477 and AA648924). These shared highest sequence
identity to the mitochondrion-targeted NHE6 isoform rather than to the
plasmalemmal NHEs (i.e. NHE1-5), suggesting that the gene
product (which we designate as NHE7) may also reside in an
intracellular compartment. Examination of the sequences of the putative
NHE7 cDNAs indicated that they lacked coding information at the 5'-
and 3'-ends. To obtain the complete nucleotide sequence, we sequenced
cDNAs isolated from a single-stranded cDNA library that had
been generated from human bone marrow poly(A+) mRNA
using rapid amplification of cDNA ends methodology and genomic
fragments cloned from a human
phage library (for details, see
"Experimental Procedures").
The deduced primary sequence of NHE7 is composed of 725 amino acids
(calculated Mr = 80,132) and exhibits high amino
acid identity (~70%) to NHE6 (Fig.
1A) but low similarity (~ 25%) to other NHEs. Based on hydropathy plot analysis, NHE7 is
predicted to contain 12
-helical hydrophobic membrane-spanning (M)
segments in the N terminus followed by a hydrophilic cytoplasmic tail
at the C terminus, similar to other NHEs (Fig. 1B). Recent
biochemical and molecular topological studies of the NHE1 isoform
partially support this structural model (26, 27), although some notable changes in the arrangement of the C-terminal transmembrane segments have been proposed (27), namely the predicted M10 segment was suggested
to reside within the lipid bilayer (the predicted M11 was renamed M10),
whereas the last extracellular loop was found to form an intracellular
loop, a new transmembrane segment (M11), and an extracellular loop.
Unlike NHE1, a recent report has suggested that a part of the
cytoplasmic C terminus of the NHE3 isoform may reside at the exoplasmic
surface (28). Whether these structural features of the plasmalemmal
NHEs also apply to the more distantly related organellar NHEs remains
to be determined.

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Fig. 1.
Primary structure and predicted membrane
topology of the human NHE7 isoform. A, amino acid
sequences of human NHE6 (HumNHE6) and human NHE7 (HumNHE7)
(GenBankTM accession numbers D87743/AF030409 and AF298591,
respectively) were aligned using the ClustalW algorithm. Gaps
(indicated by periods) were introduced in the sequence to
maintain the alignment. Positions containing identical residues are
shaded in black, and conservative amino acid
differences are shaded in gray. Predicted
membrane-spanning segments are numbered 1-12 and indicated by an
overline. B, a hydrophobicity plot determined by the
algorithm of Kyte and Doolittle (window of 11 amino acids) (63) and
corresponding model of the transmembrane organization of the NHE7
protein are shown.
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Northern blot analysis of selected human tissues using an
isoform-specific cDNA probe from the 3'-coding region revealed
three NHE7 mRNA transcripts of ~9.5, 7.5, and 3.0 kilobases in
length under high stringency hybridization conditions (Fig.
2A). More extensive RNA dot
blot analyses of human tissues showed that the gene is expressed
ubiquitously (Fig. 2B) but most prominently in certain
regions of the brain (putamen and occipital lobe), skeletal muscle, and
secretory tissues (prostate, stomach, pancreas, pituitary gland,
adrenal gland, thyroid gland, salivary gland, and mammary gland). Each
of the mRNAs is of sufficient length to contain the entire coding
region, which could result from differential processing of the
untranslated regions or alternative-splicing of a single gene product.
The former is favored since attempts to identify alternatively spliced
variants within the coding region by reverse transcriptase-PCR were
unsuccessful. Alternatively, certain transcript(s) may represent other
closely related genes that have yet to be characterized. This broad
pattern of expression is consistent with NHE7 serving a
"housekeeping" function.

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Fig. 2.
Expression of NHE7 mRNA in
human tissues. A human tissue poly(A+) RNA Northern
blot (2 µg of RNA per lane) (A) and a human
poly(A+) RNA MasterTM dot blot
(CLONTECH) (B) were hybridized with an
isoform-specific 32P-labeled NHE7 cDNA probe (0.2 kilobase pairs) under high stringency conditions. The radioactive
signals were detected using a PhosphorImager (Molecular Dynamics). The
positions and sizes (in kilobases) of the RNA markers in A
are shown on the left.
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To facilitate expression and localization of NHE7, we inserted an
influenza virus hemagglutinin (HA) epitope at the extreme C terminus
(NHE7HA). The full-length NHE7HA cDNA was
subcloned into the ecdysone-inducible expression vector pIND and stably transfected into Chinese hamster ovary cells engineered to express constitutively an ecdysone-activable receptor (EcR-CHO cells). Regulated expression of NHE7HA was verified by incubating
isolated NHE7HA-transfected EcR-CHO cell colonies in the
presence of increasing concentrations of ponasterone A, an analogue of
ecdysone. As shown in Fig. 3, ponasterone
A induced a dose-dependent increase in the expression of
NHE7HA, which migrated as two broad bands of ~180 and 80 kDa by SDS-PAGE analysis. The faster migrating band corresponds to the
predicted size of the protein. The larger band may represent the
formation of a homodimer that is modestly stable in SDS, as has been
reported for NHE1 and NHE3 (29). The bands are diffuse, suggesting the
presence of glycosylation or other post-translational modifications.
Consistent with this possibility, the N terminus contains putative
N-linked glycosylation sites in the predicted extracellular
loop between M3 and M4 (Asn145-Val-Ser) and between M9 and
M10 (Asn400-Leu-Ser). Although the latter site is highly
conserved in eukaryotic NHEs, it does not appear to be glycosylated in
other mammalian NHE isoforms (i.e. NHE1, NHE2, and NHE3)
(30-32) and therefore is unlikely to be modified in NHE7.

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Fig. 3.
Ecdysone-inducible expression of recombinant
NHE7HA in stably transfected EcR-CHO cells. Cell
lysates were prepared from an EcR-CHO cell line stably transfected with
NHE7HA following incubation with increasing concentrations
(0-10 µM) of the ecdysone analogue, ponasterone A, for
24 h. Fifty µg of each cell lysate were subjected to 7.5%
SDS-PAGE and immunoblotted with an anti-HA monoclonal antibody.
Molecular weight markers are shown on the right.
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To define the subcellular distribution of NHE7HA,
NHE7HA-transfected EcR-CHO cells were treated with a
submaximal concentration of ponasterone A (5 µM) for
24 h and then examined using immunofluorescence confocal
microscopy. Typically under these conditions, ~50% of the cells in
different stable transfectants were found to express detectable levels
of NHE7HA. The partial penetrance of NHE7HA expression upon ponasterone A induction was also observed in individual secondary and tertiary isolates obtained by dilution subcloning. The
cellular basis for this limited expression pattern is unclear but may
relate to the site of genomic integration of the ecdysone receptor gene
and/or the stage of the cell cycle.
Representative dual labeling experiments revealed that
NHE7HA accumulates predominantly in a juxtanuclear
compartment that was closely apposed but somewhat broader than the
compact structure labeled by an antibody against
-mannosidase II, an
established marker of the medial and trans-cisternae of the
Golgi (Fig. 4, A-C) (33).
Transient expression of two additional constructs, one containing an
internal HA-tag inserted at position Leu488 and the other
containing a C-terminal c-Myc epitope, gave similar results, suggesting
that the protein distribution is not influenced by the position or
sequence of the tag. The pattern was clearly distinct from those
observed with antibodies that recognize specific markers of the
endoplasmic reticulum (calnexin), lysosomes (cathepsin B and D), or
mitochondria (mito-green fluorescent protein (mito-GFP) (34) and COX
IV) (data not shown). NHE7HA also did not appear to
accumulate at the cell surface nor was it able to functionally complement a mutant strain of CHO cells lacking plasmalemmal NHE activity (35) (data not shown).

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Fig. 4.
Subcellular localization of human
NHE7HA in stably transfected EcR-CHO cells. To define
the subcellular distribution of NHE7HA, EcR-CHO cells were
treated with a suboptimal concentration of ponasterone A (5 µM) for 24 h. Subsequently, the cells were incubated
in the absence (A-C) or presence (D-F) of 5 µg/ml brefeldin A for 2 h at 37 °C. The cells were then
fixed, permeabilized, and dual labeled with a mouse monoclonal antibody
to the HA epitope (NHE7HA) followed by Cy3-conjugated
secondary antibody (A, D, and G) and a rabbit
polyclonal antibody to the endogenous Golgi cisternae marker
-mannosidase II ( -man II) followed by
Oregon Green-conjugated secondary antibody (B and
E). In certain experiments, NHE7-induced cells were
transiently transfected (24-h period) with the TGN marker CD25-TGN38,
and the chimeric protein was detected by FITC-conjugated anti-CD25
antibody (H). Composite images of the dual labels are shown
in C, F, and I. Scale bar, 10 µm.
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The comparatively broader immunofluorescence signal of
NHE7HA relative to that of
-mannosidase II suggested
that it may be present in compartments distal to the Golgi cisternae,
such as the TGN and possibly endosomes. To define further NHE7
compartmentation, cells were treated with different pharmacological
agents that are known to affect the Golgi, TGN, and endosomes
differentially. The fungal metabolite brefeldin A causes the
disassembly of the Golgi apparatus by promoting the retrograde
absorption and dispersion of Golgi cisternae resident proteins into the
endoplasmic reticulum (36, 37), thereby creating a mixed
endoplasmic reticulum/Golgi compartment. It concomitantly
induces the coalescence of the TGN with early endosomes into a dense
juxtanuclear tubulovesicular structure (38, 39). As shown in Fig. 4,
pretreatment of cells with brefeldin A (5 µg/ml for 2 h at
37 °C) dispersed the immunofluorescence signal of
-mannosidase II
(Fig. 4, E and F) into a reticular pattern,
whereas that of NHE7HA (Fig. 4, D and
F) was largely retained in a compact juxtanuclear complex,
with a minor fraction diffusely distributed throughout the cell. These
data suggest that NHE7HA is concentrated primarily in the
TGN/early endosomal structure. The location of the TGN was defined by
transiently transfecting the cells with an expression vector containing
the chimeric gene CD25-TGN38. This chimera, which is
composed of the extracellular domain of the
-chain of the
interleukin-2 receptor linked to the transmembrane and cytosolic
domains of TGN38, has been shown to accumulate in the TGN and can be
readily labeled with commercially available FITC-conjugated CD25
antibodies (15, 22). As shown in Fig. 4, G-I, the
distribution of NHE7HA precisely overlapped that of
CD25-TGN38, suggesting it is predominantly in the TGN.
To establish further the compartmentation of
NHE7HA, cells were treated with the microtubule-disrupting
agent nocodazole, which causes initial dispersion of the TGN and
endosomes (early event), followed by the redistribution of the Golgi
cisternae (late event), into discrete vesicular compartments throughout the cytoplasm (37, 40). As shown in Fig.
5, A-C, acute treatment with
nocodazole (10 µM for 1 h) caused the TGN marker
CD25-TGN38 to scatter in a pattern precisely matching that of
NHE7HA, whereas the distribution of
-mannosidase II in
the Golgi cisternae remained relatively compact (Fig. 5,
D-F). However, after a 4-h exposure to nocodazole, the
-mannosidase II signal also dispersed in a pattern that was distinct
from, but partially overlapping, that of NHE7HA (Fig. 5,
G-I). In similarly treated cells, endomembrane vesicles
containing NHE7HA also did not precisely colocalize with those containing transiently transfected, Myc-tagged NHE3, which is
known to accumulate at the cell surface but also in endocytic or
recycling endosomal vesicles of CHO cells (41) (data not shown). Taken
together, these data suggest that some NHE7 may be present in the Golgi
cisternae but most is situated in the TGN.

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Fig. 5.
Effect of nocodazole on the subcellular
distribution of NHE7HA. Expression of
NHE7HA was induced in EcR-CHO cells by 5 µM
ponasterone A for 24 h. In certain experiments, NHE7-induced cells
were transiently transfected (24-h period) with the TGN marker
CD25-TGN38. Subsequently, the cells were incubated in the presence of
10 µM nocodazole for 1 h (A-F) or 4 h (G-I) at 37 °C. The cells were then fixed,
permeabilized, and dual labeled with a mouse monoclonal antibody to the
HA epitope (NHE7HA) followed by Cy3-conjugated secondary
antibody (A, D, and G), and a FITC-conjugated
anti-CD25 antibody (B) or rabbit polyclonal antibody to the
endogenous Golgi cisternae marker -mannosidase II
( -man II) followed by Oregon Green-conjugated
secondary antibody (E and H). Composite images of
the dual labels are shown in C, F, and I. Scale bar, 10 µm.
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It is noteworthy that NHE7, unlike the closely related NHE6 isoform,
lacks an obvious mitochondrial targeting sequence at its N terminus
(i.e. ~20-60 amino acids with abundant basic residues that are predicted to form an amphipathic
-helix) (42). However, NHE7 does contain putative motifs for Golgi targeting and/or retention, including a unique Ser/Thr-phosphorylatable acidic cluster
(542EEPSEEDQNE551) (43) and a tyrosine-based
sequence (556YFRV559) (44). Differential
localization of NHE7 and NHE6 was further demonstrated biochemically by
subcellular fractionation of the endomembrane compartments using
differential centrifugation and Western blotting with antibodies that
recognize either endogenous or ectopically expressed organelle-specific
markers. As shown in Fig. 6,
NHE7HA was associated with the microsomal enriched membrane
fraction (P100 pellet) isolated from cells that were also transiently
transfected with Golgi-targeted green fluorescent protein (g-GFP), a
convenient marker for this fraction (34). By contrast,
NHE6HA (also stably expressed under the control of the
ecdysone-inducible promoter in another CHO cell line) accumulated in
the mitochondrion-enriched fraction (P10 pellet), as defined molecularly by the presence of the mitochondrion-specific marker COX
IV. Neither NHE7HA nor NHE6HA was present in
the soluble fractions (S100) isolated from cells that were transiently
transfected with an expression plasmid containing cytoplasmic GFP
(c-GFP) as a marker (34).

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Fig. 6.
Subcellular fractionation and immunoblot
analysis of NHE7HA expressed in EcR-CHO cells.
Expression of NHE7HA was induced by 5 µM
ponasterone A for 16 h. Cell lysates were prepared by mild
disruption through a 26.5-gauge needle in 250 mM sucrose,
10 mM HEPES-NaOH, 1 mM EDTA, pH 7.5, in the
presence of a proteinase inhibitor mixture (Roche Molecular
Biochemicals). Nuclei and insoluble cell debris were sedimented by
centrifugation at 500 × g. The mitochondrial and
microsomal fractions were obtained from the supernatant by sequential
centrifugation at 10,000 and 100,000 × g,
respectively, and the resulting pellets were isolated and designated
P10 and P100. Each fractionated pellet was
resuspended in an identical volume of lysis buffer. The final
supernatant fraction (S100) represented the cytosolic
fraction. For comparison, parallel subcellular fractionation
experiments were conducted using cell lysates prepared from an EcR-CHO
cell line stably transfected with human NHE6 tagged with an HA epitope
at its C terminus (NHE6HA) following exposure to 5 µM ponasterone A for 24 h. NHE6HA is
found in the P10 fraction, consistent with its known accumulation in
mitochondria. Additional controls include NHE7HA-expressing
cells transiently transfected with an expression vector containing
either a cDNA chimeric construct composed of the signal peptide
from the Golgi protein human -1,4-galactosyltransferease linked to
GFP (g-GFP) (34) or GFP lacking a membrane targeting signal
(cytoplasmic GFP or c-GFP). Equivalent volumes of each fraction were
then subjected to 10% SDS-PAGE and immunoblotted with antibodies to
HA, GFP, or COX IV.
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To assess NHE7HA activity, we adapted procedures that had
been used previously for measuring 45Ca2+
uptake and release from the Golgi (45). The plasma membrane was
selectively permeabilized with saponin, and
22Na+ uptake into intact endomembrane
compartments was compared with and without ponasterone A-induced
overexpression of NHE7HA. Preliminary experiments
demonstrated that the sequestration of 22Na+ in
choline chloride-rich buffer was dependent on the external pH
(i.e. maximal in more alkaline buffers) and linear over a
10-min period (data not shown); therefore, an external pH of 7.8 and a
5-min uptake period were chosen for the transport assay. As shown in
Fig. 7A,
NHE7HA-expressing EcR-CHO cells had ~75% higher rates of
22Na+ influx compared with uninduced cells.
Since only ~50% of cells stably overexpress NHE7HA under
these conditions, this percentage increase likely represents an
underestimate of the actual cellular flux rates due to transfected
NHE7HA. Ponasterone A had no effect on untransfected
EcR-CHO cells (data not shown). The role of pH was evaluated by
treating cells with the H+-specific ionophore carbonyl
cyanide m-chlorophenylhydrazone (CCCP), which rapidly
dissipates the organellar transmembrane H+ gradient (17).
As illustrated in Fig. 7A, 2 µM CCCP
significantly reduced 22Na+ influx in both
uninduced and ponasterone A-treated cells when compared with controls.
Likewise, alkalinization of endomembrane compartments by sustained
exposure to 30 mM NH4Cl, as previously described by Kim et al. (18), also led to decreased
22Na+ influx. By contrast, pretreatment and
rapid removal of NH4Cl, which dramatically acidifies
intracellular compartments due to the rapid efflux of ammonia (18),
significantly elevated (~ 3-fold) 22Na+
uptake in both uninduced and ponasterone A-induced cells. Taken together, these data indicate the existence of an endogenous organellar Na+ influx pathway that depends on the transmembrane
H+ gradient and that is up-regulated in
NHE7HA-overexpressing cells.

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|
Fig. 7.
Measurements of
H+-dependent 22Na+
influx into endomembrane compartments of NHE7HA-transfected
EcR-CHO cells. NHE7HA-transfected EcR-CHO cells were
grown to confluence in 24-well plates. Prior to functional
measurements, the cells were incubated in the absence ( ) or presence
(+) of ponasterone A (Pon A, 10 µM) for an
additional 24 h, followed by permeabilization of the plasma
membrane with saponin (for details, see "Experimental Procedures").
The transport rates are normalized as a percentage of control values
( Pon A) measured in choline chloride-rich buffers
(pHo 7.8). A, prior to measurements of
22Na+ influx (5 µCi of 22NaCl
(carrier-free)/ml), cells were untreated or pretreated with either the
H+ ionophore CCCP (2 µM) or the alkalizing
agent NH4Cl (30 mM) for 2 or 5 min,
respectively, and were maintained during the 5-min uptake period. In
one series of experiments, the NH4Cl was rapidly washed out
(+ w/o) of the cells prior to measurements of
22Na+ influx in order to increase the acidity
of the organellar compartments. B, initial rates of
22Na+ influx were measured in the presence of
amiloride (2 mM) or benzamil (0.1 and 1 mM).
Values represent the average of 3-4 experiments, each performed in
quadruplicate (mean ± S.D.). Differences in the experimental
groups were analyzed by a one-way analysis of variance, and comparisons
between means were carried out using the Newman-Keuls test at the 5%
significance level (asterisks = p < 0.05).
|
|
We next tested whether the activity of NHE7HA was sensitive
to amiloride derivatives that are known inhibitors of the NHEs and are
relatively membrane-permeant. As shown in Fig. 7B,
22Na+ influx was only weakly inhibited
(~25%) by 2 mM amiloride, a concentration that is
sufficient to abolish activity of the plasma membrane NHE1 isoform in
CHO cells (46). NHE7HA was also insensitive to low
concentrations (100 µM) of benzamil, an amiloride
analogue, although it was blocked significantly at high concentrations
(1 mM). Since 100 µM benzamil is known to
block the Na+-selective Na+/H+
exchanger in isolated mitochondria (47), the fluxes measured under our
conditions are unlikely to include uptake into mitochondria, more so
given that the mitochondrial matrix is alkaline under steady-state conditions.
To establish the cation selectivity of this pathway, we performed
analogous uptake experiments using 86Rb+, a
radioactive congener of K+. Unexpectedly, ponasterone
A-induced NHE7HA-expressing cells exhibited a similar
increase (~75%) in the rate of 86Rb+ influx
compared with uninduced cells (Fig.
8A). This stimulation was not
observed in untransfected EcR-CHO cells (data not shown) and, like
22Na+ uptake, was inhibited by 2 µM CCCP and 1 mM benzamil. Increasing the
concentration of external Na+, K+, or
Li+ substantially reduced 86Rb+
transport in NHE7HA-induced cells (Fig. 8B),
suggesting that all these monovalent cations compete for binding to the
same or closely associated sites. By contrast,
86Rb+ fluxes were relatively insensitive to
divalent cations such as Zn2+ (200 µM)
(Zn2+-treated cells were 92.3 ± 10.7% of controls
(n = 4); p > 0.05), which suggests
that this pathway is distinct from the Zn2+-inhibitable
H+ conductance recently identified in the Golgi complex
(17). These data indicate that NHE7 functions as a nonselective
monovalent cation/H+ exchanger. Since K+ is the
main intracellular alkali cation, the physiologically relevant mode of
transport is probably K+/H+ exchange. Thus, in
addition to serving as a H+ efflux pathway in the
trans-Golgi network, NHE7 may also participate in
controlling the luminal [K+] which could influence volume
homeostasis/morphology of this organelle.

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|
Fig. 8.
Measurements of
H+-dependent 86Rb+
influx into endomembrane compartments of NHE7HA-transfected
EcR-CHO cells. NHE7HA-transfected EcR-CHO cells were
grown to confluence in 24-well plates. Prior to functional
measurements, the cells were incubated in the absence ( ) or presence
(+) of ponasterone A (Pon A, 10 µM) for an
additional 24 h, followed by permeabilization of the plasma
membrane with saponin (for details, see "Experimental Procedures").
The transport activities are normalized as a percentage of control
values ( Pon A) measured in choline chloride-rich buffers
(pHo 7.8). A, initial rates of
86Rb+ influx (5 µCi of 86RbCl
(carrier-free)/ml) were measured in the absence or presence of CCCP (2 µM) and benzamil (1 mM). B,
initial rates of 86Rb+ influx in ponasterone
A-induced cells were measured in the presence of increasing
concentrations of KCl (closed circles), NaCl (closed
squares), and LiCl (closed triangles). Isoosmolarity
was maintained by adjusting the choline chloride concentration.
C, initial rates of 86Rb+ influx
were measured in the absence (control) or presence of increasing
concentrations of quinine. Values represent the average of 3-4
experiments, each performed in quadruplicate (mean ± S.D.).
Differences in the experimental groups were analyzed by a one-way
analysis of variance, and comparisons between means were carried out
using the Newman-Keuls test at the 5% significance level
(asterisks = p < 0.05).
|
|
The unique transport properties of NHE7 are particularly noteworthy in
light of earlier biochemical studies that revealed the presence of two
functionally distinct monovalent cation/proton exchangers in mammalian
mitochondria. One of these preferentially mediates the exchange of
matrix Na+ for intermembrane H+ generated by
respiration (i.e. a Na+-selective
Na+/H+ exchanger) (48, 49) and is inhibited by
benzamil derivatives of amiloride at micromolar concentrations (47,
50-52). It is constitutively active in respiring mitochondria and is
primarily responsible for establishing the [Na+] gradient
([Na+]i < [Na+]o) that
allows Na+-dependent extrusion of matrix
Ca2+ (53). The other monovalent cation/H+
exchanger is latent, transports all alkali cations (i.e.
Li+, Na+, K+, Rb+, and
Cs+) at similar rates, is antagonized by drugs such as
quinine, dicyclohexylcarbodiimide, and propranolol (54-56), and is
postulated to play a role in organellar volume homeostasis (57). Again,
since K+ is the predominant intracellular alkali cation, it
is simply referred to as a K+/H+ exchanger. At
present, it is unclear which of the mammalian mitochondrial NHEs
corresponds to the recently cloned mitochondrion-targeted NHE6 isoform
since it has yet to be characterized functionally. However, in view of
the high structural similarity between NHE7 and NHE6, we speculate that
NHE6 may function as the mitochondrial quinine-sensitive
K+/H+ exchanger. In this regard, we also find
that NHE7HA activity is sensitive to inhibition by quinine
(Fig. 8C), further suggestive of its functional similarity
to one of the mitochondrial NHEs (i.e. NHE6). Further
detailed pharmacological analyses are currently ongoing.
Homologues to human NHE6 and NHE7 have also been identified in lower
eukaryotes, including the yeast Saccharomyces cerevisiae gene, NHX1/NHA2 (24, 58), and the plant Arabidopsis
thaliana gene, AtNHX1 (59). Immunological analyses
showed that yeast Nhx1 and plant AtNhx1 proteins localize predominantly
to the late endosomal/prevacuolar and tonoplast/vacuolar compartments,
respectively, and were capable of conferring tolerance to cytotoxic
concentrations of NaCl (59-61). More recently, yeast Nhx1 was also
found to be important for efficient protein trafficking out of the
prevacuolar compartment (62). The latter results are particularly
intriguing in view of the localization of NHE7 to the TGN and suggest
that it may fulfill a similar physiological function. We are currently testing the hypothesis.
In summary, we describe the cloning and functional characterization of
a novel monovalent cation/H+ exchanger that localizes
predominantly to the trans-Golgi network and likely plays an
important role in maintaining the cation homeostasis and function of
this important organelle.
 |
ACKNOWLEDGEMENTS |
We thank the following individuals for
generously providing us with the reagents used in this study: J. Mort
(Shriner's Hospital, Montreal, Canada) for the anti-cathepsin
antibody; A. S. Verkman (University of California, San Francisco)
for the mitochondrial and Golgi-GFP constructs; J. Bergeron (McGill
University, Canada) for the anti-calnexin antibody; T. Hobman
(University of Alberta, Canada) and M. G. Farquhar (University of
California, San Diego) for the anti-
-mannosidase II antibodies; and
J. S. Bonifacino (National Institutes of Health, Bethesda) for the
CD25-TGN38 construct. We also thank H. Zaun and A. Boucher for
technical assistance and J. Hanrahan for critically reading the manuscript.
 |
FOOTNOTES |
*
This work was supported by the Canadian Institutes for
Health Research.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Physiology,
McGill University, McIntyre Medical Sciences Bldg., 3655 Promenade
Sir-William-Osler, Montreal, Quebec H3G 1Y6, Canada. Tel.:
514-398-8335; Fax: 514-398-7452; E-mail: orlowski@med.mcgill.ca.
Published, JBC Papers in Press, February 26, 2001, DOI 10.1074/jbc.M101319200
 |
ABBREVIATIONS |
The abbreviations used are:
V-ATPase, vacuolar-type H+-ATPase;
TGN, trans-Golgi
network;
PCR, polymerase chain reaction;
HA, hemagglutinin;
PBS, phosphate-buffered saline;
PAGE, polyacrylamide gel electrophoresis;
FITC, fluorescein isothiocyanate;
EcR, ecdysone-activated receptor;
CHO, Chinese hamster ovary;
GFP, green fluorescent protein;
CCCP, carbonyl cyanide m-chlorophenylhydrazone;
COX IV, cytochrome
oxidase subunit IV.
 |
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Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.