From the Department of Physiology, The Johns Hopkins University
School of Medicine, Baltimore, Maryland 21205
 |
INTRODUCTION |
Na+/H+ exchangers of eukaryotic cells
comprise a family of membrane proteins catalyzing the electroneutral
countertransport of Na+ and H+ (1-3). At the
plasma membrane of animal cells, the prevailing Na+
gradient generated by the Na+/K+-ATPase is used
to drive H+ equivalents from the cell. As such, these
exchangers are involved in the regulation of intracellular pH, cell
volume control, and transcellular Na+ movements in
epithelial tissue. These functions are closely related to physiological
and pathophysiological cellular events, including fertilization, cell
cycle control, differentiation, essential hypertension, gastric and
kidney disease, and epilepsies. Na+/H+ exchange
activity has been detected in virtually every cell type that has been
examined, and at least six distinct NHE isoforms have been identified
thus far. Molecular cloning of the first Na+/H+
exchanger (4) led to a predicted membrane topology based on the
hydropathy profile of the amino acid sequence: there are 12 membrane-spanning segments comprising a discrete N-terminal structural domain of approximately 500 residues, followed by a long cytoplasmic C-terminal tail of approximately 300 residues. This predicted structural subdivision mimics a partition of function: analysis of
deletion mutants has shown that the membrane-embedded domain retains
the ability to insert into the plasma membrane, is
transport-competent, and is sensitive to inhibition by amiloride and
its derivatives (5). However, it is the C-terminal domain that carries
multiple protein kinase consensus sites, binds calmodulin, and
mediates the response to a multitude of regulatory signals involved in control of cell proliferation, volume, and osmolarity changes.
All Na+/H+ exchangers that have been
characterized at a molecular level thus far localize predominantly, if
not exclusively, to the plasma membrane. Nevertheless, there has been
biochemical documentation of Na+/H+ exchange
activity in endosomal preparations from kidney, liver, zymogen granules
of pancreatic acinar cells, and chromaffin granules of adrenal glands
(6-11). In each case, the exchange activity was reported to coexist
with a distinct subset (~20%) of vesicles containing the vacuolar
H+-ATPase and to exhibit kinetic similarity with the plasma
membrane exchangers with respect to reversibility, simple hyperbolic
response to Na+, and allosteric activation by
H+. However, amiloride did not inhibit the endocytic
exchange activity, Li+ was a poor substrate but a good
inhibitor of Na+/H+ exchange, and the
Km for Na+ was somewhat lower than that
seen for plasma membrane isoforms (4.7-10 mM
versus 15-18 mM), suggesting that the endocytic
exchanger is a distinct molecular isoform.
In earlier work, we have shown that the NHX1 gene of
Saccharomyces cerevisiae mediates sequestration of
Na+ within an intracellular compartment, suggestive of a
novel intracellular localization (12). Here, we provide direct evidence
that Nhx1 localizes exclusively to a unique late endosomal compartment, thus providing a starting point to explore the molecular, cellular, and
physiological functioning of a completely novel member of this family
of transporters. We have also observed the emergence of new
exchanger homologues in other organisms, as a result of systematic
sequencing efforts worldwide, that share greater homology with yeast
Nhx1 than to the plasma membrane isoforms. We suggest that the sequence
similarites among these newly discovered isoforms is indicative of a
common intracellular, possibly endosomal
localization.
 |
EXPERIMENTAL PROCEDURES |
Yeast Strains and Recombinant DNA Techniques--
Strains K601
(wild type) and R100 (
nhx1) used in this study are
isogenic to W303 and have been described (12). A 4.5-kilobase pair
(kbp)1 SalI insert
containing the intact NHX1 gene was recovered from cosmid
C9410 (American Type Culture Collection), and a 3-kbp SalI to SpeI fragment from the 5' portion of the gene was cloned
into pRS425 (13), now called pRin72. The C terminus of Nhx1 was tagged with a triple hemagglutinin (HA) epitope using two polymerase chain
reaction (PCR) products as primers for a third PCR. The following
primers were used to amplify a 0.8-kbp product from cosmid C9410 that
extended from +1.1 kbp downstream from the initiating ATG to the end of
the NHX1 open reading frame, with the removal of the
termination codon, and addition of a NotI site and a short sequence homologous to the 5' end of the HA epitope:
5'-CTGAAGTAGAACTAGTCTATAAGCCAC-3' (sense) and
5'-AACATCGTATGGGTAAAAGATGCGGCCGCCGTGGTTTTGGGAAGAGAAATCTGCAGG-3' (antisense). The second PCR created a 1-kbp product beginning with a
short sequence homologous to the 3' end of the HA epitope, followed by
the termination codon TAG, and extending through 1 kbp of 3' noncoding
sequence of the NHX1 gene to a new SacI site at
the 3' end. The following primers were used in conjunction with C9410
as template:
5'-GACGTTCCAGATTACGCTGCTGAGTGCTAGCCGCGGGTAGACTTTAAAGTGTATGGTTTCC-3' (sense) and 5'-GGCACGAGCTCGTCTTCATCCATGACGGAAG-3' (antisense). The final PCR reaction used the PCR products, above, as primers with
the plasmid pSM491 (gift of Susan Michaelis, Johns Hopkins University)
containing the triple HA epitope as the template. The resulting 1.9-kbp
product was digested with SpeI and SacI and
cloned into pRin72 to give the full-length Nhx1::HA with the 1.9-kbp upstream sequence from the initiating codon ATG and the 1-kbp
downstream sequence from the termination codon (pRin73). The
Nhx1::GFP construct was created by digesting pEGFP-N3
(CLONTECH) with BamHI and
NotI to release the 0.7-kilobase GFP and ligating to the
same sites in pRin72. To complete the NHX1 open reading frame, a 1.3-kbp BamHI fragment from pRin73 was inserted in
the correct orientation into this plasmid, creating the full-length fusion.
Biochemical Methods--
Assays of protein,
-mannosidase
activity, Kex2 activity, and
azide-sensitive ATPase activity have been described in earlier publications (14, 15) and in references therein. Differential centrifugation of yeast lysates and nonequilibrium fractionation of
yeast lysates by sucrose gradient centrifugation was performed as
described (14, 16). SDS-polyacrylamide gel electrophoresis and Western
blotting were performed as described previously (14). Antibodies were
used as follows: mouse anti-HA antibody, 12CA5 (Boehringer Mannheim) at
1:5000, mouse anti-Vph1, monoclonal antibody 10D7-A7-B2 (Molecular
Probes) at 1:5000, mouse anti-Dpm1, monoclonal antibody 5C5-A7
(Molecular Probes) at 1:2500, mouse anti-GFP (Molecular Probes) at
1:500, rabbit anti-Pma1 (gift of Carolyn Slayman, Yale University) at
1:1000, rabbit anti-Pep12 (gift of Robert Piper, University of Iowa) at
1:1250, rabbit anti-Kex2 (gift of Robert Fuller, University of
Michigan),at 1:1000. Horseradish peroxidase-coupled goat anti-mouse
(Boehringer Mannheim) and horseradish peroxidase-coupled donkey
anti-rabbit (Amersham Pharmacia Biotech) were used at 1:1000.
Confocal Microscopy--
Cells (0.7-1.2
A600 units/ml) were labeled with 53 µM FM 4-64 (N-(3-triethylammoniumpropyl)-4-(6-(4-diethylamino)phenyl)-hexatrienzyl)pyridinium dibromide), 13 nM DiOC6
(3,3'-dihexyloxacarbocyanine iodide), and 40 nM MitoTracker
Red CMXRos (all from Molecular Probes) using a variable labeling period
(10-60 min) followed by chase in fresh medium (30-60 min). Confocal
microscopy was performed by the Noran Oz Confocal Microscope System;
single label controls for each fluorophore were captured under
identical double label conditions to eliminate any fluorescence
bleed-through.
 |
RESULTS |
Epitope-tagged and Plasmid-encoded Nhx1 Is Fully Functional and
Induced by NaCl--
Targeted disruption of the S. cerevisiae
NHX1 (YDR456w) gene leads to loss of sodium tolerance in acidic
(Fig. 1a) but not neutral or
alkaline medium (12), consistent with the expected properties of a
H+ driven Na+ transporter. The NHX1
gene was recovered from a 40-kilobase pair genomic insert in cosmid
C9410 (see "Experimental Procedures"), and the open reading frame
was tagged at the C terminus with either a triple HA epitope or the
GFP. Expression of the tagged constructs was directed from the
endogenous NHX1 promoter in a
nhx1 strain of
yeast. Both tagged constructs appeared to be fully functional, effectively complementing the Na+-sensitive phenotype of
the
nhx1 mutant in the single copy (CEN) as
well as multicopy (2µ) plasmid versions (Fig. 1a). Like
other members of the NHE family, yeast Nhx1 is predicted to be an
integral membrane protein, with an N-terminal domain of 12 transmembrane helices, followed by a C-terminal cytoplasmic tail (12).
Differential centrifugation of yeast lysates results in a substantial
enrichment of Nhx1::HA (molecular mass, 73.5 kDa) in low
speed membrane pellets (Fig. 1b); in the absence of further
fractionation, the Nhx1 polypeptide characteristically migrates as
multiple bands on SDS gels, indicative of post-translational
modifications such as phosphorylation or glycosylation. Further
evidence of the involvement of Nhx1 in halotolerance comes from salt
induction of expression (Fig. 1b).

View larger version (23K):
[in this window]
[in a new window]
|
Fig. 1.
Epitope-tagged Nhx1 confers sodium tolerance
and is induced by NaCl. a, wild type (WT) or
nhx1 strains of yeast carrying the plasmids as shown were
grown to saturation at 30 °C in the absence or presence of 400 mM NaCl in APG medium, pH 4.0, as described previously
(12). b, nhx1 cells expressing HA-tagged Nhx1
from its endogenous promoter in a 2µ plasmid (see "Experimental
Procedures") were cultured in the absence or presence of NaCl (300 mM). Clarified lysates (TL) were divided into
two aliquots and centrifuged at either 10,000 × g or
100,000 × g. Equal protein (90 µg) from pellet
(P) or supernatant (S) fractions were subjected
to SDS-polyacrylamide gel electrophoresis and Western blotting with
anti-HA antibody. Note the increase in expression level in medium
containing NaCl.
|
|
Colocalization of HA-tagged Nhx1 with Vacuolar and
Prevacuolar Markers in Subcellular Fractions--
Our measurements of
steady state intracellular 22Na levels indicated that
enhanced sequestration of Na+ via Nhx1 correlated with
salt-tolerant growth (12). By analogy with observations of vacuolar
compartmentation of salt in halotolerant plants (17, 18), we
hypothesized that Na+ transport by Nhx1 was likely to be
coupled to the vacuolar H+ pump in an acidic compartment.
Here, we show that HA-tagged Nhx1 cofractionates with markers for the
vacuole, prevacuolar compartment, and the late Golgi compartment on
sucrose density gradients (Fig. 2a), whereas it clearly
fractionated away from markers representing the endoplasmic reticulum,
plasma membrane, and mitochondria, pointing to a hitherto novel
cellular location for a Na+/H+ exchanger. To
distinguish between prevacuolar, vacuolar, and Golgi distributions,
subcellular fractions from sequential centrifugation of yeast lysates
were analyzed by gel electrophoresis (Fig. 2b). Fractionation of Nhx1 closely followed that of the vacuolar marker, Vph1 (a subunit of the vacuolar H+-ATPase), and that of
Pep12 (a prevacuolar syntaxin) but was clearly different from the late
Golgi protease, Kex2.

View larger version (30K):
[in this window]
[in a new window]
|
Fig. 2.
Subcellular fractionation of HA-tagged
Nhx1. a, nhx1 cells expressing HA-tagged
Nhx1 (2µ) were grown in APG medium, converted to spheroplasts, lysed,
and fractionated on a 10-step sucrose gradient (18-54% w/w), as
described (14, 15). Fractions were assayed for enzymatic activity of
markers of the vacuole ( -mannosidase), late Golgi (Kex2), and
mitochondria (azide-sensitive F1-ATPase). Western blots
show the distribution of markers for the plasma membrane
(Pma1, 20 µg/lane), endoplasmic reticulum
(Dpm1, 20 µg/lane), prevacuolar compartment
(Pep12, 20 µg/lane), and Nhx1::HA (90 µg/lane). b, yeast lysates from cells described in
a were sequentially fractionated at 13,000 × g and 100,000 × g as described (16). Pellet
fractions (P) were brought to the same volume as the
supernatant (S), and equal volumes (0.03 ml) were subjected
to gel electrophoresis and Western blotting as in a. Vph1 is
a subunit of the vacuolar H+-ATPase. Note that the
distribution of Nhx1 is similar to that of Vph1 and Pep12 but different
from that of Kex2.
|
|
Confocal Microscopy of GFP-tagged Nhx1 Shows Localization to
Unique, Bipolar Perivacuolar Compartments--
To further define the
cellular location of this novel exchanger, we used laser scanning
confocal microscopy to examine the distribution of GFP-tagged Nhx1 in
conjunction with the vacuolar stain FM 4-64 in exponentially growing,
unfixed cells (Fig. 3). Fluorescence from
Nhx1::GFP appears as 1-2 intensely fluorescent spots per
cell, immediately abutting the vacuolar membrane, usually with a
striking bipolar distribution. The number and size of the spots
typically increase in salt-containing media, consistent with the
observed induction of Nhx1 expression levels. The distinct perivacuolar
location of the signal is highly reminiscent of the prevacuolar
compartment (19, 20), at which the biosynthetic, autophagic, and
endosomal pathways converge for sorting of cargo before final delivery
to the vacuole. Indeed, we show that the syntaxin Pep12, which defines
the identity of the prevacuolar compartment (21), colocalizes with
Nhx1::GFP in fixed and permeabilized cells. Importantly, we
were able to show by Western blotting using anti-GFP antibodies that
the distribution of Nhx1::GFP was identical to that of
Nhx1::HA on sucrose density gradients (data not shown). Together with the functionality of the tagged constructs (Fig. 1a) and the modest levels of expression achieved from the
endogenous NHX1 promoter, the observations argue against a
potential mislocalization because of the large GFP tag.

View larger version (93K):
[in this window]
[in a new window]
|
Fig. 3.
Confocal microscopy of GFP-tagged Nhx1.
Confocal images of exponentially growing, unfixed cells of
nhx1 carrying the Nhx1::GFP (2µ) construct
(see "Experimental Procedures") in the absence
(a-d) or presence (e-h) of 300 mM NaCl. Contrast images of living yeast cells are shown in
panels a and e. Vacuoles are stained
red with FM 4-64 (panels b and f).
Nhx1::GFP fluorescence appears as one or two intense
green spots per cell (panels c and g)
that always are directly abutting the vacuolar membrane, often bipolar
in orientation relative to the vacuole (inset, panel
c). The three images are overlaid in panels d and
h. In fixed and permeabilized cells
(I-l), indirect immunofluorescence from
antibodies against Pep12 (panel j, red), a
resident of the prevacuolar compartment, colocalize with
Nhx1::GFP fluorescence (panel k,
green), as seen in the overlay (panel l).
Bar, 10 µm; inset bar, 2 µm.
|
|
The frequent distribution of Nhx1::GFP to opposing ends or
poles of the vacuole is particularly intriguing and was clearly observed in three-dimensional reconstructions of the vacuole from sequential confocal planes (not shown). Such a bipolar pattern recalls
the similar "patched" distribution of vacuolar assembly proteins,
Vam3 and Vam6, on the vacuolar membranes (34, 35) and suggests that
fusion of vacuolar precursors occurs at discrete sites. We note that
this orientation is apparently lost upon fixation of cells.
Nhx1 Does Not Colocalize with Mitochondrial Markers--
The inner
membrane of mitochondria in mammals has been shown to possess two
distinct forms of cation/H+ exchange activity: one
selective for Na+ and the other transporting all alkali
cations (22, 23). Functional studies in isolated yeast mitochondria
indicate an absence of selective Na+/H+
exchange, although a nonselective (K+/H+)
antiporter was found (24). A very recent report (25) raised the
possibility that Nhx1 localizes to mitochondria based on an overlap of
signals from the DNA-binding dye 4',6'-diaminidino-2-phenylindole dihydrochloride and Nhx1::GFP expressed at high levels from
the exogenous MET25 promoter. Evidence was also presented
for low levels (1 nmol/min/mg) of Na+/H+
exchange activity in three of five crude mitochondrial preparations, although contamination by other membranes was not assessed. Here we
show that fluorescence from Nhx1::GFP is distinct from that of two well characterized mitochondrial dyes, DiOC6 and
MitoTracker Red CMXRos (Fig. 4).
Mitochondria appear as typically elongated snake-like forms that have
no particular orientation relative to the vacuole; in contrast,
Nhx1::GFP flurorescence occurs as 1-2 spots/cell that are
always observed to directly abut the vacuolar membrane. Taken together
with the results from subcellular fractionation (Fig. 2a),
we conclusively rule out a mitochondrial localization for this
exchanger.

View larger version (66K):
[in this window]
[in a new window]
|
Fig. 4.
Nhx1 does not colocalize with mitochondrial
markers. Confocal microscopy of exponentially growing wild type
(panels a-d) or
nhx1/Nhx1::GFP-2µ (panels
e-h) cells in the absence of fixation. In panels
a-d, mitochondria stained with DiOC6
(green, panel c) are seen in relation to the
vacuole (FM 4-64; red, panel b) in the overlay
(panel d). Nhx1::GFP fluorescence (panel
g) is distinct from that of mitochondria (MitoTracker Red CMXRos;
red, panel f) as seen in the overlay (panel
h). Note that mitochondria lack the distinct perivacuolar, bipolar
distribution seen in the structures containing Nhx1::GFP.
Bar, 10 µm; inset bar, 5 µm.
|
|
 |
DISCUSSION |
Nhx1 Defines a Novel Cluster of Na+/H+
Exchanger Isoforms--
With the ongoing success of systematic genome
sequencing, genes encoding putative Na+/H+
exchangers have recently been identified in yeasts, worms, bacteria, and humans. They provide a unique opportunity to trace the phylogenetic ancestry and evolution of exchanger isoforms as well as provide clues
to the function of newly identified homologues. In our survey of all
NHE-like sequences residing in data bases, we were able to identify
previously known clusters of sequences corresponding to the plasma
membrane isoforms of Na+/H+ exchangers
(NHE1-4), as well as a previously unreported cluster of more distantly
related prokaryotic sequences. We show here that Nhx1 defines a
completely novel cluster of exchanger sequences derived from such
evolutionary divergent organisms as yeast, nematodes, and humans (Fig.
5a). In addition, pairwise
comparisons between NHE polypeptides using global alignment methods
(26) reveal significantly higher scores for members within this newly
identified cluster: average identity, 34%; global score, 1000 (Box I, Fig. 5b). Comparable scores were observed
among members outside this cluster: average identity, 36%; global
score, 1722 (Box II, Fig. 5b). In contrast,
scores for sequence pairs between the two groups were
uniformly low: average identity, 23%; global score, 373 (Box III, Fig. 5b). It should be noted that to avoid bias,
we chose representative sequences from widely divergent phyla in both
groups and that the overall length of the polypeptides varied in both groups: 540-666 (Box I) and 660-820 (Box
II).

View larger version (34K):
[in this window]
[in a new window]
|
Fig. 5.
Sequence relations and proposed function of
Nhx1-like homologues. a, phylogenetic clusters of NHE
sequences were defined using Clustalw 1.5 and PHYLIP 3.5c.
Representative examples are shown for each cluster, and accession
numbers are included for unnamed isoforms. b, pairwise
alignments between NHE sequences showing the percentage of identity
computed for each pair. In parentheses are the global
alignment scores, which reflect penalties for gaps. Box I
contains the putative intracellular cluster, and Box II
contains representative examples from the other clusters.
NHX1, S. cerevisiae; SPX1, S. pombe Z97208; NHE6, human D87743; CE1 and
CE2, C. elegans Z69646 and Z73898);
NHE1, rat; NHE2, human; CM1, C. maenas. c, patterns of amino acid homology provide the
basis for the definition of unique clusters of NHE sequences. Selected
transmembrane segments are shown, numbered according to Ref. 1.
Asterisks highlight the differences between putative
intracellular isoforms and other NHE sequences. d, model for
salt and water accumulation in vesicles. A specific colocalization of
the transporters depicted has not yet been shown but is likely.
Swelling of the vesicles is proposed to precede fusion of the
prevacuolar compartment with the vacuole.
|
|
All Na+/H+ exchanger sequences share the
highest homology within predicted transmembrane segments of the
N-terminal transporter domain. The conserved regions are presumably
important for common transport functions, whereas the C-terminal
domains are largely divergent, reflecting a diversity in modes of
regulation of different isoforms. In Fig. 5c, we show that
there are consistent differences between members of the different
clusters in sequence homology patterns within transmembrane segments
known to be important for amiloride binding and ion transport. On the
basis of these differences we suggest that members of the Nhx1-like
cluster diverged early from plasma membrane isoforms. Finally, the
length of the C-terminal hydrophilic domain is significantly shorter in
members of the Nhx1-like cluster relative to the plasma membrane
isoforms, resulting in shorter polypeptide lengths overall: 541-666
residues versus 717-832 residues. Thus, although separated
from one another by a billion years or so of evolution, members of the
newly identified Nhx1-like cluster are all recognizably related to each
other. Taken together, these observations suggest a common
intracellular, possibly endosomal location for these novel
homologues.
Functional Implications of the Prevacuolar/Endosomal Localization
of Nhx1--
There is emerging evidence that plasma membrane-derived
endosomes and Golgi-derived transport vesicles converge at a
prevacuolar compartment (PVC) equivalent to late endosomes, where cargo
is sorted prior to final delivery to the vacuole/lysosome. Proteins en route to the vacuole may be visualized in the PVC
transiently, as was observed for the
-factor receptor, Ste3, upon
coordinated internalization from the plasma membrane (27), or by
perturbation of vesicle traffic into or out of this compartment, as in
the "Class E" family of vacuolar trafficking mutants (28). The PVC itself is a discrete, rather than transient, structure that can be
isolated on density gradients from normal yeast (29) and can be shown
to have a perivacuolar distribution by immunofluorescence and electron
microscopy (20, 27). However, the only resident protein of the PVC
described in the literature is the syntaxin homologue, Pep12, which
mediates docking of this compartment with the vacuole (21). In this
context, the selective localization of Nhx1 to the prevacuolar
compartment has considerable functional significance. An intriguing
possibility is that regulation of vesicle volume and pH by endosomal
Na+/H+ exchange may be important for vacuole
biogenesis. Thus, the H+ gradient established by the
vacuolar H+-ATPase would drive Na+ accumulation
via Na+/H+ exchange, and Cl
influx via chloride channels (Fig. 5d). As osmotically
obliged water is dragged in, the vesicle swells and the hydrostatic
pressure generated provides the energy for membrane destabilization and fusion. There is evidence that osmotic swelling precedes exocytosis and
that, conversely, water loss from vesicles accompanies vesicle maturation and remodeling (30-32, 39). We suggest that the yeast chloride channel ScCLC/GEF1 also has a prevacuolar localization based
on the appearance of GFP-tagged ScCLC as 1-3 perivacuolar dots/cell
(33). Thus, colocalization of the Na+/H+
exchanger, Cl
channel, and H+ pump, together
with specialized coat proteins, syntaxins, and other docking factors
may be involved in the assembly of the vacuole/lysosome from the
prevacuolar compartment/endosomes.
Our data do not exclude the possibility that Nhx1 also localizes to
discrete patches on the vacuolar membrane. Recently, two components of
a protein complex required for vacuole biogenesis, Vam3 (34) and Vam6
(35), were shown to have an unusual bipolar patched location on the
vacuole, suggesting that specialized domains of the vacuole may be
involved in vesicle fusion. By analogy, it is known that Golgi-derived
secretory vesicles fuse at specialized regions of the plasma membrane,
resulting in oriented bud growth (36). We note the enrichment of
mammalian NheI at the leading edge and ruffles in fibroblast
cells (37), and of the unrelated Na+/H+
exchanger sod2 of Schizosaccharomyces pombe to the polarized cell tips (38), consistent with a role for
Na+/H+ exchange at these specialized sites. Our
data imply that regulation of vesicle pH and volume by endosomal
Na+/H+ exchange may be important for vesicle
maturation and fusion.
Given the multifunctionality of Na+/H+
exchangers, a variety of other cellular roles for endosomal exchangers
may be envisaged: regulation of endosomal pH via
Na+/H+ exchange can provide a functional link
between the operational diversity among endocytic compartments and the
known variability in their internal pH, intralumenal sequestration of
Na+ may serve to detoxify the cytoplasm or to drive
Ca2+ accumulation via Na+/Ca2+
exchange, and in the case of the renal proximal tubule, exocytic insertion of endosomal Na+/H+ exchangers at the
cell surface may effect rapid increases in H+ secretory
capacity. We have already demonstrated that in yeast, Nhx1 makes an
important contribution to halotolerance (12). The molecular
characterization and functional role of Nhx1-like homologues in other
organisms remains to be determined.
We thank Robert Fuller, Robert Piper,
and Carolyn Slayman for generous gifts of antibody and
acknowledge the experimental contributions of Emily Corse and Patrick
Leibovich. Michael Delannoy and Oliver Kerscher provided helpful advice
for microscopy.