From the
While most structural studies of the Na,K-ATPase support a
subunit stoichiometry of one
The Na,K-ATPase, or sodium pump, is a ubiquitous, multi-spanning
membrane protein that actively maintains a high internal K
Although
several lines of evidence seem to support an
Using the baculovirus expression system, we have
recently demonstrated the ability of the
For
immunoprecipitations, 150-200 µg of total protein from Sf-9 cells were solubilized in microcentrifuge tubes in 500
µl of 2% CHAPS in HBS or 0.1% SDS and 1% Triton X-100 in HBS for 15
min on ice, and the insoluble material was pelleted in a
microcentrifuge (10 min; 15,000
Recombinant baculoviruses containing the cDNAs coding for the
Na,K-ATPase
To
delineate the domains necessary for Na,K
Recently, a naturally
occurring truncated
In conclusion,
the data presented here imply that a peptide segment from Gly
While most structural studies of the Na,K-ATPase support an
The interaction between Na,K-ATPase
Preliminary reports of
cross-linking studies on the proteolytic fragments of the Na,K-ATPase
also indicate
To confirm the results of the cross-linking
studies in the baculovirus expression system, an
The
process of oligomerization occurs through the specific association of
complimentary surfaces from the individual subunits. Stabilization of
the oligomeric complexes typically involves both polar and nonpolar
interactions between exposed residues at the oligomerization interface;
with the charged and polar residues predicted to reside at the
periphery of the interface and hydrophobic residues positioned in the
central core(44) . Given the favorable free energy associated
with the removal of nonpolar residues from the aqueous environment to
form the hydrophobic interior of the subunit-subunit interface, one
would expect hydrophobic interactions to contribute significantly in
the oligomerization of multi-subunit water-soluble proteins. By
analogy, hydrophobic forces are also predicted to contribute
overwhelmingly to the self-association of extramembranous stretches of
intrinsic membrane proteins(45) . Interestingly, the
extra-bilayer region implicated in
The role of the Na,K-ATPase midregion
segment in mediating protein interactions does not appear to be
restricted to
The results presented here provide strong evidence that a
cytoplasmic region of the Na,K-ATPase
-subunit to one
-subunit, the
exact quaternary structure of the Na,K-ATPase and its relevance to
enzyme function is the subject of much debate. Formation of a higher
order enzyme complex is supported by our previous study demonstrating
specific
/
interactions among the rat Na,K-ATPase isoforms
(
1,
2,
3), expressed in virally infected Sf-9
insect cells and among native
isoforms in rat brain(1) .
This detergent-resistant association was not observed in insect cells
coexpressing the homologous gastric H,K-ATPase
-subunit, nor was
it dependent on the coexpression of the
-subunit. To delineate
domains necessary for
/
assembly, a series of
H,K-ATPase
Na,K-ATPase chimeras were constructed by combining the
N-terminal, cytoplasmic midregion and C-terminal segments derived from
the Na,K-ATPase(N) and the H,K-ATPase (H)
-polypeptides (HNN, HNH,
NHH, NHN, and HHN). The
-subunit chimeras were coexpressed with
the Na,K-ATPase
1-subunit in Sf-9 cells using the
baculovirus expression system. Specific and detergent-stable
association is observed between the Na,K-ATPase
-subunit and the
HNN and HNH chimeras, but not with the NHH, NHN, or HHN chimeras.
Consistent with the Na,K-ATPase cytoplasmic domain as being necessary
for
/
interactions, the full-length
-subunit stably
associates with an
N-terminal deletion mutant
(
Gly
-Leu
), but not with an
cytoplasmic deletion mutant
(
Arg
-Pro
). In addition, the
naturally occurring C-terminal truncated
1 isoform,
1T
(
Gly
to C terminus), does not associate with the
1-subunit in Sf-9 cells coexpressing both polypeptides.
Thus, a cytoplasmic region in the
-subunit
(Gly
-Pro
) is necessary for specific
/
association. The same cytoplasmic region contains a
strongly hydrophobic segment that, by analogy with oligomerization of
water-soluble proteins, may form the interface of the extramembranous
/
contact site.
concentration and a low internal Na
concentration characteristic of most eucaryotic cells. In a
chemomechanical process, the Na,K-ATPase couples the free energy from
the hydrolysis of ATP to the countertransport of sodium and potassium
ions against their respective electrochemical gradients. The
transmembrane ion gradients generated are, in turn, critical for a
number of fundamental cellular processes including regulation of cell
volume, nutrient uptake, and membrane excitability. Structurally, the
Na,K-ATPase is comprised of two, noncovalently linked subunits: a
110-kDa
-subunit and a smaller 40-60-kDa glycosylated
-subunit. All enzymatic functions of the enzyme have been assigned
to the
-subunit. The
-subunit contains the binding sites for
ATP and ouabain; it is phosphorylated by ATP and undergoes
ligand-dependent conformational changes accompanying the binding,
occlusion, and translocation of ions. The exact function of the
-subunit remains unknown; however, both the
- and
-subunits are required for functional expression of normal
Na,K-ATPase activity(2, 3) . More recently, Lutsenko et al. (4), have suggested that the extracellular domain of
the
-subunit is essential in stabilizing, or may take part in
forming, the cation occluding complex of the enzyme.
ratio of 1:1
for the functional enzyme unit(5, 6, 7) , there
is less agreement as to the higher order structure of the
membrane-bound Na,K-ATPase and the relevance of this structure to
enzyme function. Since a complete understanding of the transport
mechanism requires a detailed knowledge of the number of interacting
substrate sites, a large body of work over the last two decades has
attempted to establish the quaternary structure of the active enzyme
unit. Despite this intense focus, the question of whether the
membrane-embedded Na,K-ATPase functions as an
protomer, an
(
)
diprotomer of interacting
-subunits, or a
higher order oligomer remains unresolved. Early work examining the
two-dimensional crystalline structure of the Na,K-ATPase implicates the
protomeric
-unit as the minimum asymmetric unit of the
enzyme(8, 9) . Consistent with this model, solubilized
protomers are catalytically competent with enzyme kinetics
that approximate those of the membrane-bound enzyme(10, 11, 12, 13, 14, 15) and appear
to undergo all of the partial reactions associated with the transport
cycle(16) . In contrast, molecular weight determinations using
analytical ultracentrifugation(17) , low angle laser light
scattering (15, 18), or radiation inactivation(19) , and
evidence from cross-linking (20, 21) and ligand binding (22) studies support oligomeric models for the
Na,K-ATPase-mediated transport, with an (
)
diprotomeric structure being favored. However, in many cases the
existence of these oligomeric complexes could be due to nonspecific
collisions of the
protomers in the densely packed membrane
preparations and may not reflect specific, functional subunit
interactions.
-subunit isoforms to
stably associate into oligomers in virally infected insect
cells(1) . The specificity the of
/
interaction is
suggested by the inability of the Na,K-ATPase
-subunit to
associate with either the highly homologous gastric H,K-ATPase
-subunit or the truncated
1T isoform in cells expressing the
recombinant polypeptides. In the present work, to delineate the
domain(s) necessary for
/
assembly, we have coexpressed a
series of Na,K-H,K
-subunit chimeras and
-subunit deletion
mutants with the full-length
-subunit in Sf-9 insect
cells and assayed for detergent-resistant association. Here, we
identify a cytoplasmic region of the Na,K-ATPase
-subunit that is
necessary for stable and specific
/
association.
Mutagenesis and Cloning
The full-length cDNAs
for the -subunits from the rat Na,K-ATPase (
1) and the rabbit
gastric H,K-ATPase were cloned into the plasmid pGEM4 and used to
generate a series of
-subunit chimeras. Unique ClaI
restriction sites were introduced into the
-subunit cDNAs by
polymerase chain reaction. Restriction sites were positioned at the
junctions of the N-terminal:cytoplasmic regions (5` ClaI) and
cytoplasmic:C-terminal regions (3` ClaI) according to the
eight-transmembrane model for
-subunit
topology(23, 24) . The 5` ClaI restriction site
was engineered in the nucleic acid sequence encoding Lys
,
and the 3` ClaI site was positioned in the sequence encoding
Pro
. Polymerase chain reaction products were first
subcloned into the ddT-tailed EcoRV site of plasmid pBSSK (25) and subsequently joined via the ClaI restriction
sites to create 5` ClaI and 3` ClaI constructs with
flanking
-subunit sequences. The ClaI cassettes were then
subcloned back into context of the full-length
-subunit cDNAs in
pGEM4. Using this approach, full-length 5` ClaI, 3` ClaI, and 5`/3` ClaI constructs were created for both
-subunits. Distinct N-terminal, cytoplasmic, and C-terminal
restriction fragments were generated and interchanged between the
Na,K-ATPase and H,K-ATPase
-subunits to create the different
chimeras. As shown in Fig. 3, the chimeras are designated based
on the position of the topological regions: N for Na,K-ATPase derived,
H for H,K-ATPase derived. Polymerase chain reaction cassettes were
sequenced to verify that no other mutations were introduced during the
polymerase reaction. The
1 cytoplasmic deletion mutant
(
Gly
-Pro
) was created by excising the
cytoplasmic ClaI restriction fragment from the 5`/3` ClaI construct described above and religating the plasmid. For
the
1 N-terminal deletion mutant
(
Gly
-Leu
), site-directed mutagenesis
(Amersham Corp.) was performed to introduce a NcoI restriction
site at the position in the cDNA encoding Leu
. The mutant NcoI cassette was then subcloned back into the
1 pGEM4
construct in the NcoI recognition sequence that also encodes
the start methionine. DNA sequencing confirmed that the methionine
codon was unaltered and that no other mutations were introduced during
the mutagenesis procedure. Amino acids in the rat
1-subunit are
numbered beginning with the start methionine at position 1. Numbering
of residues from cited works is in accordance with the rat
1-subunit.
Figure 3:
Na,K-H,K -subunit chimeras.
Full-length
-subunit cDNAs from the rat Na,K-ATPase (
1) and
the rabbit H,K-ATPase were used to generate the series of chimeric
constructs depicted. Unique ClaI restriction sites were
generated at fusion junctions to effectively divide native
-subunits into distinct N-terminal, cytoplasmic, and C-terminal
regions. Positioning of fusion junctions is in accordance with the
eight-transmembrane model of
-subunit topology (23, 24). Chimeras
are designated based on the position of the structural region: N for Na,K-ATPase-derived, H for
H,K-ATPase-derived.
Cells and Viral Infections
Recombinant
baculoviruses coding for the rodent Na,K-ATPase 1- and
2-subunits,
1 deletion mutants, and Na,K-H,K
-subunit
chimeras were prepared as described previously(3, 26) .
The cDNA coding the
1T isoform (27) from rat aortic smooth
muscle was provided by Dr. Russell Medford (Emory University School of
Medicine). Recombinant baculovirus expressing the rabbit H,K-ATPase
-subunit was provided by Dr. George Sachs (University of
California, Los Angeles). Uninfected and infected Sf-9 cells
were grown in six-well culture plates in TNM:FH medium(3) ,
supplemented with 10% (v/v) fetal bovine serum, penicillin (100
units/ml), streptomycin (100 µg/ml), and Fungizone (0.25
µg/ml). Infections, using a viral multiplicity of infection ranging
from 5-10, were performed for 1 h at 27 °C. After 72 h, the
cells were lysed in 2% CHAPS
(
)in 150 mM NaCl, 25 mM HEPES, pH 7.4 (HBS), or in 0.1% SDS, 1%
Triton X-100 in HBS. Proteins from solubilized cells were
immunoprecipitated or immunoblotted as described below.
Immunoblots and Immunoprecipitations
Proteins were
separated by SDS-polyacrylamide gel electrophoresis (PAGE) (28) and transferred to nitrocellulose paper (Hybond C Plus,
Amersham Corp.). Membrane proteins from rat kidney were prepared as
described by Blanco and Beaugé(29) . Crude gastric mucosa
microsomes were prepared from the stomachs of New Zealand White rabbits
as described previously (30) but without additional microsomal
enrichment by density gradient centrifugation. Nitrocellulose blots
were blocked in Blotto (5% (w/v) nonfat dry milk, 0.1% sodium azide in
150 mM NaCl, 25 mM HEPES, pH 7.4) for 2 h at room
temperature or overnight at 4 °C. Primary antibody in 1% Blotto was
bound at 37 °C for 1-2 h on a rocking table. After two 10-min
washes in HBS and one 10-min wash in 1% Triton X-100 in HBS, 1-2
µCi of I-labeled goat anti-mouse or goat anti-rabbit
secondary antibody was added in 1% Blotto. The blots were incubated for
45-60 min at 37 °C and washed as above. After washing, the
blots were dried and exposed for autoradiography.
g). When in the buffer
containing CHAPS, the detergent was diluted to 1% by the addition of
HBS. To precipitate the
polypeptides, 50 µl of the indicated
-specific monoclonal antibody hybridoma supernatant and 70 µl
(1 mg/ml) of goat anti-mouse coated magnetic beads (BioMag, PerSeptive
Diagnostics Inc., Cambridge, MA) were added. After overnight incubation
on a rocking table at 4 °C, the magnetic beads were isolated by
holding the microcentrifuge tube to a magnet and aspirating the
supernatant. The beads were washed 3 times with 1 ml of 1% CHAPS in
HBS. When in buffer containing SDS, the beads were washed 3 times with
1 ml of 0.1% SDS and 1% Triton X-100 in HBS. The precipitated proteins
were eluted by resuspending the washed beads in sample buffer (100
mM Tris-HCl, pH 6.8, 2% SDS, 33% glycerol, 100 mM dithiothreitol) and incubating for 10 min at 65 °C. The eluted
protein was separated by SDS-PAGE (7.5% gel) transferred to
nitrocellulose, and immunoblotted with the indicated antibody. As a
control for the specificity of the immunoprecipitations, 100 µg of
protein from individually infected cells were combined in a
microcentrifuge tube, solubilized, and used for immunoprecipitations.
Unless otherwise indicated, immunoprecipitation results are from
proteins solubilized in 2% CHAPS.
Antibodies
The Na,K-ATPase 1-subunit was
identified with a monoclonal antibody (C464-6B) provided by Dr.
Michael Caplan (Yale University School of Medicine)(31) ; the
2-subunit was identified using the monoclonal antibody McB2
provided by Dr. Kathleen Sweadner (Massachusetts General
Hospital)(32) . The H,K-ATPase
-subunit was identified by
either a monoclonal antibody (12:18) provided by Dr. George Sachs
(University of California, Los Angeles, CA)(33) , or a
polyclonal antiserum (H,K-9) provided Dr. Michael Caplan(34) .
The 5
monoclonal antibody, which is specific for the Na,K-ATPase
-subunit, was provided by Dr. Doug Fambrough (Johns Hopkins
University)(35) . The anti-NASE antiserum, directed against the
1 cytoplasmic sequence
KNPNASEPKHLL
,
was provided by Dr Thomas Pressley (University of Texas Medical
School)(36) . The integrin-associated protein (IAP) was
identified with a monoclonal antibody provided by Dr. Eric Brown
(Washington University) (37). The
1 polypeptide was identified
using a polyclonal anti-
antiserum described
previously(3) .
1-subunit and highly homologous gastric H,K-ATPase
-subunit (
63% amino acid identity) were used to infect Sf-9 insect cells, a cell line derived from the ovary of the
fall armyworm, Spodoptera frugiperda. As shown in Fig. 1, Sf-9 cells express the corresponding
-subunit when infected. Insect cells infected with the Na,K-ATPase
recombinant baculovirus express high levels of
1 polypeptide,
which is recognized by the
1 monoclonal antibodies, C464 and
5
, but not by the H,K
-directed antibodies, H,K-9, and 12:18.
In Sf-9 cells infected with the H,K-ATPase recombinant
baculovirus, the expressed
-subunit is identified by the H,K
-specific antibodies, but shows no reactivity with the Na,K
1-specific antibodies. In addition, all of the
-specific
antibodies identify the respective
polypeptide in rat kidney
(
1) and rabbit mucosal (
1 and H,K
) membranes but show
no cross-reactivity with endogenous protein from uninfected Sf-9 cells. This is consistent with the low levels of
Na,K-ATPase activity and the absence of H,K-ATPase activity reported in
these cells(3, 38) .
Figure 1:
Specificity of -subunit
antibodies to the Na,K-ATPase and H,K-ATPase polypeptides. Membrane
proteins (15 µg) from rat kidney, stomach mucosa, uninfected and
1 or H,K
infected Sf-9 cells were separated by
SDS-PAGE (7.5% gel) and transferred to nitrocellulose. Na,K-ATPase
1 polypeptides were detected with the C464-6B and 5
monoclonal antibodies and H,K
polypeptides were detected with the
antibodies 12:18 and H,K-9.
We have previously demonstrated
a stable, detergent-resistant interaction among the baculovirus-induced
Na,K-ATPase isoforms (1,
2,
3) in Sf-9 cells
and among the native polypeptides in rat brain(1) . The highly
specific nature of the
/
interaction is suggested by the
inability of the
1-subunit to stably associate with the homologous
H,K-ATPase
-subunit in cells coexpressing both polypeptides. To
verify these previous findings, Sf-9 cells were infected with
the
1 virus in addition to the
2 or H,K
baculovirus.
After 72 h, the cells were solubilized in buffer containing 2% CHAPS or
0.1% SDS and 1% Triton X-100, and the proteins were subjected to
immunoprecipitation with an
1-specific monoclonal antibody. The
immunoprecipitates were electrophoresed by SDS-PAGE, transferred to
nitrocellulose, and probed with either the H,K
-specific
monoclonal antibody (12:18), or the
2-specific monoclonal antibody
(McB2). If the H,K
- or
2-subunit stably associates with the
1-subunit in coexpressing cells, then it should be identified in
the immunoprecipitated proteins. As shown in Fig. 2A,
when the
1-specific monoclonal antibody is used to
immunoprecipitate proteins from cells coexpressing the
1- and
2-subunits, the
2 polypeptide is coimmunoprecipitated.
However, when cells singly infected with either the
1 or
2
baculovirus are combined prior to solubilization, the
2
polypeptide is not identified in the immunoprecipitate. In contrast to
2-infected cells, when proteins from cells coexpressing the H,K
- and
1-subunits are immunoprecipitated with the
1-specific antibody, the H,K
polypeptide fails to
coimmunoprecipitate (Fig. 2B). The immunoreactive band
at the bottom of the gel represents the reduced form of the
immunoprecipitating antibody that reacts with the anti-mouse secondary
antibody. This lack of association is not due to differential levels of
1 protein expression since equivalent amounts of the
1
polypeptide are expressed in both
1/
2- and
1/H,K
-coinfected cells (Fig. 2C). Thus, the Na,K-ATPase
2-subunit, but not the H,K
-subunit, stably and specifically
associates with the
1-subunit when both polypeptides are expressed
together.
Figure 2:
Specific association of the Na,K-ATPase
1- and
2-subunit isoforms coexpressed in Sf -9
cells. Proteins from infected Sf-9 cells were
immunoprecipitated with the indicated antibody (IPAB), separated by SDS-PAGE, and transferred to
nitrocellulose. Coimmunoprecipitated subunits were identified on
immunoblots with the indicated antibody (IBAB). A, proteins from
1/
2-coinfected Sf-9 cells
and combined proteins from cells individually expressing
1 and
2 (
1 +
2) were solubilized in 0.1% SDS and
1% Triton X-100 in HBS. Solubilized proteins were immunoprecipitated
with an
1-specific monoclonal antibody (C464-6B) and
immunoblotted with the
2-specific antibody (McB2). The
2-subunit from coinfected Sf-9 cells (15 µg) is shown
as a standard. B, proteins from
1/H,K
-coinfected Sf-9 cells and combined proteins from cells individually
expressing
1 and H,K
(HK
+
1) were
immunoprecipitated with an
1-specific antibody (C464-6B) and
immunoblotted with the H,K
-specific antibody (12:18). The H,K
-subunit from coinfected Sf-9 cells (15 µg of
protein) is shown as a standard. The immunoreactive band at the bottom of the gel represents the reduced form of the
immunoprecipitating antibody that reacts with the anti-mouse secondary
antibody. C, proteins (20 µg) from
1/
2 and
1/H,K
-coinfected Sf-9 cells were immunoblotted with
an
1-specific antibody (C464-6B). The
1-subunit from
kidney membranes (10 µg) is shown as a standard. D, the
IAP does not selectively associate with the
1 or
2 subunit
isoforms in coexpressing Sf-9 cells. Infected Sf-9
cells were metabolically labeled with
[
S]methionine for 30 min, solubilized in 2%
CHAPS, and the protein was immunoprecipitated with the indicated
antibody (IPAB) and separated by SDS-PAGE. Proteins
from IAP/
2-coinfected Sf-9 cells and from cells
individually expressing IAP and
2 were immunoprecipitated with an
IAP-specific antiserum. Proteins from IAP/
1-coinfected Sf-9 cells and from cells individually expressing IAP and
1 were immunoprecipitated with an
1-specific antibody
(C464-6B).
As a consequence of the high levels of
baculovirus-directed protein expression, the possibility exists that
the subunit interactions in the infected Sf-9 cells may result
from the nonspecific, detergent-resistant aggregation or accidental
collision of the overexpressed recombinant polypeptides. In this
fashion, the association of the 1- and
2-subunits in
coexpressing cells may represent an artifact of the expression system
and not a specific assembly event. To address this possibility, a
recombinant baculovirus directing the expression of an unrelated
transmembrane protein, the murine IAP, was coinfected with the
1
baculovirus in Sf-9 insect cells. IAP is a 50-kDa
multi-spanning membrane protein that copurifies with the integrin
and has been implicated in the
regulation of ligand binding by the integrin receptor(37) .
After 72 h, cells were metabolically labeled with
[
S]methionine, solubilized, and cell proteins
were immunoprecipitated with an
1-specific monoclonal antibody
(C464). As shown in Fig. 2D, when the
1-subunit is
specifically immunoprecipitated from cells expressing both the IAP and
1 polypeptides, only the
1-subunit is immunoprecipitated. The
immunoprecipitated IAP from singly infected Sf-9 cells is
shown as a standard (Fig. 2D, lane3).
In addition, when IAP is coexpressed with the Na,K-ATPase
2
isoform and the labeled protein is immunoprecipitated with an anti-IAP
monoclonal antibody, no significant increase in the amount of
immunoprecipitated
2 protein is observed over background (Fig. 2D, compare lanes1 and 2). This is in contrast to our previous work demonstrating a
highly selective, detergent-stable association of the
2-subunit
with the
2 isoform in Sf-9 cells coexpressing both
subunits(39) . Thus, the association among the Na,K-ATPase
subunits in infected Sf-9 cells is specific and not a
consequence of the overexpression of the recombinant proteins.
/
assembly, a series
of five chimeras was constructed between the catalytic
-subunits
of the Na,K-ATPase
1 isoform and the homologous gastric
H,K-ATPase. Using the cDNAs, the segments corresponding to the
N-terminal transmembrane region, the cytoplasmic midregion, and the
C-terminal region were interchanged to create the fusion Na,K- and
H,K-ATPase constructs shown in Fig. 3. Positioning of the fusion
junctions is in accordance with the eight-transmembrane model for
-subunit topology(23) . The chimeras are designated based
on the position of the structural region: N for Na,K-ATPase derived, H
for H,K-ATPase derived. As we have previously demonstrated, all
chimeric
-subunits are stably expressed at high levels in the
membrane fraction of infected Sf-9 cells, and are structurally
competent, as judged by their ability to specifically and stably
associate with the coexpressed
-subunit(40) . As shown in Fig. 4A, when proteins from cells coexpressing the
1 and HNN polypeptides are immunoprecipitated with an N
terminus-directed
1-specific antibody (C464), the HNN fusion
protein is identified in the immunoprecipitate. Similarly, the
1-specific monoclonal antibody coimmunoprecipitates the HNH
subunit from cells coexpressing the
1 and HNH subunits (Fig. 4A). In both experiments, immunoprecipitated
proteins were identified with a H,K
-specific antiserum (H,K-9)
that recognizes a sequence from the extreme N terminus. In the control
lanes, proteins from cells individually expressing the corresponding
subunits were combined prior to immunoprecipitation.
Figure 4:
Association between the 1-subunit and
chimeric Na,K-H,K
-subunits coexpressed in Sf-9 cells. A, the Na,K-ATPase
1-subunit was coexpressed in Sf-9 cells with the Na,K-H,K
-subunit chimeras HNN or HNH
as indicated. Proteins were immunoprecipitated with an
1-specific
monoclonal antibody directed against the N-terminal domain
(C464-6B) and immunoblotted with an antiserum that specifically
recognizes amino acids Lys
-Ala
from the
N terminus of the H,K-ATPase
-subunit (H,K-9) (34). In lanes2 (HNN +
1) and 5 (HNH
+
1), proteins from cells individually infected with the
indicated recombinant viruses were mixed and immunoprecipitated. B, immunoblots of protein samples from coinfected cells in A probed with an
1-specific monoclonal antibody. The
Na,K-ATPase
1-subunit from rat kidney (10 µg of protein) is
shown as a standard.
In contrast to
the HNN and HNH fusion proteins, chimeric -subunits containing the
cytoplasmic midregion derived from the gastric H,K-ATPase (NHH, NHN,
HHN) do not selectively associate with the Na,K-ATPase
1
polypeptide in simultaneously infected insect cells. As shown in Fig. 5A, an
1 antibody (5
) that specifically
recognizes the cytoplasmic midregion of the Na,K
1 isoform does
not coimmunoprecipitate the NHH, NHN, or HHN polypeptides in
1/Na,K-H,K chimera coexpressing cells. This lack of association is
not due to differential levels of the
1 protein expression since
significant levels of the
1 polypeptide are expressed in
NHH/
1, NHN/
1, and HHN/
1 coinfected cells (Fig. 5B). Moreover, the absence of an observed
interaction cannot be attributed to the reduced reactivity of the
immunoprecipitating antibody, since approximately equal amounts of
1 protein from
1 infected cells is immunoprecipitated using
either the 5
antibody or the N-terminal-directed
1 antibody,
C464 ( Fig. 5C). Taken together, these results suggest
that a segment important for subunit interaction resides in the
cytoplasmic midregion portion of the
1 polypeptide
(Arg
-Pro
).
Figure 5:
Association between -subunits
requires the
1 cytoplasmic midregion. A, the Na,K-ATPase
1-subunit was coexpressed in Sf-9 cells with the Na,K-H,K
-chimeras NHH, NHN, or HHN as indicated. Proteins from infected
cells were immunoprecipitated with an
1-specific monoclonal
antibody (5
) that recognizes a region from Gly
to
Pro
in the cytoplasmic domain. Immunoprecipitated
proteins from NHH and NHN infected cells were probed with a H,K
-specific monoclonal antibody (12:18) that identifies a region in
the H,K
cytoplasmic midregion. Immunoprecipitated protein from
HHN-infected cells were probed with a H,K
-specific polyclonal
antiserum directed against the N terminus (H,K-9). In lanes2 (NHH+
1), 5 (NHN
+
1), and 8 (HHN +
1) proteins from
cells individually infected with the indicated recombinant viruses were
mixed and immunoprecipitated. Chimeras (15 µg of protein) from
coinfected cells are shown as standards. The lower immunoreactive bands
in the NHH and NHN immunoprecipitates represents the reduced form of
the immunoprecipitating antibody that reacts with the anti-mouse
secondary antibody. B, immunoblots of protein samples from
coinfected cells in A probed with an
1-specific
monoclonal antibody (5
or C464-6B). The Na,K-ATPase
1-subunit from rat kidney (15 µg of protein) is shown as a
standard. C, protein from
1-infected cells was
immunoprecipitated with the
1-specific antibody (5
or
C464-6B) and immunoblotted with the
1-specific antibody,
C464-6B.
It has previously been shown
that in the presence of the chemical cross-linker
copper-phenanthroline, Na-dependent phosphorylation of
the Na,K-ATPase induces cross-linking of two
-subunits through
disulfide bonds, and that this phenomenon is thought to be indicative
of the physical interaction between
-subunits of the
membrane-bound enzyme (41). More recently, Ganjeizadeh et al.(42, 43) have reported a phosphorylation-induced
cross-linking between the full-length
1 polypeptide and the 83-kDa
1 chymotryptic product (Ala
to C terminus)
in partially cleaved enzyme preparations. To confirm this interaction
in the baculovirus expression system, an
1 N-terminal deletion
mutant (
Gly
-Leu
), representing the
83-kDa chymotryptic fragment, was assayed for the ability to stably
associate with the full-length
1 protein in coinfected Sf-9 cells. As shown in Fig. 6A, when proteins
from insect cells coexpressing the
1 N-terminal deletion mutant
and the full-length
1 polypeptide are immunoprecipitated with an
antibody that specifically recognizes the extreme N terminus (C464),
the
1 N-terminal deletion mutant is coimmunoprecipitated. In
contrast, when proteins from cells expressing a single isoform are
mixed and subjected to immunoprecipitation, only the full-length
1
isoform is identified (Fig. 6A, lane2). These results agree with the aforementioned
cross-linking studies and are consistent with the notion that the
/
interaction involves contact between segments of the
-subunit that reside on the C-terminal side of Ala
,
possibly within the cytoplasmic midregion domain
(Arg
-Pro
).
Figure 6:
Association between -subunits
requires a region from Gly
to Pro
in the
cytoplasmic midregion. A, the full-length
1-subunit was
coexpressed in Sf-9 cells with an
1 N-terminal deletion
mutant (
Gly
-Leu
). Proteins were
immunoprecipitated with an N-terminal directed
1 antibody
(C464) and immunoblotted with an
1-specific polyclonal antiserum
(NASE) that recognizes a region from Lys
to Leu
in the cytoplasmic domain (36). In the control lane (
1
+
1(
Gly2-Leu273)), proteins from
cells individually infected with the
1 or
1(
Gly
-Leu
) recombinant virus
were mixed and immunoprecipitated. Protein (15 µg) from coinfected
cells is shown as a standard. B, the
1-subunit was
coexpressed in Sf-9 cells with an
1 cytoplasmic deletion
mutant (
Arg
-Pro
). Proteins were
immunoprecipitated with an
1 antibody (5
) that recognizes the
cytoplasmic midregion and immunoblotted with an
1 antibody (C464)
that recognizes the N-terminal region. In the control lane (
1
+
1(
Arg350-Pro785)),
combined proteins from cells individually infected with the
1 or
1(
Arg
-Pro
) recombinant
viruses were immunoprecipitated. Protein (15 µg) from
1/
1(
Arg
-Pro
)-coinfected
cells is shown as a standard. The immunoreactive band at the top of the gel represents the nonreduced form of the
immunoprecipitating antibody that reacts with the anti-mouse secondary
antibody. C, the
1-subunit was coexpressed in Sf-9 cells with the
1T isoform
(
Gly
-Tyr
). Proteins were
immunoprecipitated with an
1 antibody (5
) and immunoblotted
with the N-terminal directed antibody (C464). Protein (15 µg) from
1/
1T-coinfected cells is shown as a standard. D,
association between the
1 cytoplasmic deletion mutant and the
1 isoform coexpressed in Sf-9 cells. Protein from
1
(
Arg
-Pro
)/
1 coinfected
cells and combined proteins from cells individually expressing
1
(
Arg
-Pro
) or
1 (
1
+
1(
Arg
- Pro
)) were
immunoprecipitated with an
1-specific antibody (C464) and
immunoblotted with an anti-
1 antiserum (poly
A). For the
standards, protein (15 µg) from coexpressing cells was probed with
either an
1-specific or
1-specific antibody. The full-length
1-subunit from kidney (10 µg) is shown for
comparison.
To determine if the
-subunit interaction requires the cytoplasmic midregion or if the
C-terminal and N-terminal transmembrane segments are themselves
sufficient to promote
/
assembly, the ability of an
1
cytoplasmic deletion mutant (
Arg
-Pro
)
to selectively associate with the full-length
1 polypeptide was
determined. As shown in Fig. 6B, when the
1-subunit
from coinfected cells is specifically immunoprecipitated using an
antibody directed against the cytoplasmic midregion (5
), the
1 cytoplasmic deletion mutant is not coimmunoprecipitated with the
full-length
1 (this despite stoichiometric levels of
1 and
1(
Arg
-Pro
) expression (Fig. 6B, lane1)). Furthermore, the
1 deletion mutant appears to be structurally competent based on
its ability to selectively associate with the
1 isoform in
detergent extracts from coinfected cells (Fig. 6D).
Thus, our data are consistent with an obligatory role of the
cytoplasmic midregion in the assembly event and suggests that the
N-terminal and C-terminal transmembrane regions alone are incapable in
supporting stable
/
association.
isoform, designated
1T, was identified
as the predominant Na,K-ATPase isoform expressed in vascular carotid
and saphenous vein smooth muscle(27) . Analysis of the mRNA and
protein structure identified
1T as an RNA processing variant of
the
1 gene that encodes the first 554 amino acids of the
1
protein, terminating with a unique intron-encoded 27 residue peptide.
Previously, we have reported the inability of radiolabeled
1T
polypeptide to stably associate with the native
1-subunit in
metabolically labeled Sf-9 cells expressing both
polypeptides(1) . To confirm this finding using immunoblotting,
proteins from coinfected cells were immunoprecipitated with a
monoclonal antibody (5
) that selectively recognizes the
full-length
1 isoform, but not the truncated form(1) , and
immunoblotted with the N terminus-directed antibody, C464. As shown in Fig. 6C, when the
1-subunit is specifically
immunoprecipitated from cells expressing both the
1 and
1T
polypeptides, the
1T-subunit (
Gly
to C
terminus) fails to coimmunoprecipitate. Note that both isoforms are
expressed at approximately equal levels in the coinfected cells (Fig. 6C, lane1).
to Pro
in the cytoplasmic midregion is required for
Na,K-ATPase
/
interaction. Expression of additional Na,K-H,K
-subunit chimeras should further delineate the minimal segment
within this region that forms the
/
contact site and address
the potential importance of the
oligomerization on enzyme
function.
stoichiometry of 1:1 for the active enzyme, the exact
oligomeric structure of the Na,K-ATPase and its relevance to enzyme
function is still the subject of debate. Formation of a higher order
enzyme complex is supported by our previous study demonstrating
specific
/
interactions among the rat Na,K-ATPase
isoforms (
1,
2,
3) coexpressed in virally infected Sf-9 insect cells and among the functional
isoforms in
rat brain(1) . Here, we extend this work and identify a segment
in the cytoplasmic midregion that is necessary for detergent-stable
/
association.
-subunits is highly specific. When the Na,K-ATPase
1-subunit
is coexpressed with highly homologous
-subunit from the gastric
H,K-ATPase, the H,K
-subunit fails to coimmunoprecipitate with the
1 isoform (see Fig. 2). Expression of the Na,K
-subunit
with Na,K-H,K
-subunit chimeras demonstrates that the cytoplasmic
midregion portion of the Na,K-ATPase
1 enzyme confers the
specificity of
/
association. As shown in Fig. 4and 5,
the
1 polypeptide stably and selectively associates with the
Na,K-H,K chimeras containing the Na,K cytoplasmic midregion (HNN and
HNH), but not with Na,K-H,K chimeras containing the midregion derived
from the H,K-ATPase (NHH, NHN, HHN). Moreover, the inability of an
1 cytoplasmic deletion mutant
(
Arg
-Pro
) to associate with the
full-length
1 protein supports the conclusion that a region
necessary for
/
interaction resides in the cytoplasmic
midregion of the
1 polypeptide
(Arg
-Pro
). To further delineate the
cytoplasmic segment required for
/
interaction, the truncated
1T isoform (
Gly
to C terminus) was assayed for
its ability to stably associate with the
1 polypeptide. The
1T-subunit does not coimmunoprecipitate with the
1
polypeptide in coinfected cells (see Fig. 6). Taken together, the
data presented here demonstrate that
/
interaction requires
contact between segments of the
-subunit that reside between
residues Gly
and Pro
in the cytoplasmic
midregion. Currently, we cannot preclude the possible involvement of
additional peptide segments outside the cytoplasmic midregion in
formation of
-subunit oligomers. Although, as shown in Fig. 6B, the inability of an
cytoplasmic deletion
mutant (
Arg
-Pro
) to associate
with the full-length
-subunit strongly suggests that the
N-terminal and C-terminal transmembrane segments alone are insufficient
to promote stable
/
assembly.
/
interaction in the C-terminal
region(42) . Chymotryptic cleavage of the Na,K-ATPase in the
presence of Na
yields an 83-kDa peptide that begins at
Ala
and extends to the C terminus. When partially
proteolyzed Na,K-ATPase is phosphorylated by ATP in the presence of
Na
and exposed to the sulfhydryl cross-linker, the
induced cross-linked products are the
/
homodimer, the
/
83-kDa heterodimer, and the
83-kDa/
83-kDa
homodimer. Phosphorylation-induced cross-linking is also observed in
partially trypsinized enzyme preparations between the full-length
-subunit and the 63-kDa tryptic peptide (Ala
to C
terminus). Under these conditions, dimerization is not observed with
the smaller 48-kDa tryptic fragment (N terminus to Arg
).
These findings seemingly restrict the
/
contact site(s) to a
region C-terminal of alanine 446 and are consistent with our data,
implicating a cytoplasmic midregion segment
(Gly
-Pro
) as necessary for
/
association.
N-terminal
deletion mutant (
Gly
-Leu
),
representing the 83-kDa chymotryptic fragment, was assayed for its
ability to stably associate with the full-length
polypeptide. The
N-terminal truncated protein is also capable of forming a
detergent-resistant complex with the
-subunit in Sf-9
cells coexpressing both polypeptides (see Fig. 6A).
Interestingly, since singly infected Sf-9 cells expressing the
-subunit alone do not exhibit either ouabain-sensitive Na,K-ATPase
activity or sodium-dependent phosphorylation from ATP(3) , the
immunoprecipitation data suggest that
/
association can occur
in the absence of enzyme turnover. This is in contrast with chemical
cross-linking studies that demonstrate phosphorylation-induced
cross-linking between
-subunits from catalytically active
molecules(41) . Thus, coexpression of the
-subunit and formation of a functional enzyme is not a
prerequisite for
/
oligomerization in these cells.
/
oligomerization contains
a 44-amino acid segment with strong hydrophobic properties
(Val
-Pro
). This segment was initially
identified as an extramembranous peptide from trypsinized enzyme
preparations labeled with the lipophilic reagents adamantane diazirine (46) and
3-(trifluoromethyl)-3-(m-[
I]iodophenyl)diazarine
(47). Subsequently, it was shown to be part of a stilbene-labeled
proteolytic fragment that exhibits strong hydrophobic characteristics
in high performance liquid chromatography(48) . As this
hydrophobic segment is missing in both the
1T isoform
(
Gly
to C terminus) and the
cytoplasmic
deletion mutant (
Arg
-Pro
), and
since neither truncated protein associates with the full-length
-subunit, it is intriguing to speculate that this extramembranous
region may represent or be part of the
/
contact interface.
Additional
-subunit mutants are currently being expressed to
address this possibility.
/
assembly. It has recently been shown that two
of the five putative cytoplasmic regions of the Na,K-ATPase
-subunit, including the cytoplasmic midregion domain
(Arg
-Pro
), are capable of direct and
specific binding with both erythrocyte ankyrin (ANK1) and its
immunologic counterpart in kidney (ANK3)(49) . Of these two
sites, the cytoplasmic domain from cysteine 144 to isoleucine 294
accounts for most of the overall affinity of the Na,K-ATPase for
cytoskeletal ankyrin, while the second site in the cytoplasmic
midregion is predicted to modulate ankyrin binding through interplay
with the primary binding site. Future studies should reveal whether the
same or similar sequence motifs within the cytoplasmic midregion are
utilized for both the processes of
/
association and ankyrin
binding.
1-subunit is necessary for
association of
-subunits into stable oligomers. Although the
present study does not clarify the functional role of
/
associations, it further establishes their highly specific nature and
suggests that the oligomerization of the
protomer may be
important in the physiological regulation of the enzyme.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.