1 Department of Pharmaceutical Sciences, University of Southern California, Los Angeles 90089-9121; and 2 Department of Molecular and Cell Biology, University of California, Berkeley, California 94720
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ABSTRACT |
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The assembly of the -subunit of the
gastric H-K-ATPase (HK
) with the
-subunit of the H-K-ATPase or
the Na-K-ATPase (NaK
) was characterized with two anti-HK
monoclonal antibodies (MAbs). In fixed gastric oxyntic cells, in
H-K-ATPase in vitro, and in Madin-Darby canine kidney (MDCK) cells
transfected with HK
, MAb 2/2E6 was observed to bind to HK
only
when interactions between
- and
-subunits were disrupted by
various denaturants. The epitope for MAb 2/2E6 was mapped to the
tetrapeptide S226LHY229 of the extracellular
domain of HK
. The epitope for MAb 2G11 was mapped to the eight
NH2-terminal amino acids of the cytoplasmic domain of
HK
. In transfected MDCK cells, MAb 2G11 could immunoprecipitate HK
with
-subunits of the endogenous cell surface NaK
, as well as that from early in the biosynthetic pathway, whereas MAb 2/2E6 immunoprecipitated only a cohort of unassembled endoglycosidase H-sensitive HK
. In HK
-transfected LLC-PK1 cells,
significant immunofluorescent labeling of HK
at the cell surface
could be detected without postfixation denaturation or in live cells,
although a fraction of transfected HK
could also be
coimmunoprecipitated with NaK
. Thus assembly of HK
with NaK
does not appear to be a stringent requirement for cell surface delivery
of HK
in LLC-PK1 cells but may be required in MDCK
cells. In addition, endogenous posttranslational regulatory mechanisms
to prevent hybrid
-
heterodimer assembly appear to be compromised
in transfected cultured renal epithelial cells. Finally, the
extracellular epitope for assembly-sensitive MAb 2/2E6 may represent a
region of HK
that is associated with
-
interaction.
sodium-potassium-adenosinetriphosphatase; Madin-Darby canine kidney cells; LLC-PK1 cells
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INTRODUCTION |
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THE BIOSYNTHETIC ASSEMBLY of the Na-K-ATPase, the
best-characterized member of the P-type ion transporters, has been
extensively studied (see Refs. 8, 16, and 33 for review). The minimal functional unit of the Na-K-ATPase consists of a polytopic -subunit and a glycosylated, single membrane-spanning
-subunit. Additional tools for the investigation and characterization of interactions between
- and
-subunits in ATPases have been provided by the identification and characterization of a
-subunit from another member of the P-type ATPases, the gastric H-K-ATPase (5, 21, 36, 37,
41), and of homologs of the
-subunit of the gastric H-K-ATPase
expressed in colon (10, 11) and kidney cells (4, 24, 28).
The -subunit is typically cited as the catalytic subunit for the
H-K-ATPase and Na-K-ATPase, but enzymatic function is dependent on the
interaction between the respective
- and
-subunits (7, 15, 23).
Therefore, identification of subunit interactive sites is important to
understand the functional cycle of these cation-exchange ATPases. The
association between subunits is stable to mild detergents, such as
Triton X-100 or Nonidet P-40, whereas the forces of interaction are
clearly disrupted by denaturing detergents, such as dodecyl trimethyl
ammonium bromide (DTAB) or SDS. These detergent-sensitive properties
were first used to demonstrate the existence of a
-subunit for the
H-K-ATPase (HK
) (36) and have also been exploited to identify
specific segments of the
-subunit that interact with the
-subunit
(40). Recently, Wang et al. (43), using a heterologous yeast expression
system to monitor assembly of transfected chimeric ATPase subunits,
found a region in an extracellular domain of the
-subunit of the
H-K-ATPase (HK
) spanning Gln-905 to Val-930 that interacts with the
extracellular domain of HK
. In addition, Melle-Milovanovic et al.
(34) used the yeast two-hybrid system to probe for sites of interaction between
- and
-subunits. They reported two regions in the
ectodomain of the
-subunit, Gln-64 to Asn-130 and Ala-156 to
Arg-188, as containing sites of interaction with the
-subunit.
Geering et al. (17) demonstrated that a sequence motif
Y242Y(or F)PYY in the ectodomain of the Na-K-ATPase
-subunit (NaK
) is conserved among all
-subunits, and the
presence of P244 in this motif is essential for assembly of
Na-K-ATPase
- and
-subunits. Moreover, consistent with the
results of Wang et al., Melle-Milovanovic et al. showed that the region
Arg-898 to Arg-922 in the
-subunit interacts strongly with the
extracytoplasmic domain of the
-subunit. The ability to form hybrid
-
heterodimers between the Na-K-ATPase and the H-K-ATPase has
provided significant insight into not only the regulation of assembly
of native
-
heterodimers but also the role of the
-subunit in
the modulation of transport activities of the holoenzyme (15, 19, 22,
23, 30).
We previously reported on two monoclonal antibodies (MAbs) against
HK: MAb 2G11, which bound to the native enzyme at a site within the
cytoplasmic NH2-terminal region, and MAb 2/2E6, which bound
within the large extracytoplasmic segment, but only when the enzyme was
denatured (7). An objective for the present work was to test the
hypothesis that MAb 2/2E6 recognizes a specific site of interaction
between
- and
-subunits and to identify the epitope within HK
.
Another objective of this work was to use the MAbs against
topologically distinct regions of HK to study the regulation of differential assembly of HK
and its impact on plasma membrane delivery of P-type ATPases in epithelial cells, a topic that has received relatively little attention (6). Investigation of this
phenomenon is critically important to understanding the physiology of
transport across epithelia, inasmuch as the Na-K-ATPase is responsible
for the maintenance of transepithelial ion gradients that, in turn,
drive the transepithelial transport of other ions and nutrients.
Moreover, in the gastric acid-secreting oxyntic cell, the mechanism by
which the Na-K-ATPase and the H-K-ATPase are separately targeted to the
basolateral or apical membranes, respectively, must be precise,
notwithstanding the high degree of homology between the
- and
-subunits of the pump enzymes (6). A similar process may exist in
colon and kidney. When expressed in Xenopus, HK
has been
shown to act as a surrogate for conveying Na+-pumping
functions for the
-subunit of the Na-K-ATPase (NaK
) (23).
However, the situation appeared to be quite different when Gottardi and
Caplan (18) transfected HK
in a mammalian epithelial cell line,
LLC-PK1 cells. These authors reported that the expressed
HK
was exclusively targeted to the apical plasma membrane and
endogenous Na-K-ATPase was targeted to its standard basolateral
location, with very poor efficiency of HK
/NaK
assembly or
surrogation of activity. In the present work, HK
was transfected into two polarized kidney cell lines, MDCK cells and
LLC-PK1 cells, and the biosynthesis, assembly, and cell
surface expression of HK
were investigated.
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MATERIALS AND METHODS |
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Materials.
All chemicals were reagent grade. The anti-HK MAbs 2/2E6 and 2G11
have been previously described (7). The MAb against HK
was the kind
gift of Dr. Adam Smolka (Medical University of South Carolina,
Charleston, SC). The polyclonal anti-Na-K-ATPase antibodies were a kind
gift from Dr. W. James Nelson (Stanford University, Palo Alto, CA). The
anti-NaK
MAb 6H (18) was obtained as a hybridoma supernatant from
Dr. Alicia McDonough (University of Southern California, Los Angeles,
CA). Prestained molecular weight and secondary antibodies coupled to
horseradish peroxidase (HRP) were purchased from Bio-Rad (Hercules,
CA). Pro-mix L-[35S]amino acids in
vitro cell labeling mix, streptavidin coupled to HRP, enhanced
chemiluminescence (ECL) kit, protein A-Sepharose, protein G-Sepharose,
and Sepharose CL-2B were purchased from Amersham Pharmacia Biotech.
Trans-35S-label in vitro labeling mix was purchased
from ICN (Irvine, CA). Sulfosuccinimidobiotin (sulfo-NHS-biotin) was
purchased from Pierce (Rockford, IL). MEM was purchased from Mediatech
(Washington, DC). Cys- and Met-free DMEM, MEM with Hanks' salts,
clostripain, and streptavidin-agarose were purchased from
Sigma-Aldrich. Penicillin, streptomycin, amphotericin B (Fungizone)
cocktail, and Hanks' balanced salt solution (HBSS) were purchased from
the Cell Culture Facility at the University of California, San
Francisco, Bio-Whittaker (Washington, DC), or Mediatech. FBS was
purchased from Hyclone Laboratories (Logan, UT). G418 and
14C-labeled molecular weight markers were purchased from
Life Technologies (Bethesda, MD). Endoglycosidase H (Endo H) and
peptide N-glycosidase F (PNGase F) were purchased from
Boehringer Mannheim. Lysyl endopeptidase C (Lys C) was obtained from
Wako Chemical (Osaka, Japan). 4-(2-Aminoethyl)benzenesulfonyl fluoride
(AEBSF) · HCl was purchased from Calbiochem-Novachem (La Jolla, CA). Pepstatin, leupeptin, chymostatin, antipain, and benzamidine were purchased from Chemicon (Temecula, CA). Wheat germ
agglutinin (WGA) coupled to agarose was purchased from E-Y Laboratories
(San Mateo, CA).
Immunohistochemistry of gastric glands and cell cultures.
Gastric glands were isolated as previously described (1). The glands
were fixed in 3.7% formaldehyde in PBS for 30 min at room temperature,
then they were permeabilized by 1% Triton X-100 in PBS for 15 min. The
fixed, permeabilized glands were immunostained directly or subjected to
further treatment with denaturants before they were immunostained.
Treatment with urea or detergents was carried out at room temperature,
and the treated glands were subsequently washed with PBS before
incubation with antibodies. The treated cells were incubated for 1 h
with MAbs against HK (MAb 2/2E6 or MAb 2G11), washed three times in
PBS, and incubated with fluorescent-labeled anti-mouse IgG antibody for
1 h. Previous work has shown that the epitope on HK
for MAb 2/2E6 is
in the extracellular domain and that for MAb 2G11 is in the
NH2-terminal cytoplasmic tail (7).
Biochemical characterization of - and
-subunit interaction.
H-K-ATPase-enriched gastric microsomes were prepared as previously
described (29). As a general protocol, the microsomes were solubilized
for 1 h at room temperature in 1% Triton X-100 in suspending medium
(SM) consisting of 300 mM sucrose, Tris · HCl (pH
7.4), and 0.5 mM EDTA. The solubilized materials were applied to
different affinity matrices before or after the H-K-ATPase was
subjected to several modifying procedures.
Phage display peptide library kit. The heptapeptide display library phage kit was purchased from New England Biolabs. Phage selection, amplification, and DNA extraction were carried out according to manufacturer's instructions. Using polystyrene petri dishes coated with 2/2E6 antibody as selection plates, we carried out four rounds of selection (biopanning) to choose a pool of phages. Twenty individual clones were isolated, amplified, and analyzed with Western blot for 2/2E6. Seven of them showed that their minor coat protein strongly reacted with 2/2E6. The corresponding DNA sequences were sequenced, and the amino acids were deduced. Synthetic peptides were synthesized by BioMed. Competitive Western blots were carried out with primary antibody preincubated with the synthetic peptide for 1 h.
Construction of HK clones and transfection.
cDNA encoding rat HK
(5) was excised from the pBluescript
SK(
) vector (Stratagene) with Apa I and Avr II
and subcloned into pCB6, a vector in which the expression of the insert
cDNA is driven by a cytomegalovirus promoter (2). The Apa
I-digested end was filled in with Klenow and blunt-end ligated into the
unique Hind III site of pCB6; the Avr II-cut end was
ligated into the Xba I site. MDCK strain II cells, which are of
European Molecular Biology Lab parentage and are the clone that
delivers newly synthesized Na-K-ATPase exclusively to the basolateral
membrane, were transfected with this plasmid by the calcium phosphate
method, as described previously (35). After selection in G418, four
positive clones were identified, expanded, and maintained in MEM
supplemented with 5% FBS and antibiotic-antimycotic. The polarity of
these four clones was verified by assaying for polarized secretion of the endogenous MDCK glycoprotein gp80, as described previously (42).
The apical-to-basal ratio of gp80 secretion in all the clones described
here was
4:1. The LLC-PK cells used in this study, stably transfected
with HK
(18), were the kind gift of Dr. Michael Caplan (Yale University).
Cell surface biotinylation.
Cells grown on Costar 24-mm Transwell polycarbonate filters were cooled
to 4°C with several washes of cold HBSS supplemented with 20 mM
sodium HEPES, pH 7.4. The apical or basolateral surface of transfected
MDCK cells was biotinylated with 0.5 mg/ml sulfo-NHS-biotin in HBSS
with two 15-min incubations at 4°C. After biotinylation, cells were
washed quickly three times with cold HBSS and incubated for 15 min at
4°C with cold MEM supplemented with 0.6% BSA, 20 mM sodium HEPES,
and 50 mM glycine to quench any remaining unreacted biotin. The cells
were lysed under denaturing conditions (0.5% SDS, 100 mM NaCl, 50 mM
triethanolamine · HCl, 5 mM EDTA, 0.1 mM AEBSF, and
0.2% NaN3, pH 8.1) or nondenaturing conditions
[2.5% (wt/vol) Triton X-100, 100 mM NaCl, 100 mM
triethanolamine · HCl, 5 mM EDTA, 0.1 mM AEBSF, 0.2%
NaN3, 5 µg/ml each of pepstatin and leupeptin, 10 µg/ml
each of chymostatin and antipain, and 0.5 mM benzamidine]. After
cell lysates were precleared with Sepharose CL-2B, HK was
immunoprecipitated with MAb 2/2E6-protein A-Sepharose (specific for the
ectodomain of HK
) or MAb 2G11-protein G-Sepharose (specific for the
cytoplasmic domain of HK
), and biotinylated proteins were isolated
with streptavidin-agarose. Immunoprecipitates were run on SDS-PAGE,
transferred to nitrocellulose, and probed with MAb 2/2E6 to detect
HK
, anti-NaK
monoclonal or polyclonal antibodies to detect
NaK
, or streptavidin-HRP to detect biotinylated proteins. After
incubation with appropriate secondary antibodies coupled to HRP (if
necessary), the reactive proteins were visualized by ECL. Signals from
ECL blots were quantitated on a Bio-Rad GS-670 imaging densitometer.
Pulse-chase analysis of HK.
Transfected MDCK or LLC-PK1 cells grown on 24-mm Transwell
plates were washed with PBS containing Ca2+ and
Mg2+ and starved for 15 min at 37°C with Cys- and
Met-free DMEM supplemented with 5% dialyzed FBS, 20 mM sodium HEPES
(pH 7.4), and antibiotic-antimycotic. Cells were pulse labeled with 50 µCi of Pro-mix (New England Nuclear) or
Trans-35S-label (ICN, Irvine, CA)
L-amino acids from the basolateral surface and chased for
various periods. Cells were lysed under denaturing or nondenaturing
conditions, as described above. Lysates were processed for
immunoprecipitation, as described above. Immunoprecipitates were run on
SDS-PAGE and fluorographed. In some cases, immunoprecipitates of
pulse-labeled HK
were digested with Endo H before SDS-PAGE and
fluorography. The immunoprecipitated material was resuspended by
boiling in 0.1 M sodium citrate (pH 6.0) and 1% SDS; Endo H was added
to 3 mU, and the material was incubated overnight at 37°C.
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RESULTS |
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Immunostaining of parietal cells by 2/2E6 requires denaturation.
When we first introduced MAb 2/2E6, we pointed out that immunostaining
did not occur with conventional formaldehyde fixation and
permeabilization by Triton X-100 but "required alcohol delipidation of the glands." Figure 1
shows that H-K-ATPase of Formalin-fixed parietal cells is immunostained
by MAb 2/2E6 only when a denaturant is applied (e.g., alcohol, SDS,
DTAB, alkali). A titration with urea as a denaturant is shown in
Fig. 2. For gastric glands fixed by
Formalin and permeabilized with Triton X-100, immunostaining by 2/2E6
was barely evident up to 4.5 M urea and became uniformly apparent at 5 M urea. These data demonstrate that the epitope for 2/2E6 is not simply
related to delipidation but, rather, is buried and becomes exposed only
after denaturation.
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Dissociation of - and
-subunits
exposes the 2/2E6 epitope.
We previously reported that
-
association was stable in nonionic
detergents and disrupted by ionic detergents, such as DTAB or SDS (36).
The unmasking of the 2/2E6 epitope by DTAB or SDS in fixed gastric
glands further supported an epitopic site within the sphere of
-
interaction. To investigate the action of SDS on the
-
association and unmasking of 2/2E6 epitope, we treated purified
H-K-ATPase-containing membranes with various amounts of SDS, then added
Triton X-100 to mop up the excess SDS. The treated samples were passed
through a 2/2E6-protein A affinity column. The bound materials were
eluted with 2% SDS and analyzed by SDS-PAGE and Western blot, as shown
Fig. 3. For samples where the membranes
were treated with
0.3% SDS, HK
bound to the 2/2E6 column,
demonstrating that these levels of SDS were sufficient to expose the
binding site.
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The 2/2E6 epitope is identified as
S226LHY229.
The heptapeptide phage display library was used as described in
METHODS AND MATERIALS to screen the peptide sequence
corresponding to the epitope for MAb 2/2E6 binding. Seven individual
clones of the phage were selected for amplification and DNA sequencing. The corresponding peptide sequences are shown in Table
1. Inspection of these sequences indicates
that the common sequence for the epitope is SXHY. The most
likely site is SLHY, corresponding to amino acids 226-229 of the
rabbit HK sequence.
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MAb 2G11 interacts with the NH2-terminal eight amino
acids of the -subunit.
Earlier work showed that MAb 2G11 binds to the cytoplasmic
domain of HK
(7). Through the use of site-specific proteases, we now
provide a more definitive binding site. Figure
6, A and B, shows that,
after digestion of microsomal vesicles with Arg-specific clostripain
and subsequent deglycosylation with PNGase F, distinctly different
peptides are recognized by our two antibody probes: a peptide of ~30
kDa is recognized by MAb 2/2E6 and a peptide of ~2 kDa is recognized
by MAb 2G11. The NH2-terminal cytoplasmic domain of the
rabbit HK
has potential Arg cleavage sites at Arg-13 and Arg-18,
which would be predicted to yield NH2-terminal peptides of
~2.2 and 1.5 kDa, respectively, as indicated by the following sequence:
MAALQEK7K8SCSQR13MEEFR18HYCWN PDT. . . . .
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Immunostaining of HK expressed in
LLC-PK1 cells.
Gottardi and Caplan (18) stably transfected a kidney cell line,
LLC-PK1 cells, with a gene for HK
and subsequently
showed that HK
is synthesized in the endoplasmic reticulum (ER) and sorted to the apical plasma membrane without an accompanying
-subunit. When we employed this same stably transfected
LLC-PK1 cell line, we observed a strong reaction of MAb
2/2E6 with the apical plasma membrane, even when the antibody was added
to live cells (Fig. 7A). That is,
neither fixation nor permeabilization was required for surface staining
of the 2/2E6 epitope, although these treatments, without denaturation,
were effective in exposing additional
-subunit sites within an
intracellular membrane compartment(s) (Fig. 7B). On the other
hand, the MAb 2G11 epitope is exposed after only permeabilization of
transfected cells (Fig. 7, C and D). Thus, in the
LLC-PK1 expression system, reaction of HK
with 2/2E6 did not require denaturation, and since HK
was not being coexpressed, the results are consistent with the interpretation that the epitopic site for MAb 2/2E6 is in a region of
-
association.
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Immunostaining of HK in transfected MDCK cells.
A second cultured epithelial cell system in which to study the
expression and trafficking of HK
was generated by expressing HK
in MDCK cells (MDCK-HK
cells). MDCK-HK
cells were fixed in
formaldehyde and stained with MAb 2/2E6 without (Fig.
8A) or with (Fig. 8B)
postfixation treatment with methanol. Surprisingly, unlike the case
with transfected LLC-PK1 cells (Fig. 7), but similar to
that seen for isolated rabbit gastric glands (Fig. 1), methanol or high
concentrations of urea are required after fixation to effect staining
of HK
in MDCK-HK
cells with MAb 2/2E6. Also, in contrast to data
generated from the LLC-PK1 model in this study and from
Gottardi and Caplan (18), attempts to surface immunolabel HK
by
prebinding MAb 2/2E6 were unsuccessful (data not shown), as were
attempts to surface immunoprecipitate metabolically radiolabeled HK
(data not shown). These results imply that, in transfected MDCK cells,
access of MAb 2/2E6 to its extracellular epitope on HK
is restricted
unless HK
is further denatured. These results parallel those
obtained with native H-K-ATPase in glands or isolated gastric
microsomes. In the case of oxyntic cell membranes, access may be
limited by association of HK
with HK
; however, because HK
alone was transfected into MDCK-HK
cells, another mechanism by which
the MAb 2/2E6-binding epitope is masked must exist in MDCK-HK
cells.
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HK is expressed at the cell surface of
MDCK-HK
cells.
Analysis of the distribution of anti-HK
immunofluorescence in
MDCK-HK
cells suggested that HK
may be present at the cell surface. To assay for HK
at the cell surface of transfected MDCK cells, the apical and basolateral plasma membranes of MDCK-HK
cells
were separately biotinylated. Cell lysis was performed under denaturing
conditions, and lysates were incubated with streptavidin-agarose to
precipitate biotinylated proteins. Western blots of biotinylated proteins were probed with MAb 2/2E6. As seen in Fig.
9A for two different clones of
MDCK-HK
cells, HK
appears to be expressed at the cell membrane,
and cell surface expression of HK
is apparently predominantly
apical. In addition, in SDS gels, biotinylated HK
migrates as a
broad band of apparent molecular mass of 60-80 kDa, indicating
that its oligosaccharide chains have been modified from the
high-mannose core oligosaccharides (18, 23, 30). Alternatively, cell
surface HK
was biotinylated, and cell lysis was performed under
denaturing conditions. HK
was immunoprecipitated with anti-HK
cytoplasmic domain MAb 2G11, and the immunoprecipitate was probed on
Western blots with streptavidin-HRP. By this approach, cell surface
HK
was also observed to be predominantly apical (data not shown).
|
HK is associated with an endogenous 94-kDa MDCK cell
protein at the cell surface of MDCK-HK
cells.
To determine whether HK
was expressed alone at the cell
surface in MDCK-HK
cells, as was observed in other transfected cells (12, 14, 18, 23), the cell surface was biotinylated and cell lysis was
effected under nondenaturing conditions. HK
was subsequently
immunoprecipitated with the anticytoplasmic domain MAb 2G11, and the
immunoprecipitates were analyzed on Western blots by probing with
streptavidin-HRP. As shown in Fig. 9B, a biotinylated
endogenous 94-kDa MDCK cell protein coimmunoprecipitated with HK
,
suggesting that at least a fraction of HK
at the cell surface is
associated with another protein. This 94-kDa protein did not appear in
immunoprecipitates from cells lysed under denaturing conditions (Fig.
9A), suggesting that the 94-kDa protein and HK
are
noncovalently associated.
Noncovalent association of HK with the 94-kDa
protein occurs early in the biosynthetic pathway.
Pulse labeling of transfected MDCK-HK
cells with radiolabeled Cys
and Met for 15 min and subsequent immunoprecipitation of pulse-labeled
HK
under nondenaturing conditions with MAb 2G11 resulted in the
coimmunoprecipitation of two radiolabeled proteins: a 94-kDa protein
and a protein migrating at ~50-55 kDa (Fig.
10A). The protein migrating at
~50-55 kDa is presumably a core-glycosylated, high-mannose
("immature") form of HK
. This core-glycosylated form of HK
has been previously identified in other heterologous expression systems
(19, 23, 25, 26, 30), and the oligosaccharide chains are sensitive to
hydrolysis by Endo H (Fig. 10B). Therefore, the association
between these two proteins apparently occurs early in the secretory
pathway.
|
|
The 94-kDa protein associated with HK is endogenous
NaK
.
The identity of the 94-kDa protein associated with HK
at the cell
surface was revealed when the coimmunoprecipitating proteins from
MDCK-HK
cells were probed with anti-NaK
antibodies on Western blots. NaK
clearly coimmunoprecipitates with HK
under
nondenaturing conditions (Fig.
12A). The coimmunoprecipitation
does not occur from cell lysates harvested under denaturing conditions,
confirming that the association of HK
with NaK
is noncovalent.
Moreover, when the surface-biotinylated complex of HK
and 94-kDa
protein is probed on Western blots, the surface-biotinylated 94-kDa
protein clearly reacts with the anti-NaK
antibodies (Fig.
12B). Thus, in MDCK-HK
cells, HK
can apparently assemble
with endogenous NaK
, and a fraction of this complex can be targeted
to the apical plasma membrane.
|
HK assembles with endogenous NaK
in
transfected LLC-PK1 cells.
With the observation that HK
assembles with endogenous NaK
in
MDCK-HK
cells, we sought to reevaluate the assembly competence of
HK
expressed in LLC-PK1 cells. From LLC-PK1
cells, metabolically radiolabeled HK
could be coimmunoprecipitated
with a 94-kDa protein with MAb 2G11 (Fig. 12C). In addition,
immunoprecipitation of HK
with MAb 2G11 from cells lysed under
nondenaturing conditions clearly results in the coimmunoprecipitation
of endogenous NaK
, as shown in the Western blot in Fig.
12D. These results suggest that HK
may be generally
competent to assemble with NaK
in renal epithelial cells.
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DISCUSSION |
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Two anti-HK MAbs have been further characterized. The epitope to MAb
2/2E6 appears to include the amino acids
S226LHY229 in the extracellular domain of
HK
. In addition to evidence presented here, we have observed that
MAb 2/2E6 reacts with all known HK
containing the SLHY motif,
whereas there is no recognition of those
-subunits, dog (SLRY) or
chicken (NLHY), where substitutions have occurred (8). The SLHY epitope
appears to become masked when assembled with HK
or NaK
. The
masking of this epitope in assembled, but not unassembled, H-K-ATPase
suggests that SLHY may be part of a site for
-
interaction. Given
that this site appears to be masked in heterodimers of HK
-HK
as
well as NaK
-HK
hybrid heterodimers, this epitope may be a region
that is involved in
-
association in all heterodimeric P-type
ATPases. Alternatively, it is possible that the loss of recognition by
MAb 2/2E6 may result from the "burying" of its epitope as a
consequence of the intrinsic folding of the extracellular domain of
HK
, rather than assembly with an
-subunit. We do not favor this
interpretation, since HK
, presumably alone at the cell surface of
LLC-PK1 cells, can bind MAb 2/2E6 without requiring
denaturation or reduction of disulfide bonds. In these cells, HK
should have satisfied all the quality control mechanisms, including
proper folding, as a prerequisite to its exit from the ER for its
appearance at the cell surface in the apparent absence of an
-subunit.
Additional evidence suggests that MAb 2/2E6 binds to an epitope that is
formed at an interface between - and
-subunits. Two amino acids
in the 2/2E6-binding epitope, L227 and Y229,
are identical in all three isoforms of the
-subunit of the
Na-K-ATPase (
1,
2, and
3) as well as HK
, and the adjacent
region Y229Y(or F)P231YYG is conserved
throughout all known
-subunits (8, 31, 39). The Y229Y(or
F)P231YYG sequence is absolutely conserved in all
-subunits of heterodimeric P-type ATPases characterized to date and
overlaps with the SLHY motif in HK
. The importance of this region in
-
assembly/association is further underscored by the work of
Geering et al. (17) showing that the ability of the
-subunit to
assemble with the
-subunit is virtually abolished by mutating the
proline residue of YY(or F)PYYG. Thus these two overlapping motifs may
be one part of HK
that is responsible for the association of HK
with NaK
.
The second anti-HK MAb, MAb 2G11, was previously shown to bind to
the cytoplasmic domain of HK
(7). In this study we further defined
the epitope to reside near the eight most NH2-terminal amino acids. This region must be at or near an important site for
functional activity, and possibly a cytoplasmic K+ binding
site, inasmuch as association with MAb 2G11 inhibits H-K-ATPase
activity and alters the KA for activation of
p-nitrophenyl phosphatase activity by K+ (7). It is
also noteworthy that all mammalian species where HK
is recognized by
MAb 2G11 (mouse, rat, human, rabbit, and dog) have the same eight
NH2-terminal amino acids, whereas those
-subunits with
different NH2-terminal sequences, including chicken HK
and all isoforms of the
-subunit of the Na-K-ATPase, are not
recognized by MAb 2G11 (8).
We also showed here the utility of MAb 2G11 for immunoprecipitating
assembled as well as unassembled HK. We used MAb 2G11 in conjunction
with MAb 2/2E6 to evaluate the assembly of HK
in transfected
MDCK-HK
and LLC-PK1 cells. The simplest interpretation of our results is that there are apparently two cellular pools of
HK
. One pool is unassembled HK
. In LLC-PK1 cells,
unassembled HK
appears to transit to the plasma membrane; in
MDCK-HK
cells, unassembled HK
largely appears to remain in the
ER. The other pool of HK
assembles with endogenous NaK
in MDCK
and LLC-PK1 cells. Although the assembly of NaK
-HK
has been convincingly demonstrated in expression systems such as
Xenopus oocytes (23, 25) and yeast (14, 15, 43), our results
represent the first demonstration of assembly of HK
with NaK
in
epithelial cells, a significant result for several reasons. First,
published studies on the trafficking of transfected HK
in
LLC-PK1 and MDCK cells used the assembly-sensitive MAb
2/2E6 (18). Moreover, several studies have shown that HK
appears to
transit to the plasma membrane in the absence of an
-subunit in
other cell types (12, 18, 19, 38). All these studies used MAb 2/2E6;
thus analysis of these trafficking data would be restricted to that of
unassembled HK
. For MDCK-HK
cells, we showed that a significant fraction of HK
appears to be assembled with NaK
at steady state; thus a significant population of HK
was likely overlooked with respect to the analysis of trafficking of HK
in these earlier studies. Because HK
also assembles with NaK
in
LLC-PK1 cells, the conclusions regarding HK
trafficking
in transfected epithelial cell lines may suffer from the same
limitations (18, 38). In both previously published studies, it was
stated that HK
assembly with NaK
was not detected. These
differences in results regarding HK
assembly are not likely the
result of clonal variations, since the LLC-PK1 cell line
transfected with HK
is common to both studies. The differences,
however, are likely due to the different MAbs used by each
investigator. In the light of the present data, it will be important to
reevaluate the trafficking of HK
with respect to assembled and
unassembled HK
in these epithelial cell lines. In addition, our
preliminary unpublished data suggest that the trafficking of HK
in
MDCK-HK
cells is more complex than formerly believed and may be
regulated not only by assembly of HK
, but also by time of culture or
after induction of polarity (unpublished observations). Similarly, the
targeting of endogenous Na-K-ATPase in MDCK cells as a function of the
establishment of polarity is well documented (13, 32).
Second, these two transfected epithelial cell lines appear to display
different phenotypes relative to the relationship between assembly of
HK and its appearance at the cell surface. In LLC-PK1 cells, assembly of HK
with an
-subunit does not appear to be a
prerequisite for its exit from the ER and appearance at the cell
surface. The mechanism by which HK
appears to be able to reach the
plasma membrane alone remains to be determined; alternatively, it
remains to be determined whether HK
in LLC-PK1 cells
assembles with another protein that is not detectable by the assays
used in this study and whether the 2/2E6 epitope remains exposed in such a protein complex. Assembly with such a surrogate may then facilitate exit of this complex from the ER for delivery to the plasma
membrane. On the other hand, MDCK-HK
cells characterized here appear
to function in a manner that is more consistent with the present views
of quality control mechanisms for membrane protein assembly in the ER.
That is, HK
does not appear to exit the ER without assembly with an
-subunit. In this case, it is the
-subunit of the endogenous
Na-K-ATPase. Conversely, numerous studies have shown that the exit of
nascent
-subunits from the ER is dependent on the presence of a
-subunit (for review see Refs. 16 and 33).
Third, isoforms of the H-K-ATPase and Na-K-ATPase are clearly
coexpressed in cells other than the gastric oxyntic cell, such as
colonic (4, 11, 39) and renal medullary cells (27). The data presented
here suggest that the potential for the assembly of hybrid heterodimers
in epithelial cells is significant. Thus, in some instances, cells must
possess regulatory mechanisms to prevent hybrid heterodimer assembly,
as in the gastric oxyntic cell. One possible mechanism is to regulate
subunit expression, hence assembly, at the level of mRNA translation of
ATPase subunits, as shown recently in MDCK cells (20). Alternatively,
there may be other intrinsic posttranslational mechanisms in place to
prevent such hybrid assembly, inasmuch as subunits of the H-K-ATPase
and Na-K-ATPase appear to have a high preference for their own
respective -subunits, as demonstrated in an Sf9 insect cell
expression system (26). On the other hand, hybrid heterodimer assembly
may need to be facilitated. For example, recent evidence suggests that colonic HK
assembles with the
1-subunit of the
Na-K-ATPase in kidney and distal colon (9). Another study has localized
an Na-K-ATPase
1-
2 heterodimer at the
apical membrane of cells in developing kidney tubules (3). The
regulation of assembly of P-type ATPase in cells expressing more than
one heterodimeric P-type ATPase may ultimately involve a hierarchy of
mechanisms, as has been demonstrated for the intracellular trafficking
of Na-K-ATPase in epithelial cells (32).
One limitation of this study is that we are unable to assess functional
competence of NaK-HK
heterodimers. Although functional assembly
of NaK
-HK
heterodimers has been demonstrated in heterologous expression systems such as Xenopus (23, 25) and yeast (14, 15),
the two transfected epithelial cell lines in this study appear to
express HK
at only immunologic, rather than functional, levels. The
development of an epithelial cell culture system to test concomitantly
the assembly, trafficking, and function of hybrid and/or chimeric
P-type ATPases would be extremely valuable. Another feature of the
LLC-PK1 and MDCK-HK
cells to consider is that the
assembly (or lack thereof) of HK
and its trafficking in heterologous
systems may be a consequence of the relative overexpression of HK
as
a result of the removal of the expression of HK
from appropriate
cellular controls due to transfection. Nevertheless, the two epithelial
cell lines expressing HK
and the reagents characterized further here
may play an important role in the characterization of the regulation of
P-type ATPase assembly in epithelial cells.
![]() |
ACKNOWLEDGEMENTS |
---|
The authors acknowledge the expert technical assistance of Young Y. Jeng.
![]() |
FOOTNOTES |
---|
* C. T. Okamoto and D. C. Chow contributed equally to this work.
This work was supported in part by a grant-in-aid from the former Greater Los Angeles Affiliate of the American Heart Association (C. T. Okamoto) and by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-51588 (C. T. Okamoto), DK-38972 (J. G. Forte), and DK-10141 (J. G. Forte).
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.
Address for reprint requests and other correspondence: C. T. Okamoto, Dept. of Pharmaceutical Sciences, 1985 Zonal Ave., University of Southern California, Los Angeles, CA 90089-9121 (E-mail: cokamoto{at}hsc.usc.edu).
Received 8 June 1999; accepted in final form 2 November 1999.
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REFERENCES |
---|
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---|
1.
Berglindh, T,
and
Öbrink KJ.
A method for preparing isolated glands from the rabbit gastric mucosa.
Acta Physiol Scand
96:
150-159,
1976[ISI][Medline].
2.
Brewer, CB,
and
Roth MG.
A single amino acid change in the cytoplasmic domain alters the polarized delivery of influenza virus hemagglutinin.
J Cell Biol
114:
413-421,
1991[Abstract].
3.
Burrow, CR,
Devuyst O,
Li X,
Gatti L,
and
Wilson PD.
Expression of the 2-subunit and apical localization of the Na+-K+-ATPase in the metanephric kidney.
Am J Physiol Renal Physiol
277:
F391-F403,
1999
4.
Callaghan, JM,
Tan S-S,
Khan MA,
Curran KA,
Campbell WG,
Smolka AJ,
Toh B-H,
Gleeson PA,
Wingo CS,
Cain BD,
and
van Driel IR.
Renal expression of the gene encoding the gastric H+-K+-ATPase -subunit.
Am J Physiol Renal Fluid Electrolyte Physiol
268:
F363-F374,
1995
5.
Canfield, VA,
Okamoto CT,
Chow D,
Dorfman J,
Gros P,
Forte JG,
and
Levenson R.
Cloning of the H,K-ATPase subunit. Tissue-specific expression, chromosomal assignment, and relationship to Na,K-ATPase
subunits.
J Biol Chem
265:
19878-19884,
1990
6.
Caplan, MJ.
Gastric H+/K+-ATPase: targeting signals in the regulation of physiologic function.
Curr Opin Cell Biol
10:
468-473,
1998[ISI][Medline].
7.
Chow, DC,
and
Forte JG.
Characterization of the -subunit of the H,K-ATPase using an inhibitory monoclonal antibody.
Am J Physiol Cell Physiol
265:
C1562-C1570,
1993
8.
Chow, DC,
and
Forte JG.
Functional significance of the -subunit for heterodimeric P-type ATPases.
J Exp Biol
198:
1-17,
1995
9.
Codina, J,
Delmas-Mata JT,
and
DuBose TD, Jr.
The -subunit of the colonic H+,K+-ATPase assembles with
1-Na+,K+-ATPase in kidney and distal colon.
J Biol Chem
273:
7894-7899,
1998
10.
Cougnon, M,
Planelles G,
Crowson MS,
Shull GE,
Rossier BC,
and
Jaisser F.
The rat distal colon P-ATPase -subunit encodes an ouabain-sensitive H+,K+-ATPase.
J Biol Chem
271:
7277-7280,
1996
11.
Crowson, MS,
and
Shull GE.
Isolation and characterization of a cDNA encoding the putative distal colon H+,K+-ATPase. Similarity of deduced amino acid sequence to gastric H+,K+-ATPase and Na+,K+-ATPase and mRNA expression in distal colon, kidney, and uterus.
J Biol Chem
267:
13740-13748,
1992
12.
DeTomaso, AW,
Blanco G,
and
Mercer RW.
The and
subunits of the Na,K-ATPase can assemble at the plasma membrane into functional enzyme.
J Cell Biol
127:
55-69,
1994[Abstract].
13.
Drubin, DG,
and
Nelson WJ.
Origins of cell polarity.
Cell
84:
335-344,
1996[ISI][Medline].
14.
Eakle, KA,
Kabalin MA,
Wang S-G,
and
Farley RA.
The influence of -subunit structure on the stability of Na+/K+-ATPase complexes and interaction with K+.
J Biol Chem
269:
6550-6557,
1994
15.
Eakle, KA,
Kim KS,
Kabalin MA,
and
Farley RA.
High-affinity ouabain binding by yeast cells expressing Na+,K+-ATPase subunits and the gastric H+,K+-ATPase
subunit.
Proc Natl Acad Sci USA
89:
2834-2838,
1992[Abstract].
16.
Geering, K.
The functional role of the -subunit in the maturation and intracellular transport of Na,K-ATPase.
FEBS Lett
285:
189-193,
1991[ISI][Medline].
17.
Geering, K,
Jaunin P,
Jaisser F,
Mérillat A-M,
Horisberger J-D,
Mathews PM,
Lemas V,
Fambrough DM,
and
Rossier BC.
Mutation of a conserved proline residue in the -subunit ectodomain prevents Na+-K+-ATPase oligomerization.
Am J Physiol Cell Physiol
265:
C1169-C1174,
1993
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,
and
Caplan MJ.
Molecular requirements for the cell-surface expression of multisubunit ion-transporting ATPases. Identification of protein domains that participate in Na,K-ATPase and H,K-ATPase subunit assembly.
J Biol Chem
268:
14342-14347,
1993
20.
Grindstaff, KK,
Blanco G,
and
Mercer RW.
Translational regulation of Na,K-ATPase 1- and
1-polypeptide expression in epithelial cells.
J Biol Chem
271:
23211-23221,
1996
21.
Hall, K,
Perez G,
Anderson D,
Gutierrez C,
Munson K,
Hersey SJ,
Kaplan JH,
and
Sachs G.
Location of the carbohydrates present in the H,K-ATPase vesicles isolated from hog gastric mucosa.
Biochemistry
29:
701-706,
1990[ISI][Medline].
22.
Hasler, U,
Wang X,
Crambert G,
Béguin P,
Jaisser F,
Horisberger J-D,
and
Geering K.
Role of -subunit domains in the assembly, stable expression, intracellular routing, and functional properties of Na,K-ATPase.
J Biol Chem
273:
30826-30835,
1998
23.
Horisberger, J-D,
Jaunin P,
Reuben MA,
Lasater LS,
Chow DC,
Forte JG,
Sachs G,
Rossier BC,
and
Geering K.
The H,K-ATPase -subunit can act as a surrogate for the
-subunit of Na,K-pumps.
J Biol Chem
266:
19131-19134,
1991
24.
Jaisser, F,
Horisberger J-D,
Geering K,
and
Rossier B.
Mechanisms of urinary K+ and H+ excretion: primary structure and functional expression of a novel H,K-ATPase.
J Cell Biol
123:
1421-1429,
1993[Abstract].
25.
Jaunin, P,
Jaisser F,
Beggah AT,
Takeyasu K,
Mangeat P,
Rossier BC,
Horisberger J-D,
and
Geering K.
Role of the transmembrane and extracytoplasmic domain of subunits in subunit assembly, intracellular transport, and functional expression of Na,K-pumps.
J Cell Biol
123:
1751-1759,
1993[Abstract].
26.
Koenderink, JB,
Swarts HGP,
Hermsen HPH,
and
De Pont JJHHM
The -subunits of Na+,K+-ATPase and gastric H+,K+-ATPase have a high preference for their own
-subunit and affect the K+ affinity of these enzymes.
J Biol Chem
274:
11604-11610,
1999
27.
Kone, BC
Renal ATPase: structure, function, and regulation.
Miner Electrolyte Metab
22:
349-365,
1996[ISI][Medline].
28.
Kraut, JA,
Starr F,
Sachs G,
and
Reuben M.
Expression of gastric and colonic H+-K+-ATPase in the rat kidney.
Am J Physiol Renal Fluid Electrolyte Physiol
268:
F581-F587,
1995
29.
Lee, HC,
and
Forte JG.
A study of H+ transport in gastric microsomal vesicles using fluorescent probes.
Biochim Biophys Acta
508:
339-356,
1980.
30.
Lemas, MV,
Yu H-Y,
Takeyasu K,
Kone B,
and
Fambrough DM.
Assembly of Na,K-ATPase -subunit isoforms with Na,K-ATPase
-subunit isoforms and H,K-ATPase
-subunit.
J Biol Chem
269:
18651-18655,
1994
31.
Malik, N,
Canfield VA,
Beckers M-C,
Gros P,
and
Levenson R.
Identification of the mammalian Na,K-ATPase 3-subunit.
J Biol Chem
271:
22754-22758,
1996
32.
Mays, RW,
Siemers KA,
Fritz BA,
Lowe AW,
van Meer F,
and
Nelson WJ.
Hierarchy of mechanisms involved in generating Na/K-ATPase polarity in MDCK epithelial cells.
J Cell Biol
130:
1105-1115,
1995[Abstract].
33.
McDonough, AA,
Geering K,
and
Farley RA.
The sodium pump needs its subunit.
FASEB J
4:
1598-1605,
1990
34.
Melle-Milovanovic, D,
Milovanovic M,
Nagpal S,
Sachs G,
and
Shin JM.
Regions of association between the - and
-subunit of the gastric H,K-ATPase.
J Biol Chem
273:
11075-11081,
1998
35.
Okamoto, C,
Shia S-P,
Bird C,
Mostov KE,
and
Roth MG.
The cytoplasmic domain of the polymeric immunoglobulin receptor contains two internalization signals that are distinct from its basolateral sorting signal.
J Biol Chem
267:
9925-9932,
1992
36.
Okamoto, CT,
Karpilow JM,
Smolka A,
and
Forte JG.
Isolation and characterization of gastric microsomal glycoproteins. Evidence for a glycosylated -subunit of the H+/K+-ATPase.
Biochim Biophys Acta
1037:
360-372,
1990[ISI][Medline].
37.
Reuben, MA,
Lasater LS,
and
Sachs G.
Characterization of a subunit of the gastric H+/K+-transporting ATPase.
Proc Natl Acad Sci USA
87:
6767-6771,
1990[Abstract].
38.
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
39.
Sangan, P,
Kolla SS,
Rajendran VM,
Kashgarian M,
and
Binder HJ.
Colonic H-K-ATPase -subunit: identification in apical membranes and regulation by dietary K depletion.
Am J Physiol Cell Physiol
276:
C350-C360,
1999
40.
Shin, JM,
and
Sachs G.
Identification of a region of the H,K-ATPase subunit associated with the
subunit.
J Biol Chem
269:
8642-8646,
1994
41.
Shull, GE.
cDNA cloning of the -subunit of the rat gastric H,K-ATPase.
J Biol Chem
265:
12123-12126,
1990
42.
Urban, J,
Parczyk K,
Leutz A,
Kayne M,
and
Kondor-Koch C.
Constitutive apical secretion of an 80 kD sulfated glycoprotein complex in the polarized epithelial Madin-Darby canine kidney cell line.
J Cell Biol
105:
2735-2743,
1987[Abstract].
43.
Wang, S-G,
Eakle KA,
Levenson R,
and
Farley RA.
Na+-K+-ATPase -subunit containing Q905-V930 of gastric H+-K+-ATPase
preferentially assembles with H+-K+-ATPase
.
Am J Physiol Cell Physiol
272:
C923-C930,
1997