Download ATPase in the plasma membrane of HeLa cells

JCS ePress online publication date 3 June 2008
Research Article
2159
Fast degradation of the auxiliary subunit of Na+/K+ATPase in the plasma membrane of HeLa cells
Shige H. Yoshimura1,*, Shizuka Iwasaka1, Wolfgang Schwarz2 and Kunio Takeyasu1
1
Graduate School of Biostudies, Kyoto University, Yoshida-konoe-cho, Sakyo-ku, Kyoto, 606-8502, Japan
Max-Planck-Institute for Biophysics, Max-von-Laue-Str. 3, 60438, Frankfurt, Germany
2
*Author for correspondence (e-mail: [email protected])
Journal of Cell Science
Accepted 14 April 2008
Journal of Cell Science 121, 2159-2168 Published by The Company of Biologists 2008
doi:10.1242/jcs.022905
Summary
The cell-surface expression and function of multisubunit plasma
membrane proteins are regulated via interactions between
catalytic subunits and auxiliary subunits. Subunit assembly in
the endoplasmic reticulum is required for the cell-surface
expression of the enzyme, but little is known about subunit
interactions once it reaches the plasma membrane. Here we
performed highly quantitative analyses of the catalytic (α1) and
auxiliary (β1 and β3) subunits of Na+/K+-ATPase in the HeLa
cell plasma membrane using isoform-specific antibodies and a
cell-surface protein labeling procedure. Our results indicate that
although the β-subunit is required for the cell-surface expression
of the α-subunit, the plasma membrane contains more αsubunits than β-subunits. Pulse-labeling and chasing of the cellsurface proteins revealed that degradation of the β-subunits was
Introduction
The functional expressions of multisubunit membrane proteins in
the plasma membrane require proper folding of the nascent
polypeptide chain and assembly with other subunits in the
endoplasmic reticulum (ER). The subunit assembly in the ER is,
in some cases, a prerequisite for the functional protein complex to
exit the ER. The proper assembly of the subunits allows the complex
to exit the ER and appear on the cell surface after a series of
modifications in the Golgi apparatus. The unassembled subunits
are retained in the ER and, in some cases, degraded via a quality
control system. This assembly-dependent expression mechanism
plays a critical role in controlling the quantity of the enzyme in the
plasma membrane.
There is an increasing number of reports on the structure and
function of the auxiliary subunits of membrane proteins, including
ion channels, receptors and ion-motive ATPases (Chow and Forte,
1995; Flucher et al., 2005; Geering, 2006; Hanlon and Wallace,
2002; Isom, 2001; Mayer, 2005). The structures of the auxiliary
subunits are extremely diverse. The β-subunits of the voltage-gated
Ca2+ channel (Catterall, 1995) and the K+v channel (Rettig et al.,
1994) do not carry any membrane-spanning domains and exist in
the cytoplasm, whereas the β-subunits of the voltage-gated Na+
channel and the KATP channel have 1 and 17 membrane-spanning
regions, respectively. The functions of the auxiliary subunits are
also diverse. The auxiliary subunit of the KATP channel (SUR) affects
the biophysical properties of the channel in many ways and is
regarded as an intricate component of the channel (Shi et al., 2005;
Teramoto, 2006). However, the auxiliary subunits of Na+/K+ATPase and Na+ and Ca2+ channels do not play a central role in
much faster than that of the α1-subunit. Ubiquitylation, as well
as endocytosis, was involved in the fast degradation of the β1subunit. Double knockdown of the β1- and β3-subunits by RNAi
resulted in the disappearance of these β-subunits but not the
α1-subunit in the plasma membrane. All these results indicate
that the α- and β-subunits of Na+/K+-ATPase are assembled in
the endoplasmic reticulum, but are disassembled in the plasma
membrane and undergo different degradation processes.
Supplementary material available online at
http://jcs.biologists.org/cgi/content/full/121/13/2159/DC1
Key words: Auxiliary subunit, Ion pump, Membrane protein, Subunit
assembly
the catalytic or ion-transport functions, but rather modulate the
expression and/or the activity of the catalytic subunits in the plasma
membrane (Chow and Forte, 1995; Hanlon and Wallace, 2002; Isom
et al., 1992; Isom et al., 1995; Perez-Reyes et al., 1992; Shi et al.,
1996).
Na+/K+-ATPase is an ion pump that translocates Na+ and K+ ions
against the electrochemical gradients across the plasma membrane
(Skou, 1957). The enzyme is composed of a large catalytic subunit
(α-subunit), a membrane-spanning auxiliary subunit (β-subunit) and
a small regulatory subunit (γ-subunit). The α-subunit contains the
binding sites for ATP, a specific inhibitor (ouabain) and ions
(Jorgensen and Andersen, 1988; Lingrel and Kuntzweiler, 1994;
Skou and Esmann, 1992). The β-subunit is a single-membranespanning polypeptide with a small cytoplasmic domain and a large
extracellular portion, usually containing three disulfide bridges and
several sugar modifications (Chow and Forte, 1995; Fambrough et
al., 1994; Okamura et al., 2003; Yu et al., 1994). In the current
model, the functional expression of the enzyme in the plasma
membrane requires assembly of the α- and β-subunits in the ER
(Fambrough et al., 1994; Geering, 1991; Geering et al., 1989). The
assembled enzyme can exit the ER, pass through the Golgi complex
and finally reach the plasma membrane. Thus, the β-subunit is
indispensable for the structural and functional maturation of the
enzyme, as well as its intracellular transport to the plasma
membrane.
Because the 1:1 assembly of the α- and β-subunits is necessary
for the functional expression of Na+/K+-ATPase in the plasma
membrane, it has been an a priori concept that the both subunits
remain as a 1:1 complex in the plasma membrane. In this report,
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Journal of Cell Science 121 (13)
however, we demonstrate, by utilizing a cell-surface protein
labeling method and an antibody-based protein quantifying
procedure, that the plasma membrane contains more α-subunits
than β-subunits.
Results
HeLa S3 cells express one catalytic subunit isoform and two
auxiliary subunit isoforms
Journal of Cell Science
Four isoforms of each α- and β-subunit of Na+/K+-ATPase (α1α4, official protein symbols ATP1A1-ATP1A4; β1-β4 or ATP1B1ATP1B4) have been identified in humans (Okamura et al., 2003).
RT-PCR using isoform-specific primers indicated that HeLa S3 cells
used in this study expressed only one isoform of the α-subunit (α1),
which is a ubiquitous α-isoform expressed in almost all tissues and
cell types (Fig. 1A). The α2-isoform (found in brain and skeletal
muscle), α3-isoform (found in brain) and α4-isoform (found in
testis) could not be detected in HeLa cells, consistent with a previous
report (Shamraj and Lingrel, 1994). However, two β-isoforms (β1
and β3) were found in HeLa cells, but the other two isoforms (β2,
found in brain and testis, and β4, found in skeletal muscle) could
not be detected (Fig. 1A).
Characterization of isoform-specific antibodies and
establishment of an immunoblot-based protein quantification
method
To examine the subunit stoichiometry in HeLa cells, an immunoblotbased subunit-quantification method was established by utilizing
isoform-specific antibodies. First, isoform-specific antibodies for
each isoform (α1, β1 and β3) were prepared and carefully
characterized (see Materials and Methods). The band patterns from
the anti-β1 and anti-β3 antibodies were apparently different,
indicating that they do not crossreact. Furthermore, these antibodies
detected both glycosylated and nonglycosylated forms of the βsubunits in the immunoblot analysis; treatment of the cell lysate
with N-glycosidase F completely removed sugar moieties and
produced a single band at ~32 kDa for β1 and ~28 kDa for β3. It
should be noted that the total immunoreactive signal was not
changed by the glycosidase treatment, indicating that
immunodetection by these antibodies was not affected by the sugar
modification of the β-subunits. An additional experiment examining
crossreactivity using HA-tagged β1- and β3-subunits and
immunoprecipitation, confirmed the specificity of these antibodies
(Fig. 1C).
As each antibody has a different sensitivity in the immunoblot
analysis, the intensity ratio of the immunoreactive bands does not
necessarily reflect the molecular ratio of the protein. Therefore, the
relative sensitivity of these isoform-specific antibodies was
examined before they were used in the quantitative analysis. In brief,
tandem HA-tagged α1-, β1- and β3-isoforms were expressed in
HeLa cells and immunopurified using anti-HA polyclonal antibody.
Each purified sample was then subjected to immunoblot analysis
using both anti-HA antibody and isoform-specific antibody (Fig.
1C). The immunoreactive signals on the immunoblot were carefully
quantified and the sensitivity ratios of the antibodies were obtained
by dividing the signal from the isoform-specific antibody by that
of the corresponding anti-HA antibody. Based on five
independent experiments, the sensitivity ratio obtained was
1.0:9.6(±0.7):7.2(±1.1) (mean ± s.d., n=5) for anti-α1: anti-β1:antiβ3 antibodies, respectively, in our experimental condition. The
sensitivity of the anti-α-subunit antibody was lower than that of
the anti-β-subunit antibodies because the anti-α1 antibody was
Fig. 1. Characterization of the cells and antibodies used in this study.
(A) Detection of isoform-specific mRNA in HeLa S3 cells by RT-PCR. The
total RNA isolated from HeLa S3 cells used in this study was subjected to
RT-PCR to examine the expression of four α-isoforms (α1-α4) and four βisoforms (β1-β4) of Na+/K+-ATPase. Primers used were designed to amplify
an approximately 300 bp DNA fragment of each isoform cDNA (for the
primer sequences, see supplementary material Table S1). The amplified
fragment was subcloned into the cloning vector pT7blue (Takara) and the
nucleotide sequences were confirmed by nucleotide sequencing. The
expression patterns of these isoforms in human whole brain, human skeletal
muscle and human testis were also examined. (B) Immunoblotting of HeLa S3
cell lysate using isoform-specific antibodies. The HeLa cell lysate was
subjected to SDS-PAGE analyses followed by immunoblotting using isoform
specific antibodies (anti-α1, -β1 and -β3 antibodies). For the analyses of the
N-linked glycosylation states, the lysate was pretreated with (+) or without (–)
N-glycosidase F before the SDS-PAGE analysis. The positions of the
molecular size markers (in kDa) are indicated on the right.
(C) Characterization of the sensitivities of the isoform-specific antibodies in
immunodetection. HAx2-tagged α1-, β1- and β3-subunits of human Na+/K+ATPase were transiently expressed in HeLa cells and immunoprecipitated with
anti-HA polyclonal antibody. These purified HA-tagged proteins were then
subjected to SDS-PAGE and immunoblot analyses using anti-α1, anti-β1 or
anti-β3 isoform-specific antibodies. The exposure time was adjusted to avoid
signal saturation. To normalize the number of protein molecules in each
sample, the same samples were subjected to immunoblot analyses using antiHA monoclonal antibody. The sensitivity ratio of the antibodies was obtained
by dividing the signal intensity from the specific antibody by that from the
anti-HA antibody (see Materials and Methods for the detailed procedure).
Because the anti-β3-antibody is a rabbit polyclonal antibody, the signal from
the immunoglobulin (rabbit anti-HA polyclonal antibody) used in the
immunoprecipitation was detected on the immunoblot slightly overlapping
with the bands from the β3-subunit. To obtain an accurate signal from the
β3-subunit, the immunoglobulin signal was obtained from the control
immunoprecipitation, in which the cell lysate was replaced with buffer and
was subtracted from the total signal (immunoglobulin + β3-subunit).
originally raised against the α-subunit of chicken Na+/K+-ATPase
(Takeyasu et al., 1988).
Subunit disassembly of sodium pump
2161
The sensitivity ratio of the antibodies obtained above was then
applied to examine the molecular ratio of α1-, β1- and β3subunits in HeLa cells. The total HeLa cell lysate was subjected
to immunoblot analysis using isoform-specific antibodies under
the same conditions as described for Fig. 1C (Fig. 2A). The
molecular ratio of these subunits was obtained by adjusting the
intensities of these immunoreactive signals with the sensitivity
ratio obtained in Fig. 1C. The careful quantification of
immunoreactive signals revealed that the molecular ratio of the
α1-, β1- and β3-subunits in the whole HeLa cell lysate was
1.0:0.21(±0.058):0.41(±0.065) (mean ± s.d., n=4), indicating that
HeLa cells express more of the α-subunit than the total amount
of the β-subunits.
Journal of Cell Science
HeLa cell plasma membrane shows unbalanced subunit
stoichiometry
To distinguish the cell-surface protein fraction from the intracellular
ones, the above-mentioned procedure was utilized with a membraneimpermeable biotinylation reagent to quantify the protein on the
cell surface (see supplementary material Fig. S1 for details of the
labeling method). HeLa cells were incubated with a membraneimpermeable biotinylation reagent, sulfo-NHS-SS-biotin, for 1
hour at 4°C. The membrane impermeability and cell-surface
specificity of this biotin labeling were confirmed by both western
blot analysis and immunofluorescence microscopy (see
supplementary material Fig. S1). Biotinylation of the cell surface
did not affect the affinity for ouabain, a specific inhibitor of Na+/K+ATPase, and the number of ouabain-binding sites on the cell surface
(data not shown). The biotinylated cell-surface proteins were
purified with streptavidin-agarose and subjected to western blot
analysis using isoform-specific antibodies. The efficiency of the
labeling and affinity purification was optimized so that more than
95% of the cell-surface proteins could be recovered (see
supplementary material Fig. S1).
The biotin-labeled cell-surface proteins were subjected to
immunoblot analysis by the same procedure described in Fig. 1C.
Quantification of the immunoreactive bands revealed that the
molecular ratio of α1-, β1- and β3-subunits in the plasma membrane
was measured as 1.0:0.030(±0.0021):0.22(±0.062) (mean ± s.d.,
n=4) (Fig. 2), indicating that the plasma membrane possesses an
extremely small number of the β1-subunit compared with the α1subunit. The amount of the β3-subunit was larger than that of the
β1-subunit but still much smaller than that of the α1-subunit,
demonstrating that the subunit stoichiometry of Na+/K+-ATPase in
the plasma membrane is not 1:1.
A summary of the quantitative analyses is shown in Fig. 2B.
The relative amounts of the α1-, β1- and β3-subunits in
intracellular compartments (ER, Golgi complex, and other
intracellular vesicles) were obtained by subtracting the cellsurface amount from the total amount of each subunit (see the
figure legend for the detailed procedure). When the total amount
of the α1-subunit was counted as 1, the molecular ratio of
α1:β1:β3 was 0.74:0.20:0.35 in the intracellular organelles and
0.26:0.01:0.06 in the plasma membrane. The amount of the
intracellular α-subunit (0.74) was slightly larger than that of the
total β-subunits (0.20 + 0.35), suggesting the existence of α1subunits free of the β-subunits in the ER. Approximately one
quarter (0.26) of the total α1-subunits exist in the plasma
membrane. This surface-intracellular ratio of the α1-subunit
varies among different cell types: ~50% in chick sensory neurons
(Tamkun and Fambrough, 1986), 40% in chick myogenic culture
Fig. 2. Quantification of the Na+/K+-ATPase subunits in the plasma
membrane and the whole cell fraction of HeLa cells. The amount of the α1-,
β1- and β3-subunits in the whole cell lysate and in the plasma membrane of
HeLa cells at 80% confluence was quantified by immunoblot analysis using
isoform-specific antibodies. The detailed procedures of cell-surface protein
labeling and purification are described in supplementary material Fig. S1.
(A) The total HeLa cell lysate (total) and the purified cell-surface proteins
(surface) were subjected to SDS-PAGE and immunoblot analysis using the
isoform-specific antibodies (α1, β1 and β3) described in Fig. 1C. Note that
the cell-surface fraction contains only matured forms of the β-subunits,
whereas the total cell lysate contains the molecules with various sugar
modifications. The positions of the molecular size markers (in kDa) are
indicated on the right. Three different amounts of the samples (1, 5 and 10 μl)
were subjected to the analyses to obtain a linear relationship between the
amount of protein and the intensity of the immunoreactive signal on the
immunoblot. The surface and total samples were blotted to the same
membrane filter and subjected to exactly the same procedure to avoid
experimental error. (B) A summary of the quantitative analyses. The
immunoreactive bands in A were quantified, avoiding signal saturation. To
compare the ratio of three subunits in each fraction, the band intensity was
multiplied by the mean value of the antibody ‘sensitivity ratio’ obtained in
Fig. 1C (1:9.6:7.2 for α1:β1:β3). To compare the surface and total samples,
the difference in the sample volume (50 μl surface and 500 μl total) was
considered. The intracellular fraction of each subunit was obtained by
subtracting the surface amount from the total amount. The surface and the
intracellular fractions of α1-, β1- and β3-subunits are represented with the
total amount of α1 as 1. The values in the figure were the mean values
obtained from 4-5 independent experiments.
(Wolitzky and Fambrough, 1986) and 50% in mouse L-cells (our
unpublished results). Although the α- and β-subunits are
assembled in the ER with a 1:1 ratio and then travel to the plasma
membrane, the result here demonstrated that the amount of the
β-subunits in the plasma membrane is much smaller (0.01+0.06)
than that of the α1-subunit (0.26).
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Journal of Cell Science
Different lifetimes of the α- and β-subunits in the plasma
membrane
The results of the quantitative analyses of the cell-surface proteins
suggest that the fates of the α- and β-subunits might be different
in the plasma membrane. To examine this possibility, the lifetime
of the α- and β-subunits in the plasma membrane were investigated
by pulse-chase experiments using cell-surface biotin labeling. HeLa
cells were pulse-labeled with sulfo-NHS-SS-biotin for 1 hour at
4°C. The labeled proteins were then chased for 1, 3 or 5 hours at
37°C in normal medium. At the end of the chase period, the cells
were either directly lysed or lysed after treatment with glutathione
(GSH), which would cleave off the biotin moieties only on the cell
surface (supplementary material Fig. S1). Subsequent affinity
purification with streptavidin-agarose and immunoblot analysis
characterized the stability of the cell-surface proteins.
The amount of the Na+/K+-ATPase α1-subunit was fairly
constant, and internalization of the subunit was not detected during
the first 5-hour chase period (Fig. 3). Thus, the α1-subunit, once
expressed on the cell surface, stably remains in the plasma
membrane at least for 5 hours and slowly undergoes internalization
and degradation processes, which is consistent with previous pulsechase experiments using [35S]Met which showed that the half-life
of the α-subunit in cultured neurons is about 40 hours (Tamkun
and Fambrough, 1986). When cells were chased in the presence of
1 μM ouabain, the α1-subunit was swiftly internalized within 1
hour and remained inside the cell for the next few hours without
degradation (data not shown). These data match well those of
previous studies in which ouabain treatment induces the
internalization of the enzyme (Cook et al., 1982; Griffiths et al.,
1983; Lamb and Ogden, 1982; Nunez-Duran et al., 1996; NunezDuran et al., 1988).
In contrast to the α1-subunit, the β1-subunit was swiftly
internalized and degraded within 5 hours (Fig. 3). After 5 hours,
the total amount of the labeled β1-subunit was reduced to about
50% (Fig. 3B). There was also accumulation of the labeled β1subunit in the intracellular compartment (Fig. 3, +GSH), indicating
that the β-subunit is internalized before being degraded. The
degradation of the β3-subunit was much slower than that of the β1subunit, but still faster than the α1-subunit; approximately 80% of
the β3-subunit remained on the cell surface after 5 hours (Fig. 3B).
These results suggest that the degradation of each type of subunit
is independently regulated in the plasma membrane.
The ubiquitin-proteasome pathway is involved in the quick
degradation of the β-subunit
To examine the degradation mechanism of the Na+/K+-ATPase
subunits, the biotin-labeled cell-surface proteins were chased in the
presence of various inhibitors. Treatment of HeLa cells with
chloroquine, an inhibitor of the endocytotic pathway, completely
abolished the internalization and the degradation of both β1- and
β3-subunits (Fig. 3B), indicating that endocytosis is indeed involved
in the degradation of the β-subunits. Interestingly, in the presence
of an inhibitor for proteasomal degradation (LLnL), the degradation
of the β1-subunit was significantly blocked (Fig. 3B). A similar
result was obtained with another proteasomal inhibitor, lactacystin
(data not shown). The effect of the proteasome inhibitors on the β3
degradation was relatively small compared with that on β1 (Fig.
3B).
Since the proteasome-dependent protein degradation requires
the ubiquitylation of the target protein, we examined whether the
β-subunits are ubiquitylated. All ubiquitylated proteins were
immunoprecipitated with anti-ubiquitin antibody and the
existence of the α- and β-subunits was detected by
immunoblotting. Under normal conditions, none of the subunit
types could be detected in the immunoblot. However, when the
cells were pretreated with the proteasome inhibitor lactacystin
for 5 hours and all of the immunoprecipitation procedures were
performed in the presence of the inhibitor, the anti-β1-antibody
detected a smear signal over 50 kDa, suggesting
polyubiquitylation on the β1-subunit (Fig. 4). However,
the ubiquitylation of the α- and β3-subunits was hardly
detected even in the presence of lactacystin, possibly
because the amounts of the internalized proteins were too
small. These results match the result in Fig. 3B well,
indicating that the ubiquitin-proteasome pathway is
involved in the fast degradation of the β1-subunit.
Fig. 3. The differential degradation of the Na+/K+-ATPase subunits
in the plasma membrane. HeLa cells at 80% confluence were pulselabeled with membrane-impermeable biotinylation reagent (sulfoNHS-SS-biotin) at 4°C for 1 hour, placed back in DMEM and
incubated at 37°C for 0, 1, 3 or 5 hours in the absence or presence
of inhibitors for degradation pathways. At the end of the chase
period, the cells were harvested before (–GSH) and after (+GSH)
treatment with glutathione to remove biotin moieties remaining on
the cell surface. The labeled proteins were purified with
streptavidin-agarose and analyzed by immunoblotting using
antibodies against α1-, β1- and β3-subunits. (A) A typical result of
the immunoblot analyses. (B) Quantitative analyses of the
immunoblot. The results from at least three independent
experiments are summarized.
Subunit disassembly of sodium pump
The α-subunit remains in the plasma membrane in the
β-subunit double-knockdown cells
Journal of Cell Science
The fast degradation of the β-subunits in the plasma membrane
suggests that a significant amount of the α-subunit in the plasma
membrane is not being assembled with the β-subunit. To examine
this possibility, RNA interference was employed to knock down
the β-subunits in HeLa cells. Immunoblot analysis demonstrated
that 24 hours after the introduction of siRNA (for β1 or β3), the
amount of the target protein in the plasma membrane was reduced
to less than 5% of that of the nontransfected cells (Fig. 5A). The
amount of each subunit (α1, β1 and β3) in the total cell lysate
fraction and in the plasma membrane fraction was examined by
immunoblot analysis.
When the β1-isoform was knocked down, the amount of the β3subunit in the plasma membrane was increased, whereas the total
amount of the β3-isoform in the cell remained constant (Fig. 5A,B).
Similarly, when the β3-isoform was knocked down, the amount of
the β1-isoform in the plasma membrane was increased without
changes in the total cellular amount of the β1-isoform. The amount
of the α-subunit in the whole cell and in the plasma membrane was
not changed (Fig. 5A,B).
Fig. 4. Ubiquitylation of the Na+/K+-ATPase subunits. The HeLa cells in
DMEM were incubated with or without the proteasome inhibitor lactacystin
for 5 hours. The ubiquitylated proteins were immunoprecipitated from the cell
lysate with anti-ubiquitin antibody (polyclonal antibody for α1, β1, ubiquitin
and IgG immunoblots and monoclonal antibody for β3 immunoblot) in the
presence of the inhibitor and then subjected to immunoblotting using the
antibodies indicated. Mouse IgG was used in the immunoblotting as a negative
control (IgG). The positions of the molecular size markers (in kDa) are
indicated on the right.
Fig. 5. The effect of specific β-isoform knockdown by RNAi on the amount of
other isoforms. siRNA for β1 or β3 or both was introduced into HeLa cells.
(A) After 24 hours, the cells were either directly harvested as the total cell
lysate or harvested after the surface labeling with sulfo-NHS-SS-biotin as
described in Fig. 2. The total cell lysate (whole cell) and the affinity-purified
biotinylated proteins (surface) were analyzed by immunoblotting using α1-,
β1- or β3-specific antibodies. A typical result of the immunoblot is shown.
The positions of the molecular size markers (in kDa) are indicated on the right.
(B) The band intensity in A was quantified and represented as the ratio to that
of the nontransfected cells. More than three independent experiments were
performed to obtain error bars (s.d). (C) The amount of Na+/K+-ATPase
subunits in the plasma membrane in β1/β3-double-knockdown cells. The
siRNAs for both β1- and β3-subunits were introduced into the HeLa cells.
After the indicated time, the cell surface proteins were labeled with biotin and
purified. These samples were subjected to immunoblot analysis as in A and the
amount of the α1-, β1- and β3-subunits were quantified and represented
relative to the amount at the beginning of the chase. The loading volumes were
adjusted based on the total protein amount in the sample. The results from
three independent experiments are summarized (mean ± s.d.).
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When both β1- and β3-isoforms were knocked down
simultaneously, the amount of the α-subunit in the whole cell and
in the plasma membrane decreased (Fig. 5A,B), confirming that at
least one β-isoform is necessary for the expression of the α-subunit
Journal of Cell Science
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Journal of Cell Science 121 (13)
in the plasma membrane. However, even when the amounts of both
β1- and β3-subunits were reduced to less than 5% of the
nontransfected cells (24 hours), a significant amount of the α1subunit (~80%) still remained in the plasma membrane. An
examination of this situation by following the time course after
transfection indicated that the amounts of β1- and β3-subunits in
the plasma membrane were swiftly reduced after transfection, and
reached the minimum level after 24 hours, whereas more than 80%
of the α1-subunit still remained in the plasma membrane after 24
hours (Fig. 5C). It should be noted that the degradation rate of the
α-subunit after the complete reduction of the β-subunits in the whole
cell fraction was similar to the normal degradation rate of the αsubunit examined by a pulse-chase experiment using [35S]Met (~30
hours, data not shown). These results indicate the existence of the
α-subunit in the plasma membrane that is not assembled with the
β-subunits.
To investigate whether disassembled α-subunits are functional,
the activity of Na+/K+-ATPase was estimated by the cell viability.
The β1 and β3 double-knockdown cells maintained normal viability
up to 32 hours, judged from a conventional cell proliferation assay
(data not shown). As a comparison, the treatment of HeLa cells
with 100 μM ouabain (KI of Na+/K+-ATPase is ~0.1 μM) started
to abolish the cell viability after several hours and completely killed
the cells after 24 hours. This result indicates that the disassembled
α-subunits in the plasma membrane have a pump function, at least
partially, if not completely.
The amount of β-subunits but not α-subunits in the plasma
membrane is affected by cell density
The existence of disassembled α-subunits in the plasma membrane
suggests a dynamic rearrangement of the subunit assembly state in
the plasma membrane and the possibility of the involvement of the
β-subunit in cellular processes other than the regulation of the pump
activity. Because previous studies demonstrated that the rat β2subunit is involved in cell-cell-adhesion in neuronal cells (Gloor et
al., 1990; Muller-Husmann et al., 1993; Schmalzing et al., 1992),
the possible involvement of the β-subunits in cell-cell adhesion is
also most likely in HeLa cells.
HeLa cells cultured to different confluencies (20, 40, 60, 80 and
100%) were labeled with sulfo-NHS-SS-biotin and the amount of
α1-, β1- and β3-subunits in the cell-surface fraction, as well as in
the total cell lysate, was quantified by immunoblot analysis as
described in Fig. 2 (Fig. 6). The amount of the α1-subunit both in
the total cell lysate and in the plasma membrane fraction was almost
constant at different cell densities. However, the amount of the cellsurface β-subunits (β1 and β3) increased as the cell confluency
became higher. This result suggests that the β-subunits, but not the
α-subunit, of Na+/K+-ATPase are likely to be involved in cell-cell
adhesion.
Discussion
In this study, we performed quantitative analyses of the turnover
of the Na+/K+-ATPase subunits in HeLa cells using subunit- and
isoform-specific antibodies combined with cell-surface protein
labeling. Our quantitative analyses on the cell-surface amounts of
the Na+/K+-ATPase subunits suggested that once expressed in the
plasma membrane, the α- and β-subunits show different behavior.
The examination of cell-surface proteins using a membraneimpermeable biotinylation reagent demonstrated that there is a large
excess of the α-subunit relative to the amount of β-subunits in the
plasma membrane. We also found that this unusual stoichiometry
Fig. 6. The number of β-subunits but not α-subunits in the plasma membrane
is dependent on the cell density. HeLa cells with different confluencies (20, 40,
60, 80 and 100%) were prepared. The amount of individual isoforms in the
whole cell lysate (A), as well as in the plasma membrane (B), were examined
by quantitative immunoblotting as described in Fig. 2. The loading volumes
were adjusted based on the total protein amount. The immunoreactive bands
were quantified and plotted as described in Fig. 2, with the amount of the α1subunit at 20% confluence adjusted to 1. Three independent experiments were
performed to obtain mean ± s.d. The immunoblotting results are also shown as
insets.
results from the faster degradation of the β-subunits and that the
α-subunit can exist in the plasma membrane without the β-subunit.
The different behavior of the catalytic and auxiliary subunits raises
several critical issues regarding the dynamics of the subunit
assembly and disassembly in the plasma membrane.
Disassembly of the auxiliary subunit from the catalytic subunit
occurs in the plasma membrane
The Na+/K+-ATPase β-subunit ensures the correct folding of the αsubunit upon assembly in the ER. Only the α/β-assembled enzyme
with the correct folding may exit the ER, follow a maturation process
in the Golgi complex and reach the plasma membrane (Beguin et
al., 1998; Fambrough et al., 1994; Geering, 1991). Previous
experiments using Xenopus oocytes have demonstrated that injection
of cRNA for the α-subunit does not increase the cell-surface amount
Journal of Cell Science
Subunit disassembly of sodium pump
of the enzyme, but the co-injection of β-subunit cRNA drastically
increases the amount of cell-surface enzyme (Geering et al., 1989;
Yoshimura et al., 1998; Zhao et al., 1997). Many types of cells
express excess α-subunits (as seen in Fig. 2) and the biosynthesis
of β-subunits is the rate-limiting step for the production of functional
α/β-complex (Takeyasu et al., 1989; Taormino and Fambrough,
1990). Our results from RNAi experiments (Fig. 5) also
demonstrated that the β1- and β3-isoforms are complementary in
delivering the α-subunit to the plasma membrane and that at least
one of the β-isoforms is required for the expression of the α-subunit
in the plasma membrane. Therefore, it was thought that the α/β
subunit stoichiometry in the plasma membrane would be 1:1.
However, our quantitative analysis using isoform-specific
antibodies and the cell-surface labeling technique demonstrated that
the amount of the α-subunit is larger than that of the β-subunits in
the plasma membrane (Fig. 2). Since there has been no evidence
of unassembled α-subunits being transported from the ER to the
plasma membrane in mammalian cells, this unusual stoichiometry
is simply due to the longer lifetime of the α-subunit in the plasma
membrane than the β-subunit (Fig. 3). Our RNAi experiment clearly
demonstrated that when almost all of the β-subunits (β1 and β3)
were knocked down, a significant amount of the α-subunit still
existed in the plasma membrane (Fig. 5), indicating that the αsubunit possesses a longer lifetime than the β-subunits, and that
even after disassembly from the β-subunit, it can exist in the plasma
membrane without the β-subunit.
The present result seems to be contrary to the previous notion
that the α:β ratio is 1:1 in the plasma membrane, even considering
possible oligomeric forms of the enzyme, such as α2β2 (Taniguchi
et al., 2001). However, most previous biochemical assays often used
crude membrane preparations that included not only the plasma
membrane fraction but also significant amounts of other intracellular
membrane organelles, especially ER and Golgi complex. In this
study, we clearly separated the cell-surface fraction from the
intracellular fraction and performed highly quantitative analyses for
each subunit and isoform. Since the cell-surface fraction of the
enzyme is much smaller than the intracellular fraction (Fig. 2), the
crude membrane fraction seems to primarily contain the intracellular
fraction of the enzyme.
The β-subunit-independent function of the α-subunit
Our results obtained from the RNAi experiment demonstrated that
a significant number of the α-subunits are separated from the βsubunits on the cell surface while maintaining significant enzymatic
functionality (Fig. 5). The functionality of the unassembled αsubunit was also demonstrated in previous studies. Blanco et al.
have demonstrated that the α-subunit may not necessarily require
the β-subunit for the basal ATPase activity (Blanco et al., 1994).
Furthermore, the amino acid sequence analysis of the Na+/K+ATPase catalytic subunits demonstrated that α-subunits lacking the
assembly domain with the β-subunit (Lemas et al., 1994; Lemas et
al., 1992) exist in some lower eukaryotes (Okamura et al., 2003).
Thus, it is possible that the free α-subunit in the plasma membrane
also has some pump activity. The reassembly of the α- and βsubunits (DeTomaso et al., 1994) may occasionally occur in the
plasma membrane. Because the different β-isoforms confer different
pump activities and ATPase activity to the enzymes (Blanco and
Mercer, 1998; Sweadner, 1989), this disassembly-reassembly of the
subunits may regulate the pump activity in the plasma membrane.
Na+/K+-ATPase reportedly functions as a signal transducer.
Na+/K+-ATPase has been shown to interact with multiple signaling
2165
proteins to transmit ouabain signal to various intracellular
compartments via multiple signaling pathways (Aydemir-Koksoy
et al., 2001; Kometiani et al., 1998). Binding of ouabain to Na+/K+ATPase induces the binding of the cytoplasmic tyrosine kinase Src,
resulting in the formation of an active complex that phosphorylates
other signaling molecules (Haas et al., 2002). Na+/K+-ATPase also
directly interacts with ankyrin and adductin, as well as proteins in
caveolae. Xie and colleagues have recently reported that cultured
cells contain a significant amount of non-pumping Na+/K+-ATPase
in the plasma membrane (Liang et al., 2007), indicating that there
are functionally different pools of Na+/K+-ATPase. These ‘nonpumping’ pumps may consist of some portion of the disassembled
α-subunits and be mainly involved in signal transduction.
The auxiliary subunit undergoes a different degradation
pathway from the catalytic subunit
The polyubiquitylation of the cytosolic proteins has been known
to function as a signal for proteasome-dependent protein degradation
(Laing et al., 1997; Levkowitz et al., 1998). The stability of the
cell-surface receptors and channels are also regulated by
ubiquitylation (Hicke, 1999; Kolling and Losko, 1997; Luscher and
Keller, 2001; Rotin et al., 2001; Ward et al., 1995). For the
membrane proteins, monoubiquitylation is implicated in the
regulation of the endocytic pathway (Aguilar and Wendland, 2003;
Haglund et al., 2003; Hicke, 2001; Rotin et al., 2000; Strous and
Govers, 1999). In the case of the β1-subunit of Na+/K+-ATPase in
HeLa cells, the polyubiquitylation seems to function as a signal for
proteasomal degradation (Fig. 4).
Interestingly, although degradation of the β1-subunit was
significantly blocked by LLnL, which is an inhibitor of proteasomal
degradation (Fig. 3B), both the endocytosis and the degradation of
the β-subunit were almost completely blocked by chloroquine,
which is an inhibitor of endocytosis (Fig. 3B). These results indicate
that endocytosis is a prerequisite for the ubiquitin-dependent
degradation of the β-subunit and that this rapid endocytosis is
independent of the α-subunit turnover. The entire life cycle of the
Na+/K+-ATPase subunits are depicted in Fig. 7.
Possible involvement of the β-subunit in cell-cell adhesion
In addition to the involvement in the intracellular transport of the
α-subunit and the regulation of the pump activity, the β-subunit has
been suggested to play a role in other cellular functions. The β2isoform of mouse Na+/K+-ATPase has been identified as a celladhesion molecule (Gloor et al., 1990; Muller-Husmann et al., 1993;
Schmalzing et al., 1992). Our results in Fig. 6 show that the amounts
of the cell surface β-subunits increase when the cell density
becomes higher, whereas the amount of the α-subunit does not
change significantly. These results indicate that the quantity of the
β-subunit in the plasma membrane is regulated depending on the
cell density, and suggest the general involvement of the β-subunits
in cell-cell adhesion.
The amino acid sequence analysis of the β-subunits and other
adhesion molecules also supports this notion. One of the structural
characteristics of the cell adhesion molecules (e.g. cadherin) is
the existence of at least one Ig-fold domains in the extracellular
region (Nollet et al., 2000; Takeichi, 1995). The β-subunit of the
Na+-channel has an Ig-fold domain in the extracellular region
and was demonstrated to be involved in cell-cell adhesion via a
direct interaction with the extracellular matrix (Srinivasan et al.,
1998; Undrovinas et al., 1995; Xiao et al., 1999). Recent
bioinformatic analysis and x-ray crystallography demonstrated
2166
Journal of Cell Science 121 (13)
Materials and Methods
Cell culture
HeLa S3 cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Sigma)
containing 10% fetal bovine serum (Hyclone) at 37°C with 5% CO2. All of the
experiments described in this paper were performed using the cells at 80% confluency,
unless otherwise indicated.
Antibodies
The antibody against the Na+/K+-ATPase α-subunit was previously raised against
chicken Na+/K+-ATPase α-subunit (α5) (Takeyasu et al., 1988). This monoclonal
antibody recognizes the human Na+/K+-ATPase α-subunit in western blot analysis.
The human β1-subunit-specific antibody (clone M17-P5-F11) was purchased from
Affinity Bioreagents. The epitope of this antibody (LETYP) exists only in the β1subunit and not in the β3-subunit of human Na+/K+-ATPase. The rabbit polyclonal
antibody specific to the β3-subunit was raised against the C-terminal region of the
β3-subunit (SQDDRDKFLGRVMFKITARA). The amino acid sequence of the
corresponding region of the β1-subunit is SEKDRFQGRFDVKIEVKS, which shows
less than 40% amino acid identity. A rabbit was immunized with the synthetic
polypeptide and the antibody was affinity-purified from the antiserum by protein-GSepharose beads. The final concentration of the anti-β3 antibody was 1 mg/ml. The
specificities of these antibodies were confirmed by immunoprecipitation and western
blot analysis. Anti-ubiquitin polyclonal antibody (rabbit) was a kind gift from Dr
Yokota (Yamanashi University, Japan) and anti-ubiquitin monoclonal antibody was
purchased from Nippon Bio-Test Laboratories. Anti-HA polyclonal (HA11) and
monoclonal (16B12) antibodies were purchased from Babco. The antibody against
lamin was a kind gift from Dr Yamaguchi (Nagoya University, Japan) and antiBiP/GRP78 antibody was purchased from Stressgen.
Journal of Cell Science
DNA constructs
Fig. 7. Model of the life cycle of the Na+/K+-ATPase subunits in HeLa cells.
The results obtained in this study, combined with those of other previous
studies, are summarized. The α- and β-subunits first co-translationally
assemble in the ER. The assembled enzymes leave the ER for the Golgi
network, where they are subjected to sugar modifications for maturation.
Once they reach the plasma membrane, the subunits dissociate and the
unassembled β-subunit undergoes ubiquitylation, endocytosis and fast
degradation, whereas the α-subunit remains in the plasma membrane and is
subjected to slow degradation. As a result, the plasma membrane contains
more α-subunit than β-subunit. Reassembly of the subunits may occur in the
plasma membrane, although it is not clear how this assembly-disassembly is
regulated.
that the Ig-fold structure can be found in various proteins (Halaby
et al., 1999; Pearl et al., 2005). Each Ig fold is composed of
sequential and antiparallel short β-sheets, some of which are
relatively hydrophobic (Halaby et al., 1999). Although the threedimensional structure of the Na+/K+-ATPase β-subunit has not
been elucidated, the prediction of the secondary structure from
the primary structure demonstrated that the human β-subunits
possess sequential short β-sheets in the extracellular region, some
of which are relatively hydrophobic (supplementary material Fig.
S2). The extracellular domain of the β-subunit may possess the
Ig-fold structure for cell-cell adhesion. The β-subunit can be
found only in multi-cellular eukaryotes (Okamura et al., 2003).
Furthermore, our BLAST and FASTA searches of unicellular
eukaryotes and prokaryotes whose genome projects have been
completed did not find any proteins homologous to the β-subunit
(our unpublished data).
In this report, we demonstrated that the stoichiometry of the
Na+/K+-ATPase α- and β-subunits is not necessarily 1:1 in HeLa
cells. Our results obtained here, together with the previous findings,
support the idea that the α/β complex is necessary for intracellular
transport, but becomes highly mobile once it reaches the plasma
membrane. The α- and β-subunits might independently be involved
in cellular functions other than ion pumping. The evolutionary
process of the P-type ATPases strongly supports this possibility.
cDNA fragments encoding the human Na+/K+-ATPase α1-, β1- and β3-subunits were
amplified by PCR from a cDNA pool of HeLa cells (Clontech) and subcloned into
pBluescript II SK– vector (Stratagene). The nucleotide sequences were confirmed by
a genetic analyzer ABI310 (Applied Biosystems). A tandem HA tag
(YPYDVPDYAAAYPYDVPDYA) was introduced at Ala14 in the α1-subunit and
Met1 in the β1- and β3-subunits. These recombinant cDNA fragments were subcloned
into the mammalian expression vector pCDNA3.1(+) (Invitrogen). The standard
lipofection procedure was used to introduce these expression vectors into HeLa cells
(Effectene, Qiagen).
RT-PCR
The total RNA was purified from HeLa cells with a commercial total RNA isolation
kit (Qiagen) following the manufacturer’s protocol. The first strand was synthesized
with reverse transcriptase (ReverTra-PlusTM, TOYOBO). The complementary strand
of the mRNA from human brain, skeletal muscle and testis were purchased from
Clontech (Quick-clone). Amplification by PCR was performed using LA-taq DNA
polymerase (Takara), single-stranded DNA template and isoform-specific primers (α1,
α2, α3, α4, β1, β2, β3 and β4 of human Na+/K+-ATPase; the nucleotide sequences
of these primers are listed in supplementary material Table S1), which amplify an
~300bp DNA fragment.
Determining the sensitivity ratio of the isoform-specific antibodies
Each HAx2-tagged human α1-, β1- and β3-subunit was transiently expressed in HeLa
cells in a 60 mm culture dish. The cells were lysed on ice with 500 μl RIPA buffer
(50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% Triton X-100, 0.5% sodium
deoxycholate and 0.1% SDS) containing protease inhibitors and the nucleus was then
removed by centrifugation (3300 g for 10 minutes at 4°C). The supernatant was
incubated with anti-HA polyclonal antibody (HA11, Babco) and protein-A-Sepharose
beads (25 μl bead volume) at 4°C for 4 hours. The beads were washed with RIPA
buffer and finally resuspended with 50 μl PBS. 5 μl of each bead suspension was
subjected to SDS-PAGE and subsequent immunoblot analysis using anti-HA
monoclonal antibody (16B12, Babco). The band intensity was carefully measured
with image analysis software (Scion Image) to obtain the intensity ratio
(α1:β1:β3=xα1:xβ1:xβ3). Different amounts of the sample were applied to the gel to
ensure that the amount of protein and the signal intensity were in a linear relationship.
Equal amounts of each immunoprecipitated protein were also subjected to immunoblot
analysis using isoform-specific antibody [anti-α1 (1:5000 dilution of ascitic fluid),
anti-β1 (1:20,000 dilution of ascitic fluid), or anti-β3 (1:5000 dilution from the purified
stock, 1 mg/ml)]. The band intensities were carefully measured as described above
to obtain the intensity ratio (α1:β1:β3=yα1:yβ1:yβ3). The sensitivity ratio of these
antibodies was obtained as anti-α1:anti-β1:anti-β3=(yα1/xα1):(yβ1/xβ1):(yβ3/xβ3). All
the quantitative immunoblot analyses using these specific antibodies described in
this study were performed with the same procedure and conditions described here.
Labeling of cell surface proteins by biotin
HeLa cells were cultured in a 60 mm culture dish. The cells were washed three
times with ice-cold PBS (10 minutes each) and incubated with 1% sulfo-NHS-SSbiotin or sulfo-NHS-LC-biotin (Pierce) in PBS(2+) (PBS containing 0.9 mM CaCl2
and 0.5 mM MgCl2) at 4°C for 30 minutes with gentle agitation. The biotinylation
Subunit disassembly of sodium pump
was terminated by the addition of PBS containing 50 mM glycine and 5 mg/ml
bovine serum albumin. After repeated washes with PBS, the cells were either
harvested or incubated in DMEM with 10% FBS for indicated periods in the presence
or absence of inhibitors (1 μM ouabain, 10 μg/ml LLnL, 100 μM chloroquine) at
37°C. After 1, 3 and 5 hours, cells were either directly harvested or treated with
glutathione (GSH) to strip biotins on the cell surface. For GSH stripping, the cells
were washed three times with PBS and subsequently incubated for 15 minutes on
ice with a solution containing 50 mM glutathione, 75 mM NaCl, 10% FBS and 75
mM NaOH.
The harvested cells were washed with PBS three times and resuspended in a buffer
containing 0.5% Triton X-100, 0.1% SDS and 0.1% deoxycholate (500 μl) and
incubated for 10 minutes at 4°C. The nucleus and unsolubilized cells were removed
by low-speed centrifugation (800 g, for 5 minutes). The supernatant was then incubated
with streptavidin-agarose beads (Pierce) (25 μl bead volume) for 4 hours at 4°C with
gentle agitation. The beads were washed with PBS containing 0.5% Triton X-100
four times and resuspended in SDS-PAGE sample buffer (total 50 μl). 5 μl of the
bead suspension was analyzed by SDS-PAGE and immunoblotting using the antibody
against the α1-, β1- or β3-subunit of Na+/K+-ATPase. The dilutions of the antibodies
were always constant in all experiments: anti-α1, 1:5000; anti-β1, 1:20,000; anti-β3,
1:5000. The bands were detected by an ECL® system (GE healthcare), captured as
a digital image and quantified by using image analysis software (Scion Image).
Journal of Cell Science
Immunoprecipitation of ubiquitylated proteins
HeLa cells were harvested and washed with PBS three times. The cells were lysed
with RIPA buffer containing protease inhibitors on ice for 10 minutes and the
solubilized fraction was collected by centrifugation (1000 g at 4°C for 5 minutes).
The lysate was pre-cleared with Protein-A-Sepharose beads (GE healthcare) for 4
hours at 4°C with rotation. The resin was removed by brief centrifugation and the
supernatant was then incubated with polyclonal antibody or monoclonal antibody
against ubiquitin together with Protein-A-Sepharose beads at 4°C for 2 hours with
rotation. The beads were washed with ice-cold RIPA buffer containing protease
inhibitors four times. The bound proteins were eluted with 1% SDS and analyzed by
SDS-PAGE and immunoblotting.
RNA interference
The siRNAs against human Na+/K+-ATPase β1- and β3-subunits were purchased
from Invitrogen (Stealth siRNA, the nucleotide sequence of these siRNAs is described
in supplementary material Table S2). Double-stranded siRNA was introduced into
HeLa cells by the standard lipofection method by following the manufacturer’s
protocol (Lipofectamine2000, Invitrogen).
This work was supported by a Grant-in-aid for the basic science
research (B) from the Japanese Ministry of Education, Culture, Sports,
Science and Technology. We thank Dr Ohniwa for the analysis of the
β-subunit structure and Dr Yokota for providing the polyclonal antibody
for ubiquitin.
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