1 Department of Physiology, University of Münster, 48149 Münster, Germany; and 2 Department of Cellular and Molecular Physiology, Yale University, School of Medicine, New Haven, Connecticut 06520
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ABSTRACT |
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The human
H+-K+-ATPase, ATP1AL1, belongs to the subgroup
of nongastric, K+-transporting ATPases. In concert with the
structurally related gastric H+-K+-ATPase, it
plays a major role in K+ reabsorption in various tissues,
including colon and kidney. Physiological and immunocytochemical data
suggest that the functional heteromeric ion pumps are usually found in
the apical plasma membranes of renal epithelial cells. However, the low
expression levels of characteristic nongastric ion pumps makes it
difficult to verify their spatial distribution in vivo. To investigate
the sorting behavior of ATP1AL1, we expressed this pump by stable
transfection in MDCK and LLC-PK1 renal epithelial cell
lines. Stable interaction of ATP1AL1 with either the endogenous
Na+-K+-ATPase -subunit or the gastric
H+-K+-ATPase
-subunit was tested by
confocal immunofluorescence microscopy and surface biotinylation. In
cells transfected with ATP1AL1 alone, the
-subunit accumulated
intracellularly, consistent with its inability to assemble and travel
to the plasma membrane with the endogenous
Na+-K+-ATPase
-subunit. Cotransfection of
ATP1AL1 with the gastric H+-K+-ATPase
-subunit resulted in plasma membrane localization of both pump
subunits. In cotransfected MDCK cells the heteromeric ion pump was
predominantly polarized to the apical plasma membrane. Functional
expression of ATP1AL1 was confirmed by 86Rb+
uptake measurements. In contrast, cotransfected LLC-PK1
cells accumulate ATP1AL1 at the lateral membrane. The distinct
polarization of ATP1AL1 indicates that the
-subunit encodes
sorting information that is differently interpreted by cell
type-specific sorting mechanisms.
proton-potassium-adenosinetriphosphatase; sorting
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INTRODUCTION |
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RENAL POTASSIUM SECRETION and reabsorption is mediated by various ion transport mechanisms and plays a major role in maintaining the systemic K+ equilibrium. To carry out these processes, ion-transporting proteins must be differentially expressed along the segments of the nephron, and their subcellular distributions in either the apical or basolateral membranes of renal epithelial cells must be rigorously controlled. Active secretion of K+ into the lumen of the renal collecting tubule requires the participation of the basolateral Na+-K+-ATPase and apically polarized K+ transporting ion channels. Previous studies revealed that under conditions of low dietary intake, luminal potassium secretion is reduced and is instead superseded by active K+ reabsorption (46). A large body of evidence indicates that active K+ reabsorption in the renal collecting tubule is mainly attributable to the activity of several H+-K+-ATPases. However, the molecular mechanisms that regulate these pump activity, expression and subcellular distributions have yet to be identified.
The H+-K+-ATPases belong to the family of
P-type ion-transporting ATPases and are structurally related to the
Na+-K+-ATPases (32). These
heterodimeric proteins are composed of a catalytic - and an
associated
-subunit (26). The polytopic
-subunit
spans the membrane 10 times, is not glycosylated, and includes the
enzyme's sites for ATP binding and ion translocation. Assembly with
the single membrane spanning and highly glycosylated
-subunit is a
prerequisite for posttranslational processing and transport of the
newly synthesized ion pump from the endoplasmic reticulum (ER) to the
plasma membrane (16). Although members of the
H+-K+-ATPase family show strong structural
homology to one another, their unique functional characteristics
clearly differentiate two subgroups. One subgroup is defined by the
gastric H+-K+-ATPase, which is also responsible
for acid secretion in the parietal cells of the stomach. The other
class is composed of the nongastric H+-K+-ATPases. Both types are expressed along
different nephron segments and show characteristic pharmacological
sensitivities (13, 46). Recent studies
suggest that nongastric H+-K+-ATPases can
transport protons in exchange for potassium but act primarily as
Na+-K+-ATPases under physiological conditions
(10, 21, 22).
Interactions with different -subunits are known to modify the
biochemical and cell biological properties of P-type ATPases (7, 17). It was recently shown that the rat
colonic nongastric H+-K+-ATPase can assemble
with the
-subunit of the Na+-K+-ATPase, with
the
-subunit of the gastric H+-K+-ATPase and
with a newly identified H+-K+
-subunit from
colon (8, 9, 28). The human
nongastric H+-K+-ATPase, ATP1AL1, shows
different
-subunit affinities (21, 35).
The ATP1AL1
-subunit will interact with the gastric
H+-K+-ATPase
-subunit but not with the
Na+-K+- ATPase
-subunit isoforms when
expressed by transfection in HEK-293 cells. Gastric
H+-K+-ATPase
-subunit expression has been
detected in epithelial cells of the renal collecting tubule
(5).
Both functional and structural localization studies have detected the
gastric H+-K+-ATPase in the luminal membranes
of stimulated gastric parietal cells (25) and renal tubule
epithelial cells (46). In gastric parietal cells under
resting conditions, the H+-K+-ATPase is stored
in the membranes of tubulovesicular elements (TVEs). After stimulation
of the cells, the TVEs fuse with the apical membrane and expose the
H+-K+-ATPase to the gastric lumen. Ion pump
inactivation and restoration of the TVEs is regulated by endocytosis
(11). The requisite endocytosis signal is localized on the
short N-terminus of the gastric
H+-K+-ATPase -subunit. Interestingly, it was
recently shown that similar mechanisms also influence the
H+-K+-ATPase-mediated K+
reabsorption in renal cells (46). Although the majority of functional and pharmacological studies predict an apical distribution for the nongastric H+-K+-ATPases, their actual
cellular localizations and modes of regulation are not clear. Except
for the rat colonic nongastric H+-K+-ATPase
subtype, which was immunohistochemically localized in the apical
membrane of rat colon and renal principal cells (PC) (29, 38), none of the various nongastric
H+-K+-ATPase subtypes has been structurally
localized in renal epithelial cells. Moreover, it is conceivable that
nongastric H+-K+-ATPases can also be
differentially localized in epithelial cells.
Sorting information that directs the gastric
H+-K+-ATPase to the apical membrane has been
identified in the fourth transmembrane domain of the -subunit and is
restricted to a sequence of 8 amino acids (14). In the
nongastric H+-K+-ATPases, the sequence in this
region is essentially identical to that present in the
-subunit of
the basolateral sodium pump. Thus either the sorting behaviors or the
sorting signals manifest by the nongastric
H+-K+-ATPases must differ substantially from
those employed by their gastric counterpart. To elucidate the sorting
properties of nongastric H+-K+-ATPases,
we investigated the spatial distribution of the human nongastric
H+-K+-ATPase, ATP1AL1, expressed by
transfection in polarized renal epithelial cells.
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EXPERIMENTAL PROCEDURES |
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Tissue culture. MDCK cells were maintained in minimal essential medium supplemented with Earle's salts (EMEM) and LLC-PK1 cells were maintained in Dulbecco's modified Eagle's medium (DMEM). Both media contained 10% fetal bovine serum (Sigma), 2 mM L-glutamine, and 50 U/ml penicillin and streptomycin (Life Technologies, Grand Island, NY). Stably transfected cells were grown in the presence of 0.9 g/l Geneticin (Life Technologies). Cells were cultured under standard conditions (37°C, 5% CO2) and passaged twice a week with 0.05% trypsin and 0.5 mM EDTA (Life Technologies).
Stable transfection.
ATP1AL1 -subunit cDNA and H+-K+-ATPase
-subunit cDNA were subcloned in the mammalian expression vector
pJB20 and pCB6, respectively, as previously described
(21). Single transfections of MDCK and LLC-PK1
cells with ATP1AL1
-subunit and cotransfections with the
H+-K+-ATPase
-subunit were performed
with the PerFect transfection kit from Invitrogen (San Diego, CA) and
DOTAP liposomal transfection reagent (Roche, Mannheim, Germany)
according to the manufacturer protocols. Both cell types do not express
ATP1AL1 or the gastric H+-K+-ATPase
-subunit
endogenously. After Geneticin selection for 3 wk, ATP1AL1
-subunit
and H+-K+-ATPase
-subunit cotransfected
cells were cultured in ouabain (0.5-1 µM) containing
medium for 3 days. Only cells functionally expressing the
H+-K+-ATPase, ATP1AL1, survived this additional
selection. Afterwards, transfected cells were cloned by single cell dilution.
Cell surface biotinylation.
Transfected cells were grown on 24-mm polycarbonate Transwell filter
inserts (0.4 µm pore, Costar) for 10 days. Medium was replaced daily. Before biotinylation, expression of the transfected cDNAs was enhanced by incubation for 12 h with media containing 10 mM sodium butyrate. Cell surface biotinylation was performed as
previously described (19) using NHS-SS-Biotin (Pierce,
Rockford, IL). Apical and basolateral biotinylation was performed for
40 min, at pH 9.0, and biotinylated proteins were separated
with streptavidin-agarose beads (Pierce). Protein concentration was determined (12), and similar amounts (0.2-1 µg) of
biotinylated proteins were analyzed by SDS-polyacrylamide gel
electrophoresis and Western blotting using a rabbit polyclonal,
affinity-purified anti-ATP1AL1 (XG) antibody (1:1,000;
Ref. 21), a monoclonal mouse anti H+-K+-ATPase
-subunit antibody (1:250; kindly provided by J. Forte and D. Chow,
University of California at Berkeley, Berkeley CA), and a mouse
monoclonal anti Na+-K+-ATPase
-subunit
antibody (Upstate Biotechnology). Detection was performed using either
goat anti-mouse or goat anti-rabbit antibodies (1:1,000) conjugated to
horseradish peroxidase (Sigma, St. Louis, MO) and developed by the
enhanced chemiluminescence technique (ECL; Amersham, Arlington Heights, IL).
Immunofluorescence.
Transfected MDCK and LLC-PK1 cells were seeded at low
densities (1 × 105 cells/24-mm filter) on
polycarbonate Transwell filter inserts (Corning Costar) and grown for
at least 7 days. Medium was changed daily, and 12 h before
fixation supplemented with 10 mM sodium butyrate. Cells were washed
with PBS+ (150 mM NaCl, 10 mM
NaH2PO4, 0.1 mM CaCl2, and 1 mM
MgCl2; pH 7.4) and fixed with ice-cold methanol for 7 min.
Blocking (30 min) and antibody dilution was performed with GSDB [16%
goat serum (Sigma), 0.3% Triton X-100, 0.1% bovine serum albumin
(Sigma), 0.45 M NaCl, and 20 mM NaH2PO4, pH 7.4 (6)]. Diluted primary antibodies [XG, 1:200;
H+-K+-ATPase
-subunit, 1:50;
anti-Na+-K+-ATPase
-subunit, 1:100; PDI
(Dianova)] were incubated for 1 h at room temperature.
After several washing steps using PBS+, secondary
anti-mouse FITC- or Rhodamine Red-labeled and anti-rabbit TRITC- or
fluorescein-labeled antibodies (1:100) were incubated with the samples
for 1-6 h at room temperature. After additional washing
steps, filters were mounted on coverslips with Vectashield (Vector
Laboratories, Burlingame, CA). Confocal images were generated on a
Zeiss model LSM 410 and on an Olympus Fluoview laser-scanning microscope. Images are the product of eightfold line averaging, and
xz cross sections were generated with a 0.2-µm motor step.
86Rb+ uptake measurements.
86Rb+ uptake measurements with
H+-K+-ATPase - and gastric
H+-K+-ATPase
-subunit transfected and
untransfected MDCK wild-type cells were performed as previously
described for transfected HEK-293 cells (21,
22). 86Rb+ uptake was determined
in the presence of an extracellular Na+ concentration of 10 mM and in the presence of ouabain concentrations ranging from 0.1 nM
and 1 mM. Data are reported as means ± SE.
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RESULTS |
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The subcellular steady-state localization of the human
ATP1AL1-ATPase was assessed in stably transfected MDCK and transfected LLC-PK1 wild-type cells. We examined the assembly and
plasma membrane expression of the transfected ATP1AL1 -subunit with
the endogenous Na+-K+-ATPase
-subunit by
confocal immunofluorescence microscopy and surface biotinylation.
Previous studies in our lab showed that the strong endogenous
expression of Na+-K+-ATPase in MDCK and
LLC-PK1 cells provides sufficient pools of sodium pump
-subunit for assembly and targeting of transfected
-subunits
(14, 18). Therefore, the epithelial cells
were stably transfected only with the ATP1AL1
-subunit, which is not endogenously expressed. To enhance the expression of the transfected
-subunit, clonal filter-grown cell lines were treated with sodium butyrate (10 mM) for 12 h. Colocalization of both proteins was monitored using an affinity-purified polyclonal ATP1AL1 antibody, a
monoclonal Na+-K+- ATPase
-subunit
antibody, and TRITC- and FITC-labeled secondary antibodies,
respectively. Our confocal immunofluorescence results revealed no
plasma membrane colocalization for these two subunits.
The en face (xy) view in Fig.
1A revealed predominantly
perinuclear ATP1AL1 staining (arrow in Fig. 1A), consistent
with ATP1AL1 accumulation in the ER. Cross sections in the
xz direction confirmed the intracellular ATP1AL1
distribution and the absence of colocalizations with the laterally
concentrated Na+- K+-ATPase -subunit (Fig.
1B). The intracellular accumulation of ATP1AL1 was confirmed
by colocalization analysis with an antibody against the protein
disulfide isomerase (PDI), a marker of the ER. The transfected ion pump
(Fig. 1C; fluorescein-labeled) and the ER marker (Fig.
1D; Rhodamine Red-labeled) were localized in the perinuclear
region. Merging a representative detail of both stainings
(yellow-orange color) revealed partial colocalization in the ER and
ER-derived vesicle structures (Fig. 1E). The same results
were obtained by analyzing ATP1AL1 transfected LLC-PK1 cells (data not shown). Because the strong intracellular ATP1AL1
-subunit accumulation could possibly mask a small degree of surface colocalization with the Na+-K+-ATPase
-subunit, we performed surface biotinylation experiments with
ATP1AL1 transfected cells. We used only transfected MDCK cells, because
LLC-PK1 cells have been shown to be unsuitable for
quantitative surface biotinylation analysis (19). Similar amounts of apically or basolaterally biotinylated proteins (Fig. 2; lanes 1 and 2,
respectively) were separated on polyacrylamide gels, and the
corresponding Western blots were probed with ATP1AL1 and
Na+-K+-ATPase
-subunit-specific antibodies.
Although the ATP1AL1
-subunit is highly enriched in intracellular
compartments (as seen in Fig. 1), the
-subunit could not be found in
either the apical or in the basolateral biotinylated membrane protein
fraction. Only the Na+-K+-ATPase
-subunit
(~50-60 kDa) could be detected in the basolateral membrane (Fig.
2, lane 2), which reflects its colocalization with the
endogenous Na+-K+-ATPase
-subunit. Therefore
ATP1AL1 does not reach the plasma membrane together with the
-subunit of the related Na+-K+- ATPase in
MDCK and LLC-PK1 cells when expressed by itself.
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Based on previous studies, which showed interaction between ATP1AL1 and
the -subunit of the gastric H+-K+-ATPase
(21, 22) in unpolarized HEK-293 cells,
assembly and targeting of both subunits was tested in cotransfected
MDCK cells. Double-transfected cells were cloned by functional
selection. In contrast to untransfected or to monotransfected cells, in
cells expressing ATP1AL1 and the gastric
H+-K+-ATPase
-subunit the activity of the
heteromeric ion pump enables the cells to grow in media containing up
to 1 µM ouabain (21). Although the proliferation rate
seems to be reduced during ouabain selection, the transfected ATPase is
able to compensate for the inhibited endogenous
Na+-K+-ATPase (Ki for
ouabain ~10
7 M).
To further document the presence of pump activity in cells
expressing ATP1AL1 in association with the gastric
H+-K+-ATPase -subunit, we measured the
ouabain sensitivity of 86Rb+ uptake in
cotransfected MDCK cells. 86Rb+ uptake
measurements (Fig. 3) were performed as
previously described (21). In these studies it was shown
that single transfection of the gastric
H+-K+-ATPase
-subunit in HEK-293 cells does
not alter the ouabain sensitivity and the 86Rb+
uptake activity of the endogenously expressed
Na+-K+- ATPase. By extrapolating the data
from HEK-293 cells to the MDCK system, we compared
86Rb+ uptake activity of three different
ATP1AL1/gastric H+-K+- ATPase
-subunit
cotransfected MDCK cell lines with untransfected MDCK wild-type cells.
Cotransfected MDCK cells are characterized by threefold higher uptake
in the presence of a ouabain concentration (1 nM) that does not
influence the endogenous influx. Ouabain concentrations up to 1 µM
inhibit the endogenous Na+-K+-ATPase activity
in wild-type cells but have only a marginal effect on the activity of
the ATP1AL1-ATPase, which is fully blocked only at ouabain
concentrations exceeding 1 mM. The viability of ATP1AL1/gastric
H+-K+-ATPase
-subunit transfected MDCK cells
in the presence of micromolar ouabain concentrations and the ouabain
sensitivity profile are in line with previous findings from transfected
HEK-293 cells (21, 22). These data confirm
functional ATP1AL1- ATPase expression in the MDCK renal epithelial
cell line.
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Confocal immunofluorescence analysis of the cotransfected MDCK cells
reveals that the ATP1AL1 -subunit accumulates exclusively at the
apical plasma membrane, which is shown in the xz cross section of Fig. 4A. There is
no intracellular ATP1AL1
-subunit accumulation as found in single
transfected cells (xz cross section in Fig. 1B).
In parallel, the cotransfected gastric
H+-K+-ATPase
-subunit was predominantly
apical but also laterally localized (Fig. 4B and
xz cross section). The subcellular localization of both
subunits was independent of their expression level, as found by
analyzing more than 10 independent, stable cotransfected and clonal
MDCK cell lines. Merging both staining patterns revealed clear apical
colocalization of ATP1AL1 and the H+-K+-ATPase
-subunit (Fig. 5, top). The
additional lateral
-subunit staining (Fig. 5, top; FITC)
is due to the high
-subunit expression level and reflects the
distribution of unassembled H+-K+-ATPase
-subunit protein. This result is in line with previous findings that
documented gastric H+-K+-ATPase
-subunit
targeting to the basolateral membrane of singly transfected MDCK cells
(37). In contrast, in the same ATP1AL1/gastric H+-K+- ATPase
-subunit doubly transfected
cells, the endogenous Na+-K+-ATPase
-subunit
(FITC) was entirely basolateral and showed no overlapping distribution
with the apically sorted ATP1AL1 protein (TRITC), confirming the lack
of plasma membrane colocalization of these two polypeptides (Fig. 5,
bottom). It is interesting to note that, despite the fact
that we generated clonal cell lines, the expression level of ATP1AL1
and the gastric H+-K+-ATPase
-subunit varied
considerably among neighboring cells (xy section in Fig. 4,
A and B). Similar results have been obtained in
transfections of MDCK cells with other transport proteins (T. R. Muth, personal communication). We assume that the heterogenous expression levels are due to differences in the transcription and/or
translation efficiencies of both proteins. Nevertheless, the
steady-state polarization of the transfected
- and
-subunits were
shown to be independent of their protein expression level as found by
analyzing more than 10 different clonal cell lines.
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Further confirmation of our immunofluorescence data was obtained
through surface biotinylation of ATP1AL1 -subunit and gastric H+-K+-ATPase
-subunit transfected MDCK cells
(Fig. 6). The ATP1AL1
-subunit (~110
kDa) was present in the apical membrane (Fig. 6A), whereas
the Na+-K+-ATPase
-subunit was not found in
the apically biotinylated plasma membrane protein fraction (Fig.
6B). This results were reproducible with four different
cotransfected MDCK cell lines. Western blots incorporating serial
dilutions of biotinylated proteins for purposes of quantitation
indicated that the steady-state distribution of the ATP1AL1 protein was
~80% apical and 20% basolateral (not shown). This ratio is in line
with the polarity ratios determined by other groups for apically
polarized marker proteins in MDCK cells, such as the influenza virus
hemagglutinin (31). Contrary to the predominantly apical
ATP1AL1
-subunit expression, the gastric
H+-K+-ATPase
-subunit seems to be equally
distributed in both plasma membrane domains (Fig. 6A). Taken
together, the biotinylation and immunofluorescence data clearly show
that ATP1AL1 is predominantly polarized to the apical plasma membrane
of transfected MDCK cells.
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Surprisingly, cotransfected LLC-PK1 cells showed a
completely different behavior. Both the ATP1AL1 subunit (Fig.
7, A and B) and the
gastric H+-K+-ATPase -subunit (Fig.
7C) accumulated in the lateral plasma membrane. Although
technical limitations precluded the acquisition of quantitative
biotinylation data, the analysis of six different transfected
LLC-PK1 cell clones strongly supports this result. Previous
studies on LLC-PK1 cells in our lab showed that the gastric H+-K+-ATPase
-subunit is differentially
polarized to the apical membranes of LLC-PK1 cells and to
the basolateral membranes of monotransfected MDCK cells
(37). Based on these results, the basolateral localization of the gastric H+-K+-ATPase
-subunit in
ATP1AL1-expressing LLC-PK1 cells and its apical
localization in MDCK cells provides strong evidence for sorting signals
residing on the ATP1AL1
-subunit playing a dominant role in the
sorting of the complex.
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DISCUSSION |
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The human P-type ATPase, ATP1AL1, belongs to the subgroup of nongastric H+-K+-ATPases and is involved in K+ reabsorption in various tissues, including colon and kidney. Functional characterizations reveal that ATP1AL1 and the related rat colonic nongastric H+-K+-ATPase mediate K+ reabsorption in exchange for either sodium ions or protons. In addition to their functional properties, the establishment and regulation of the spatial distributions of nongastric H+-K+-ATPases in polarized renal epithelial cells must play a critical role in determining their physiological activity. Little is known of the cell biologic properties of this pump subfamily. It is known that the nongastric H+-K+-ATPases undergo upregulation after K+ depletion (for review see Ref. 27) and that their functional plasma membrane expression is modulated by endocytosis (46). However, the molecular signals and pathways responsible for this behavior in renal epithelial cells remain to be determined.
Although five functionally distinguishable K+-dependent
K+-ATPase activities are detected in renal epithelial cells
(for review see Refs. 27 and 46), the cellular distributions of only
two, the gastric H+-K+-ATPase and the
nongastric colonic H+-K+-ATPase, have been
investigated. The gastric subtype was identified in the apical
membranes of -type intercalated cells (IC), in PC, and in the
basolateral membranes of rat
-type ICs (2, 46), whereas the nongastric colonic
-subunit protein
was apically localized in renal PCs and in the colonic epithelium
(29, 38). Studies of the distributions of
H+-K+-ATPase isoforms have been complicated by
the possible existence of multiple species-specific subtypes as well as
by low expression levels of the nongastric ATPases under normal
circumstances. Previous studies showed that the mRNA of the human
ATP1AL1 ion pump, for example, was only detectable in sensitive RT-PCR
experiments (20, 34). Furthermore, although
this pump may be upregulated in response to K+ deprivation,
this effect cannot be practically exploited to examine its distribution
in its native human renal tissue. To examine the subcellular targeting
and functional activity of ATP1AL1, we expressed this ATPase by stable
transfection in MDCK and LLC-PK1 cells. These cell lines
manifest characteristics of cells from the distal nephron and the
proximal nephron segments, respectively, and thus are suitable
expression systems for H+-K+-ATPase characterization.
We have previously shown that the strong expression of the endogenous
Na+-K+-ATPase in these cells provides
sufficient Na+-K+-ATPase -subunit to support
the assembly, proper folding and targeting of exogenous sodium pump and
sodium pump chimeras expressed by transfection (14,
18). Nevertheless, our immunofluorescence and
biotinylation data demonstrate that in both transfected MDCK and
LLC-PK1 cells, the ATP1AL1
-subunit expressed by itself
does not reach the plasma membrane. This result corresponds to previous findings that documented poor assembly between ATP1AL1 and the Na+-K+-ATPase
-subunit in in vitro
expression studies (21, 35). The present data
further demonstrate, therefore, that the Na+ pump
1-subunit is not an effective partner for ATP1AL1. Instead, ATP1AL1
once again exhibits a strong preference for functional assembly with
the gastric H+-K+-ATPase
-subunit
(21). In this context it is interesting to note that the
related rat colonic H+-K+-ATPase protein and
its guinea pig ortholog appear to associate with the Na+
pump
1-subunit and the gastric H+-K+-ATPase
-subunit, both in vivo and in Xenopus oocyte expression studies (1, 9, 28). Previous
elegant studies identified in an extracellular loop of the
Na+ pump, of the gastric
H+-K+-ATPase, and in the colonic
H+-K+-ATPase a stretch of 26 amino acids that
is highly conserved and plays a major role in
-subunit specificity
(30, 33). Amino acid residues that were shown
to prefer assembly between the colonic H+-K+-ATPase and the gastric
H+-K+-ATPase
-subunit (46) are
identical in the human ATP1AL1
-subunit. Whether the shown
-subunit specificity is a property unique among the
nongastric H+-K+- ATPases to the ATP1AL1
subtype or whether it is due to species-specific preferences remains to
be determined.
We find that ATP1AL1 is differentially sorted to opposite membrane
domains when expressed in transfected MDCK or LLC-PK1
cells. The apical polarization of ATP1AL1 in MDCK cells corresponds to predictions based on physiological and pharmacological studies, which
reveal the presence of functionally similar ATPase activities in the
luminal membranes of outer medulla and cortical collecting duct cells
after K+ deprivation (4). The basolateral
distribution observed in LLC-PK1 cells lacks similar
functional correlations. It should be noted, however, that at least the
related gastric H+-K+-ATPase -subunit
protein has also been detected basolaterally in situ (2).
Thus it is possible that these pumps are differentially sorted by the
multiple epithelial cell types that line the nephron.
The distinct ATP1AL1 localization patterns raise the question as to
which molecular signals are responsible for the disparate steady-state
distributions of this protein in MDCK and LLC-PK1 cells.
Since the gastric H+-K+-ATPase -subunit
accumulates basolaterally in monotransfected MDCK cells
(37) but is colocalized to the apical membrane after cotransfection with ATP1AL1 and vice versa in LLC-PK1
cells, the
-subunit cannot contain the dominant sorting signals
responsible for the distribution of the holoenzyme complex. It has been
shown that N-linked glycosylation can play a functional role in apical targeting of transmembrane proteins (24, 46).
It is conceivable, therefore, that cell-type-specific glycosylation of
the gastric H+-K+-ATPase
-subunit could
influence the cellular distribution of the complex in different
epithelial cell types. To test this possibility, we blocked N-linked
glycosylation in transfected MDCK cells using tunicamycin (20 µM for
12 h). Unglycosylated
-subunit and ATP1AL1 were still
strictly polarized to the apical membrane, excluding a functional role
for N-linked glycosylation in apical sorting of ATP1AL1 (data not
shown). Furthermore, association of ATP1AL1 with apically sorted,
detergent-insoluble glycosphingolipid-rich membrane domains (for review
see Ref. 3) appears not to occur in either cell type based on detergent
solubilization and density gradient centrifugation experiments (data
not shown).
Based on these observations, we suggest that the ATP1AL1 -subunit
embodies plasma membrane sorting information that is differentially recognized by cell type-specific mechanisms. Similar conclusions have
also been drawn in studies of the gastric
H+-K+- ATPase
-subunit (37)
and the human dopamine transporter expressed in MDCK and
LLC-PK1 cells (23). Very recently it was shown
that LLC-PK1 cells lack the epithelial cell-specific
clathrin adaptor subunit µ1B, which can recognize basolateral sorting
signals (15). In LLC-PK1 cells, the absence of
µ1B leads to apical sorting of some basolateral membrane proteins. In
context with the basolateral ATP1AL1
-subunit/gastric
H+-K+-ATPase
-subunit localization
in LLC-PK1 cells and the apical distribution of
H+-K+-ATPase
-subunit in single transfected
LLC-PK1 cells (37), µ1B cannot be
involved in the basolateral steady-state localization at least of the
functional holoenzyme complex.
Comparison of the sequences of the apical gastric
H+-K+-ATPase -subunit, the basolateral
Na+-K+- ATPase
-subunit, and ATP1AL1 may
shed some light on the molecular nature of the sorting information. A
signal sufficient to ensure the apical sorting of the gastric
H+-K+-ATPase resides within the fourth
transmembrane domain (TM4) of this pump's
-subunit
(14). The sequence of the gastric
H+-K+-ATPase and
Na+-K+-ATPase
-subunit TM4s differ by only 8 amino acids. The residues associated with apical pump sorting are not
conserved in ATP1AL1 and, instead, are highly homologous to the
corresponding sequence of the basolaterally sorted
Na+-K+-ATPase. Presumably, the
Na+-K+-ATPase-like TM4 sequence of ATP1AL1 is
recognized by cell type-specific sorting mechanisms leading to this
protein's basolateral delivery in LLC-PK1 cells. According
to this model, additional or distinct information must be responsible
for this protein's apical distribution in MDCK cells. The preparation
and expression of chimeric pump constructs will be necessary to test
this hypothesis.
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ACKNOWLEDGEMENTS |
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We thank the members of the entire Caplan and Oberleithner laboratories for helpful discussions for the exceptional working atmosphere. Our special thanks go to L. Dunbar, T. R. Muth, and M. Mense. We appreciate the technical assistance of Vanathy Rajendran and Helga Bertram.
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FOOTNOTES |
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These studies were supported by the National Institutes of Health Grants DK-17433 and GM-42136. J. Reinhardt was supported by a postdoctoral fellowship of the Deutsche Forschungsgemeinschaft (Re1284).
Address for reprint requests and other correspondence: M. Caplan, Dept. of Cellular and Molecular Physiology, Yale Univ., School of Medicine, New Haven, Connecticut 06520 (E-mail: michael.caplan{at}yale.edu).
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. §1734 solely to indicate this fact.
Received 22 October 1999; accepted in final form 4 May 2000.
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REFERENCES |
---|
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---|
1.
Asano, S,
Hoshina S,
Nakaie Y,
Watanabe T,
Sato M,
Suzuki Y,
and
Takeguchi N.
Functional expression of putative H+-K+-ATPase from guinea pig distal colon.
Am J Physiol Cell Physiol
275:
C669-C674,
1998[Abstract].
2.
Bastani, B.
Colocalization of H+-ATPase and H+,K+-ATPase immunoreactivity in the rat kidney.
J Am Soc Nephrol
5:
1476-1482,
1995[Abstract].
3.
Brown, DA,
and
London E.
Functions of lipid rafts in biological membranes.
Annu Rev Cell Dev Biol
14:
111-136,
1998[ISI][Medline].
4.
Buffin-Meyer, B,
Younes-Ibrahim M,
Barlet-Bas C,
Cheval L,
Marsy S,
and
Doucet A.
K+ depletion modifies the properties of Sch-28080-sensitive K+-ATPase in rat collecting duct.
Am J Physiol Renal Physiol
272:
F124-F131,
1997
5.
Campbell-Thompson, ML,
Verlander JW,
Curran KA,
Campbell WG,
Cain BD,
Wingo CS,
and
McGuigan JE.
In situ hybridization of H-K-ATPase beta-subunit mRNA in rat and rabbit kidney.
Am J Physiol Renal Fluid Electrolyte Physiol
269:
F345-F354,
1995
6.
Cameron, PL,
Sudhof TC,
Jahn R,
and
De Camilli P.
Colocalization of synaptophysin with transferrin receptors: implications for synaptic vesicle biogenesis.
J Cell Biol
115:
151-164,
1991[Abstract].
7.
Caplan, MJ.
Ion pumps in epithelial cells: sorting, stabilization, and polarity.
Am J Physiol Gastrointest Liver Physiol
272:
G1304-G1313,
1997
8.
Codina, J,
Kone BC,
Delmas-Mata JT,
and
DuBose TD, Jr.
Functional expression of the colonic H+,K+-ATPase -subunit.
J Biol Chem
271:
29759-29763,
1996
9.
Codina, J,
Delmas-Mata JT,
and
DuBose TD, Jr.
The alpha-subunit of the colonic H+,K+-ATPase assembles with beta1- Na+,K+-ATPase in kidney and distal colon.
J Biol Chem
273:
7894-7899,
1998
10.
Cougnon, M,
Bouyer P,
Planelles G,
and
Jaisser F.
Does the colonic H+,K+-ATPase also act as an Na+,K+-ATPase?
Proc Natl Acad Sci USA
95:
6516-6520,
1998
11.
Courtois-Coutry, N,
Roush D,
Rajendran V,
McCarthy JB,
Geibel J,
Kashgarian M,
and
Caplan MJ.
A tyrosine-based signal targets H+/K+-ATPase to a regulated compartment and is required for the cessation of gastric acid secretion.
Cell
90:
501-510,
1997[ISI][Medline].
12.
Dieckmann-Schuppert, A,
and
Schnittler HJ.
A simple assay for quantification of protein in tissue sections, cell cultures, and cell homogenates, and of protein immobilized on solid surfaces.
Cell Tissue Res
288:
119-126,
1997[ISI][Medline].
13.
Doucet, A.
H+, K+-ATPase in the kidney: localization and function in the nephron.
Exp Nephrol
5:
271-276,
1997[ISI][Medline].
14.
Dunbar LA, Roush DL, Courtois-Coutry N, Muth TR, Gottardi CJ, Rajendran
V, Geibel J, Kashgarian M, and Caplan MJ. Sorting of ion pumps in
polarized epithelial cells. Ann NY Acad Sci 834:
514-23, 514-523, 1997.
15.
Fölsch, H,
Ohno H,
Bonifacino JS,
and
Mellman I.
A novel clathrin adaptor complex mediates basolateral targeting in polarized epithelial cells.
Cell
99:
189-198,
1999[ISI][Medline].
16.
Geering, K.
Posttranslational modifications and intracellular transport of sodium pumps: importance of subunit assembly.
FEBS Lett
285:
189-193,
1991[ISI][Medline].
17.
Geering, K,
Theulaz I,
Verrey F,
Hauptle MT,
and
Rossier BC.
A role for the beta-subunit in the expression of functional Na+-K+-ATPase in Xenopus oocytes.
Am J Physiol Cell Physiol
257:
C851-C858,
1989
18.
Gottardi, CJ,
and
Caplan MJ.
An ion-transporting ATPase encodes multiple apical localization signals.
J Cell Biol
121:
283-293,
1993[Abstract].
19.
Gottardi, CJ,
Dunbar LA,
and
Caplan MJ.
Biotinylation and assessment of membrane polarity: caveats and methodological concerns.
Am J Physiol Renal Fluid Electrolyte Physiol
268:
F285-F295,
1995
20.
Grishin, AV,
Sverdlov VE,
Kostina MB,
and
Modyanov NN.
Cloning and characterization of the entire cDNA encoded by ATP1AL1, a member of the human Na+,K+/H+,K+-ATPase gene family.
FEBS Lett
349:
144-150,
1994[ISI][Medline].
21.
Grishin, AV,
Bevensee MO,
Modyanov NN,
Rajendran V,
Boron WF,
and
Caplan MJ.
Functional expression of the cDNA encoded by the human ATP1AL1 gene.
Am J Physiol Renal Fluid Electrolyte Physiol
271:
F539-F551,
1996
22.
Grishin, AV,
and
Caplan MJ.
ATP1AL1, a member of the non-gastric H+,K+-ATPase family, functions as a sodium pump.
J Biol Chem
273:
27772-27778,
1998
23.
Gu, HH,
Ahn J,
Caplan MJ,
Blakely RD,
Levey AI,
and
Rudnick G.
Cell-specific sorting of biogenic amine transporters expressed in epithelial cells.
J Biol Chem
271:
18100-18106,
1996
24.
Gut, A,
Kappeler F,
Hyka N,
Balda MS,
Hauri HP,
and
Matter K.
Carbohydrate-mediated Golgi to cell surface transport and apical targeting of membrane proteins.
EMBO J
17:7:
1919-1929,
1998.
25.
Hersey, SJ,
and
Sachs G.
Gastric acid secretion.
Physiol Rev
75:
155-189,
1995
26.
Horisberger, JD.
The Na-K-ATPase: Structure-Function Relationship. Austin, TX: Landes, 1994.
27.
Jaisser, F,
and
Beggah AT.
The nongastric H+-K+-ATPases: molecular and functional properties.
Am J Physiol Renal Physiol
276:
F812-F824,
1999
28.
Kraut, JA,
Hiura J,
Shin JM,
Smolka A,
Sachs G,
and
Scott D.
The Na+-K+-ATPase beta 1 subunit is associated with the HK alpha 2 protein in the rat kidney.
Kidney Int
53:
958-962,
1998[ISI][Medline].
29.
Lee, J,
Rajendran VM,
Mann AS,
Kashgarian M,
and
Binder HJ.
Functional expression and segmental localization of rat colonic K+-adenosine triphosphatase.
J Clin Invest
96:
2002-2008,
1995[ISI][Medline].
30.
Lemas, MV,
Hamrick M,
Takeyasu K,
and
Fambrough DM.
26 Amino acids of an extracellular domain of the Na,K-ATPase alpha-subunit are sufficient for assembly with the Na,K-ATPase beta-subunit.
J Biol Chem
269:
8255-8259,
1994
31.
Lin, S,
Naim HY,
Rodriguez AC,
and
Roth MG.
Mutations in the middle of the transmembrane domain reverse the polarity of transport of the influenza virus hemagglutinin in MDCK epithelial cells.
J Cell Biol
142:
51-57,
1998
32.
Lingrel, JB,
Orlowski J,
Shull MM,
and
Price EM.
Molecular genetics of Na+,K+-ATPase.
Prog Nucleic Acid Res Mol Biol
38:
37-89,
1990[ISI][Medline].
33.
Melle-Milovanovic, D,
Milovanovic M,
Nagpal S,
Sachs G,
and
Shin JM.
Regions of association between the and the
subunit of the gastric H,K-ATPase.
J Biol Chem
273:
11075-11081,
1998
34.
Modyanov, NN,
Petrukhin KE,
Sverdlov VE,
Grishin AV,
Orlova MY,
Kostina MB,
Makarevich OI,
Broude NE,
Monastyrskaya GS,
and
Sverdlov ED.
The family of human Na+,K+-ATPase genes. ATP1AL1 gene is transcriptionally competent and probably encodes the related ion transport ATPase.
FEBS Lett
278:
91-94,
1991[ISI][Medline].
35.
Modyanov, NN,
Mathews PM,
Grishin AV,
Beguin P,
Beggah AT,
Rossier BC,
Horisberger JD,
and
Geering K.
Human ATP1AL1 gene encodes a ouabain-sensitive H-K-ATPase.
Am J Physiol Cell Physiol
269:
C992-C997,
1995
36.
Pathak, RK,
Yokode M,
Hammer RE,
Hofmann SL,
Brown MS,
Goldstein JL,
and
Anderson RG.
Tissue-specific sorting of the human LDL receptor in polarized epithelia of transgenic mice.
J Cell Biol
111:
347-359,
1990[Abstract].
37.
Roush, DL,
Gottardi CJ,
Naim HY,
Roth MG,
and
Caplan MJ.
Tyrosine-based membrane protein sorting signals are differentially interpreted by polarized Madin-Darby canine kidney and LLC-PK1 epithelial cells.
J Biol Chem
273:
26862-26869,
1998
38.
Sangan, P,
Rajendran VM,
Mann AS,
Kashgarian M,
and
Binder HJ.
Regulation of colonic H+-K+-ATPase in large intestine and kidney by dietary Na+ depletion and dietary K+ depletion.
Am J Physiol Cell Physiol
272:
C685-C696,
1997
39.
Sangan, P,
Kolla SS,
Rajendran VM,
Kashgarian M,
and
Binder HJ.
Colonic H-K-ATPase -subunit: identification and regulation by dietary K depletion.
Am J Physiol Cell Physiol
276:
C350-C360,
1999
40.
Scheiffele, P,
Peranen J,
and
Simons K.
N-glycans as apical sorting signals in epithelial cells.
Nature
378:
96-98,
1995[ISI][Medline].
41.
Silver, RB,
and
Soleimani M.
H+-K+-ATPases: regulation and role in pathophysiological states.
Am J Physiol Renal Physiol
276:
F799-F810,
1999
42.
Wang, SG,
and
Farley RA.
Valine 904, tyrosine 898, and cysteine 908 in Na,K-ATPase subunits are important for assembly with
subunits.
J Biol Chem
273:
29400-29405,
1998
43.
Wang, T,
Courtois-Coutry N,
Giebisch G,
and
Caplan MJ.
A tyrosine-based signal regulates H+-K+-ATPase-mediated potassium reabsorption in the kidney.
Am J Physiol Renal Physiol
275:
F818-F826,
1998
44.
Wingo, CS,
Madsen KM,
Smolka A,
and
Tisher CC.
H+-K+-ATPase immuno-reactivity in cortical and outer medullary collecting duct.
Kidney Int
38:
985-990,
1990[ISI][Medline].
45.
Wingo, CS,
and
Smolka AJ.
Function and structure of H+-K+-ATPase in the kidney.
Am J Physiol Renal Fluid Electrolyte Physiol
269:
F1-F16,
1995
46.
Wright, FS,
and
Giebisch G.
Regulation of potassium secretion.
In: The Kidney: Physiology and Pathophysiology, edited by Seldin DW,
and Giebisch G.. New York: Raven, 1992, p. 2209-2248.