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Oligomerization of the Na,K-ATPase in Cell Membranes.

Melissa Laughery, Matthew Todd and Jack H. Kaplan

Department of Biochemistry and Molecular Genetics

University of Illinois at Chicago

Molecular Biology Research Building

900 S. Ashland Avenue

Chicago, IL 60607-7170

Short title: Na,K-ATPase αβ heterodimer oligomerization

Key words: Na,K-ATPase, oligomerization, trafficking, assembly

This work was supported by NIH grants GM39500 and HL30315 to JHK.

JBC Papers in Press. Published on June 18, 2004 as Manuscript M402778200

Copyright 2004 by The American Society for Biochemistry and Molecular Biology, Inc.

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Abstract

The higher order oligomeric state of the Na,K-ATPase αβ heterodimer in cell membranes is the

subject of controversy. We have utilized the baculovirus-infected insect cell system to express

Na,K-ATPase with α-subunits bearing either (His)6 or FLAG epitopes at the carboxyl terminus.

Each of these constructs produced functional Na,K-ATPase αβ heterodimers that were delivered

to the PM. Cells were simultaneously co-infected with viruses encoding α−His,β and

α−FLAG,β Na,K-ATPases. Co-immunoprecipitation of the His tagged α−subunit in the ER and

PM fractions of co-infected cells by the anti-FLAG antibody demonstrates that protein-protein

associations exist between these heterodimers. This suggests the Na,K-ATPase is present in cell

membranes in an oligomeric state of at least (αβ)2 composition. Deletion of 256 amino acid

residues from the central cytoplasmic loop of the α-subunit results in the deletant α−4,5LL, that

associates with β but is confined to the ER. Co-immunoprecipitation demonstrates that when

this inactive α−4,5LLβ heterodimer is co-expressed with wild type αβ, oligomers of wild-type

αβ and α−4,5LLβ form in the ER, but the α−4,5LL mutant remains retained in the ER and the

wild-type protein is still delivered to the PM. We conclude that the Na,K-ATPase is present as

oligomers of the monomeric αβ heterodimer in native cell membranes.

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Introduction

The Na,K-ATPase is the primary active transport system responsible for maintaining Na

and K ion concentrations in almost all animal cells. It utilizes the energy of hydrolysis of the

terminal phosphate bond of ATP to accomplish the coupled transport of Na and K ions against

steep electrochemical gradients (1). This enzyme, along with its close mammalian homolog, the

gastric H,K-ATPase is unique among P2-type ATPases because it requires a β-subunit in

addition to the catalytic α−subunit. The α-subunit is composed of about 1000 amino acids with

ten transmembrane segments and encodes all of the essential domains for ATP hydrolysis and

ion transport. The β-subunit is a glycoprotein of around 300 amino acids with a single

transmembrane segment and an intracellular amino-terminus. Recent data from a number of

systems has established that the β-subunit probably plays an influential role in the catalytic

activity of the system (2,3) in addition to its essential role in the delivery of the heterodimers to

the plasma membrane (PM), from the site of synthesis in the ER (4-6). The subunit assembly

takes place in the ER and assembled heterodimers are delivered to the PM (for review, see ref.

6).

Although the heterodimeric state of the Na,K-ATPase is well established, the organization

and possible higher order oligomerization of heterodimers in native membranes remains a

controversial subject (1,7,8). It has been claimed that the Na,K-ATPase αβ heterodimer in

native membranes exists as an oligomer of multiple heterodimer units (7,9,10). The existence of

these oligomers has been postulated to account for kinetic observations that are difficult to

explain with a simple monomeric unit (11-13). Evidence for the existence of such multimers, or

for associations among monomers has derived from cross-linking experiments (10,14-16), single

particle images (17), thermal inactivation studies (18), and association among different isoforms

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(9,19,20). Other recently published work demonstrates that the major cytoplasmic loop between

the M4 and M5 membrane segments associates in an ATP-dependent way when expressed in

isolation (21). Conversely, it has been claimed that the monomeric solubilized enzyme is

capable of normal enzymatic activity (22). Furthermore, cross-linking between α-subunits was

not observed in cell membranes containing a low density of Na,K-ATPase molecules (23), or

after detergent solubilization (24), leading to the suggestion that higher-order associations may

only be a result of unusually high Na,K-ATPase densities in some enriched preparations.

In the present work, we have utilized the expression of recombinant Na,K-ATPase

molecules in baculovirus-infected insect cells, where viruses contain the cDNA that encodes for

α-subunits bearing either (His)6 or FLAG-epitopes. Expressed in this system, the level of Na,K-

ATPase has been estimated to reach up to 1-2% of the total membrane protein (5). We show that

each of these epitope tagged α−subunits expressed with wild-type β-subunits results in the

synthesis and delivery of functional Na,K-ATPase αβ heterodimers to the PM of the insect cells.

We show that co-infection of cells with both viruses and subsequent immunoprecipitation of

either ER or PM fractions from infected insect cells by an anti-FLAG antibody results in co-

immunoprecipitation of the α-subunit bearing the (His)6 epitope. We also show that co-

expression of wild-type Na,K-ATPase heterodimers with non-functional heterodimers containing

a deletant α−subunit, lacking most of the nucleotide binding and phosphorylation domains,

produces oligomeric molecules. These data provide strong evidence for association of native αβ

heterodimers in cell membranes.

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Experimental Procedures

Plasmids and Construction of Mutants. The mutants were constructed by overlapping

PCR extension mutagenesis using sheep α1 cDNA as a template. The (His)6 (HHHHHH) or

FLAG (MDYKDDDD) epitope tag was inserted on the carboxyl-terminal of the α1-subunit to

create α−His and α−FLAG respectively. The α−4,5-loop-less (α−4,5LL) mutant is a deletion

(ΔAsp443-Val707) of the sheep α1 M4M5 loop. As a result of mutagenesis, a Glu residue was

inserted at the junction of the N and C-terminal regions such that the M4M5 loop in the α−4,5LL

sequence is M4…G442ET708…M5.

Mutated α1 cDNA was sub-cloned in the pOCUS-2 vector (Novagen) and subsequently

ligated into the pFBD (Invitrogen) vector in the multicloning site (MCS) 2. The wild-type sheep

β1−subunit cDNA was placed at MCS1 in the same pFBD. The Bac to Bac system (Invitrogen)

was used create recombinant baculovirus particles each of which contain cDNA for α and

β−subunits under separate promoters (5). Mutations in the α1-subunits were confirmed by

sequencing of DNA isolated from recombinant baculovirus using Easy-DNA Kit (Invitrogen).

Protein expression of the α−4,5LL mutant was also confirmed by its mobility in SDS PAGE

visualized via Western Blot analysis using the anti-KETYY Antibody.

Protein Expression and Isolation. Log-phase high viability (≥98% viability, determined

by trypan blue exclusion) High Five cells were infected with recombinant baculovirus in the

presence of 1% ethanol (v/v). The progress of each infection was monitored by recording the

cell density and viability. Infections were harvested by centrifugation (500 X g, 10 min.)

between 3 to 5 days post viral infection, when cells retained 60-75% viability. The harvested

High Five cells were homogenized, whole cells removed, and lysate was either ultracentrifuged

to collect total membranes, or separated on a five-step sucrose gradient into fractions enriched in

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ER, Golgi apparatus (G), or PM as previously described (5). Membrane pellets were

resuspended in HB (250 mM sucrose, 2 mM EDTA, 10 mM Tris, pH 7.4) and stored on ice or at

–20°C for long-term storage. Protein concentration was determined by the Lowry assay using

bovine serum albumin as a standard (25).

ATPase Activity. ATPase activity assay of isolated membranes was performed in

triplicate for 30 min at 37°C with 16 µg of membrane protein as previously described (25).

Na,K-ATPase activity was determined as the difference in the amount of phosphate liberated in

the presence and absence of 0.3 mM ouabain, expressed as µMol Pi per mg protein per h.

Western Blot Analysis. Equal protein concentrations of ER, Golgi and PM-enriched

fractions were subjected to SDS PAGE in 7.5 or 10% acrylamide/bis gels in the presence of

0.75% β-ME and transferred to nitrocellulose membranes. Western blots were blocked and

probed with anti-α1, anti-β1, anti-FLAG, anti-KETTY, anti-(His)6-HRP, or anti-hCTR1

antibody as previously described (4). Goat anti-mouse HRP (Pierce #31430) or goat anti-rabbit

HRP (Pierce #31460) secondary antibodies were used when appropriate. Chemilumenscent

reagents (Pierce) were used for signal detection.

Antibodies. The anti-KETYY antibody, which was raised in rabbit against the sheep α1

C-terminal amino acid sequence 1111KETYY1116, (gift from Dr. Jack Kyte, University of

California, San Diego) was used for both Western blot and co-immunoprecipitation. Likewise,

the anti-α1 antibody (Affinity Bioreagents #MA3-929), which recognizes an epitope in the

region of amino acid residues 496-506 in the M4M5 loop of the α1-subunit was used for both

Western blot and co-immunoprecipitation experiments. The anti-β1 antibody (Affinity

Bioreagents #MA3-930) was used to detect the β1-subunit on Western blots. The anti-FLAG

antibody (Sigma #F7425) was used for both Western and co-immunoprecipitation. The anti-

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(His)6 antibody (Affinity Bioreagents #PA1-983) was used for co-immunoprecipitation and anti-

(His) 6-HRP conjugate antibody (Qiagen #34460) was used for Western blot detection of the

(His)6 tag. A rabbit polyclonal antibody raised against an epitope in the C-terminal region of

hCTR1 was used for Western blot detection of hCTR1.

Protein Solubilization. Membrane preparations (500 to 1000 µg) were pelleted in a TLA

45 rotor at 45,000 rpm for 30 min. Pellets were resuspended in 200 µL immunoprecipitation

buffer (IPB) (150 mM NaCl, 10 mM KCl, 2.5 mM MgCl, 25 mM Hepes, pH 7.4, 1 mg/mL

leupeptin, 2 mg/mL antipain, 1 mg/mL pepstatin, 100 mg/mL TPCK, and 100 mg/mL PMSF)

supplemented with detergent. For subsequent immunoprecipitation of C-terminal

tagged α−subunits, 2% n-Dodecyl-β-D-maltoside (DDM) was used as the solubilizing detergent,

as previously described (4). Solubilization of the α−4,5LL construct required 1% Anagrade Fos-

Choline-12 (Anatrace #F308). Initial suspensions were passed through a 28 Ga. X 0.5 inch

needle until homogenous and incubated at room temperature (∼25°C) for 30 min. Suspensions

were centrifuged at 14,000 x g for 5 min to remove insoluble material. In the absence of

detergent, no α− or β−subunit is detected by Western blot in the supernatant after this

centrifugation step. In the presence of detergent, a significant portion of each subunit is

detectable in the supernatant. After solubilization, the supernatant was removed and an aliquot

(1 to 5%) used for Western blot analysis (S sample). Remaining soluble material (supernatant)

was used for immunoprecipitation. The insoluble pellet was resuspended in 200 µL IPB using a

28 Ga. X 0.5 inch needle until homogenized and an equal volume (1 to 5%) subjected to Western

blot analysis (IS sample).

Co-immunoprecipitations. Supernatant from solubilization (~200 µL: see above) was

transferred to a fresh tube containing IPB and antibody (anti-FLAG, anti-(His)6, anti-α1 or anti-

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KETYY) for a total volume of 1 mL. Two sets of controls were done in parallel. Only soluble

protein (no antibody) was added to one control and only antibody (no protein) was included in

the other control. The samples were incubated at 4°C with end-over-end rotation overnight.

Protein-G-Sepharose beads (50 to 100 µl at 1:1 IPB) were added to all samples and incubated at

4°C with rotation for 4 h. Beads were pelleted at 500 X g for 5 min and supernatant removed by

aspiration. The beads were washed 3 to 5 times for 5 min with rotation at 4°C in 1 mL IPB

followed by 500 X g for 5 min and aspiration of supernatant. Precipitated protein was removed

from the beads by incubation for 30 min to overnight at room temperature with 30µL 4X SDS

sample buffer (100 mM Tris-Cl, pH 6.8, 10% glycerol, 8% SDS, 0.75% β-ME, 0.0001%

bromophenol blue). Beads were removed by centrifugation at 21,000 X g for 5 min, then the

supernatant was resolved on a 7.5% SDS-PAGE and Western blot analysis performed.

Trypsin Digestion. Membrane preparations in HB (see above) were supplemented with

20mM NaCl, 20mM KCl, 2mM ATP-Tris, or no ligand. Volumes were adjusted such that the

final protein concentration was 4mg/mL after addition of trypsin at 1mg/mL. Digestion was

stopped by the addition of denaturing sample buffer after 2 hr at room temperature and

proteolyzed fragments analyzed by Western blot analysis.

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Results

In our initial experiment, High Five cells were infected with recombinant baculovirus

encoding the wild-type sheep β1-subunit and sheep α1-subunit bearing either the (His)6 or

FLAG epitope tag at the carboxyl terminus (α−His,β or α−FLAG,β). Cells were harvested,

Dounce homogenized, and ER, G, and PM enriched fractions separated on a 5-step sucrose

gradient, as previously described (5) Western blot analysis of the membrane fractions by the

anti-α1 antibody confirms that, like the wild-type α−subunit, both tagged α-subunit constructs

are delivered to the PM (Fig 1A, top panel). Only the appropriately tagged constructs are

detectable with the anti-FLAG and anti-(His)6–HRP antibodies and there is no cross-reactivity

(Fig 1A, bottom panels). Fig 1A also demonstrates the expression and trafficking of the

β−subunit. Previous work in the baculovirus expression system has established that delivery of

the α−subunit to the PM is dependent upon the heterodimeric assembly with the β−subunit (4,5).

The appearance of the α−subunit in the PM fraction (Fig 1A) demonstrates that αβ heterodimers

containing these tagged α−subunits are sorted like wild-type protein and delivered to the PM of

the cells. Fig 1B shows the ouabain-sensitive ATPase activities in each of the membrane

fractions of the tagged α−subunit construct expressions. These ATPase activities are

comparable to the ATPase activities of wild-type protein with equivalent protein expression

levels (data not shown). Likewise, the distribution of the Na,K-ATPase activity between the

membrane fractions is the same as we have previously reported with the wild-type protein (4), in

which the PM fraction contains the greater proportion of Na,K-ATPase activity. The data

presented in Fig 1 establish that attachment of the epitopes to the carboxyl terminus of the α-

subunit has no detrimental effect on the normal αβ heterodimer synthesis, assembly, delivery to

the PM, or activity of the Na,K-ATPase.

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Next, High Five cells were simultaneously infected with the α−FLAG,β and α−His,β

baculoviruses. Each virus was at a concentration one order of magnitude higher than the cell

density of the suspension. This level of each virus is sufficient to independently infect the entire

population of cells. Co-infection for the purpose of simultaneously expressing Na,K-ATPase

subunits in insect cells has been utilized previously (26). The top two panels of Fig 2A show

Western blot reactivity by both the anti-(His)6–HRP and anti-FLAG antibodies, confirming that

both tagged α−subunits are present in the indicated membrane fractions. Likewise, the

β−subunit is present in all fractions (Fig 2B bottom panel). Co-infection with the α−FLAG,β

and α−His,β baculoviruses does not alter the level or cellular distribution of Na,K-ATPase

activity (compare Fig 1B and Fig 2B).

Fig 2C demonstrates solubilization of the β−subunit in 2% DDM prior to co-

immunoprecipitation. Note that a significant portion of the β−subunit is present in the

supernatant. In the absence of detergent, the β−subunit is not observed in the supernatant

fraction, indicating that the presence of detergent is necessary to separate the β−subunit from the

membrane pellet (i.e. solubilized the β−subunit) under these conditions (data not shown). The

lane in Fig 2C marked S represents 5% of the solubilized fraction subsequently used as protein

input for co-immunoprecipitation. Since the β−subunit expresses at a higher level than the

α−subunit, a significant portion of the β−subunits are not assembled in heterodimers with

α−subunits (4,5). Therefore, only a portion of this solubilized β−subunit is available for co-

immunoprecipitation by antibodies against the α−subunit. Fig 2D shows co-

immunoprecipitation of the wild-type β−subunit by either the anti-(His)6 (lane 5) or the anti-

FLAG antibodies (lane 4). It should be noted that in control immunoprecipitations lacking either

precipitating antibody (lane 1) or protein input (lane 2 and 3) we were unable to reproduce this

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immunoprecipitation, demonstrating the specificity of the co-immunoprecipitation. These results

confirm that each of the tagged α−subunits assemble with the β−subunit as well as

demonstrating that the anti-FLAG and anti-(His)6 antibodies are capable of immunoprecipitating

the FLAG and (His)6 tagged α−subunits respectively. Fig 2D shows that the β-subunits which

are co-immunoprecipitated the most efficiently with the α−subunit tend to be on the higher side

of the molecular weight range for the β−subunit, indicating higher glycosylation and a more

mature β−subunit.

Since the immunoprecipitation efficiency of the anti-FLAG antibody appeared slightly

higher than that of the anti-(His)6 antibody (compare Fig 2D lanes 4 and 5), the ER and PM

fractions of co-infected cells were subjected to immunoprecipitation with anti-FLAG antibody.

The immunoprecipitates were then probed with the anti-(His)6–HRP antibody to investigate the

presence of oligomers containing both of the epitope tagged α−subunits. Prior to

immunoprecipitation, membranes were solubilized in 2% DDM. Fig 3A shows the Western blot

analysis detecting the (His)6-tagged α−subunit from the ER and PM fractions prior to

solubilization, and in the soluble and insoluble fractions. Interestingly, Fig 3A reveals that more

of the Na,K-ATPase α−(His)6 subunit is solubilized from the PM fraction than from the ER

fraction under these solubilization conditions. The soluble ER and PM fractions were used as

input for the co-immunoprecipitation experiment in Fig 3B. The anti-FLAG antibody was able

to precipitate the α−(His)6 subunit from ER and PM membrane fractions, as indicated by

Western blot detection of the immunoprecipitate with the anti-(His)6-HRP antibody (Fig 3B

lanes 4 and 5). Control precipitations lacking the anti-FLAG antibody confirm that the positive

co-immunoprecipitation result is not due to nonspecific binding to beads (Fig 3B lanes 1 and 2).

Since there is no cross-reactivity between the epitope antibodies (Fig 1A), this result indicates

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the α−FLAG subunits must be in stable association with α−His subunits in the DDM-solubilized

ER and PM fractions.

The results from Fig 3A & B imply that higher order oligomerization of the Na,K-

ATPase occurs. However, this is only true if the DDM solubilization liberates the α−tagged

Na,K-ATPase from co-localization in membrane patches. If DDM solubilization does not

separate the heterodimers from the membrane, the differently tagged subunits might be expected

to co-immunoprecipitate whether or not protein-protein associations occurred due to their co-

localization in membrane patches. To test the ability of DDM-solubilized membrane proteins to

sufficiently disrupt non-associated membrane proteins, we performed an additional control

experiment. Baculovirus-mediated co-expressions in High Five cells were carried out with the

tagged α−subunits and the unrelated membrane protein, hCTR1. hCTR1 is the human copper

transporter that has been functionally expressed recently in insect cells using the baculovirus

system (27). The hCTR1 protein does not specifically associate with the Na,K-ATPase, but it is

delivered to the PM, like the Na,K-ATPase. Fig 3C demonstrates the cellular localization by

Western blot analysis of fractionated membranes co-expressing hCTR1 and either α−His,β or

α−FLAG,β. Like wild-type Na,K-ATPase, hCTR1 is delivered to the PM. The complex pattern

of bands seen with hCTR1 is due to glycosylation and the presence of some degradation products

(18 kD and below) of this protein (27). Total membrane preparations of cells co-expressing

hCTR1 with α−His,β or α−FLAG,β were subjected to the same 2% DDM solubilization protocol

as used in Fig 3A & B. The first lane of each blot in Fig 3D shows 2.5% of the soluble protein

input for immunoprecipitation experiments. Due to preferential solubilization of higher

glycosylated hCTR1, slightly altered gel conditions, and reduced levels of secondary antibody

for Western blot detection, the hCTR1 signal in Fig 3D appears tighter and of only the higher

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molecular species observed in Fig 3C. Immunoprecipitation experiments in Fig 3D (second lane

of each blot) shows that neither the anti-FLAG or the anti-(His)6 antibodies co-

immunoprecipitated hCTR1 with the tagged Na,K-ATPase. Note that there is a protein G band

present in all immunoprecipitation and control lanes which runs at a higher molecular weight

(about 31 kD) than hCTR1 (about 26 kD) . The control experiment presented in Fig 3D

demonstrates that solubilization in 2% DDM is sufficient to separate non-associated but co-

localized membrane proteins. In summary, the data presented in Fig 3 demonstrate the

oligomeric association of the Na,K-ATPase αβ heterodimers after solubilization from

membranes and the lack of association of the subunit with hCTR1.

Recently published work has shown that the large cytoplasmic loop between the M4 and

M5 transmembrane segments, when expressed in isolation, form oligomers in an ATP-dependent

fashion (21). This loop contains the phosphorylation and nucleotide binding domains (28), for

which significant structural information is provided by crystal structures of SERCA and EM

structures of the Na,K-ATPase (29-31). Furthermore, crosslinking experiments (16) and chimera

studies (9,20) using the intact protein have suggested that regions of association between

oligomers occur in the M4M5 loop. We investigated the potential oligomerization of the Na,K-

ATPase wild-type αβ heterodimer with a mutant of the α−subunit lacking a significant portion

of the M4M5 loop. This anomalous Na,K-ATPase, termed the α−4,5LL (for M4M5-loopless) is

shown in Fig 4. The M4M5 loop, which is normally composed of the stretch of amino acid

residues from about 354 to 774, lacks amino acid residues 443-708 in the α−4,5LL mutant. This

eliminates about 65% of the loop, with regions missing from both the P and N domains (see Fig

4). However, the α−4,5LL retains the essential site of enzymatic phosphorylation, D369, and the

ATP-binding motif GCGxNDxP.

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Fig 5A demonstrates the α−4,5LL,β baculovirus encoded expression of the α−4,5LL

mutant in High Five cells. For comparison, a wild-type expression is shown in adjacent lanes.

Western blot detection with the KETYY antibody, which recognizes the C-terminus of the

α−subunit, shows the α−4,5LL mutant migrates at the predicted mass of about 73kD (top panel

of Fig 5A). Due to the elimination of the epitope within the M4M5 loop, the α−4,5LL mutant is

not detected by the α1 antibody (Fig 5A bottom panel). Fig 5A also shows that although, the

β−subunit expressed from the α−4,5LL,β virus is targeted to the PM (Fig 5A middle panel), the

α−4,5LL mutant is predominantly retained in the ER, unlike the wild-type αβ heterodimers. ER

retention of the α−subunit can occur for a number of reasons including the loss of a PM targeting

signal, recognition of misfolded protein by the ER quality control machinery, or failure to

associate with the β−subunit. It is interesting that we do not observe extensive degradation of

the α−4,5LL mutation, unlike some other α−subunit mutations we have characterized previously

(32). Although it would be undetectable by these methods if the KETYY moiety were removed

first, the absence of extensive degradation suggests that the α−4,5LL is not greatly denatured and

is integrated appropriately into the membrane.

To further explore the possibility that the α−4,5LL mutant might be retained in the ER

due to the presentation of misfolded protein, we looked at ligand-dependent digestion of the

α−subunit. It has been well established that the Na,K-ATPase assumes different conformational

states when bound to different ligands. We therefore tested the trypsin sensitivity of the wild-

type and the α−4,5LL mutant in the presence of Na, K, ATP, or no ligand. Mild trypsin

treatment reveals increased trypsin sensitivity of the α−4,5LL(Fig 5B). The C-terminal α−4,5LL

tryptic product migrating at approximately 32 kD is predicted to result from a cleavage within

the M4M5 loop. Fig 5B shows that the presence of ATP provides protection against tryptic

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cleavage at this site. Although the α−4,5LL deletant fails to produce ouabain-sensitive Na,K-

ATPase activity (data not shown), Fig 5B demonstrates that the α−4,5LL mutant achieves a

reasonably folded state, although it is retained in the ER. Furthermore, the α−4,5LL does not

show increased tryptic sensitivity at further C-terminal sites, suggesting that the C-terminal of

the α−4,5LL mutant has a fairly normal fold.

Co-infection with the wild-type α,β and the α−4,5LL,β baculoviruses does not alter the

sub-cellular localization of the wild-type or mutant α−subunits, as demonstrated by Western blot

analysis of fractionated membranes in Fig 6A. Likewise, the presence of the α−4,5,LL subunit

did not greatly inhibit or modify the distribution of the Na,K-ATPase activity of the wild-type

protein (Fig 6B). We have shown previously that α−subunits not associated with the β−subunit

are retained in the ER (5). Therefore, if the α−45LL mutant were not able to assemble with the

β−subunit, the ER retention of the α−45LL would be explained. Our initial attempts at

immunoprecipitation of the α−4,5LL subunit revealed that, unlike the wild-type protein, this

mutant is not soluble in 2% DDM. We therefore tried solubilizing the co-expressed wild-type

and α−4,5LL mutant in a number of detergents that have been previously used for the

solubilization of the Na,K-ATPase and/or other membrane proteins. As Fig 6C shows, at least

50% of the wild-type α−subunit was solubilized by all of the detergents tested. From the six

detergents shown in Fig 6C, only SDS was able to solubilize the α−4,5LL subunit.

Unfortunately, presence of SDS during immunoprecipitation caused nonspecific binding of the

Na,K-ATPase to the Protein-G Sepharose beads in the absence of antibody. Therefore, we

turned to less widely used detergents. We found that the α−4,5LL subunit could be solubilized

using 1% Fos-Choline (see Fig 8A).

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Fig 7 shows the results of β−subunit co-immunoprecipitation experiments after

solubilization in 1% Fos-Choline. Fig 7A demonstrates the relative amount of the β−subunit

solubilized from ER membranes containing either wild-type α− and β−subunits or the α−4,5LL

mutant and wild-type β−subunits. Only the ER fractions were used because the α−4,5LL is

retained in the ER (Fig 6A). The soluble protein fractions shown in Fig 7A were used as input

for immunoprecipitation by the anti-α1 (top panel Fig 7B) or anti-KETYY (bottom panel Fig

7B) antibodies, followed by Western blot detection for the β−subunit. The anti-α1 antibody was

able to co-immunoprecipitate the β−subunit from wild-type α,β (Fig 7B, top panel, lane 5)

containing membranes, but not from the α−4,5LL,β expression (Fig 7B, top panel, lane 4). This

was expected because the α−4,5LL does not contain the epitope recognized by the anti-α1

antibody. Note that antibody bands are also detected on the Western blot due to reactivity of the

anti-mouse-HRP secondary antibody with the anti-α1 antibody (Fig 7B top panel, lanes 1,4 and

5). Negative controls lacking antibody or protein input confirm that the association is specific

(Fig 7B top panel, lanes 2 and 3). The lower panel of Fig 7B shows the anti-KETYY antibody,

which recognizes the C-terminus of the α−subunit, was able to co-immunoprecipitate the

β−subunit from both expressions (Fig 7B bottom panel, lanes 4 and 5). Again, negative control

lacking antibody or protein input produced no signal (Fig 7B bottom panel, lanes 1-3). These

results demonstrate that although the α−4,5LL lacks a major part of the cytoplasmic loop, it is

still able to associate with the β-subunit. The failure of the α−4,5LL subunit to exit the ER (Fig

5A) is not then due to a lack of association with the β-subunit. Heterodimerization with the

β−subunit further suggests the correct membrane insertion of the α−4,5LL mutant because the

region of the α−subunit which has been implicated in assembly with the β−subunit (the M7M8

loop) is C-terminal to the deleted M4M5 loop.

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We next conducted co-immunoprecipitation experiments with the ER membranes

containing both wild-type and α−4,5LL,β protein (Fig 6A). Fig 8A shows solubilization of two

different ER preparations in 1% Fos-Choline. Whereas nearly all of the wild-type α−subunit

was present in the soluble fraction, less than half of the α−4,5LL mutant subunits were

solubilized. Each soluble lane in Fig 8A shows 5% of the protein input which was subject to

immunoprecipitation. Western blot detection by the anti-KETYY antibody after precipitation

with the anti-α1 antibody detects both the wild-type α−subunit and the α−4,5LL mutant in the

precipitated material (Fig 8B lanes 4 and 5), indicating that the wild-type α−subunit and

α−4,5LL mutant are associated in the Fos-Choline solubilized preparation. Control experiments

utilizing co-infected hCTR to test for protein solubilization were not performed because we

found hCTR solubilized in Fos-Choline nonspecifically associates with protein G-sepharose

beads. However, control precipitations of the wild-type and α−4,5LL mutant lacking antibody

or protein input confirm that the co-immunoprecipitation result in Fig 8B lane 4 and 5 are not

due to nonspecific binding to beads or antibody cross-reactivity (Fig 8B lanes 1-3).

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Discussion

We have shown in the present work that the Na,K-ATPase, which forms a stable

heterodimer of αβ subunits, exists in a higher oligomeric state of at least two α−subunits. We

demonstrate the existence of stable protein-protein associations between heterodimers by co-

immunoprecipitation with anti-FLAG antibody of α-subunits that bear the (His)6 epitope along

with α−subunits that bear the FLAG epitope. Control experiments established that this result is

not accounted for by cross-reactivity of the antibodies or due to opportunistic co-localization of

the tagged α−subunits within unsolubilized membrane patches. We also demonstrate that such

epitope-tagged α−subunits also associate with β−subunits. Our findings thereby imply the

existence of a higher oligomeric state of at least α2β2 in native membranes. These oligomeric

heterodimers are present in enriched fractions of the ER and PM of High Five cells, so it is likely

that the oligomeric unit is formed following synthesis in the ER and then delivered (via the Golgi

network) to the PM. Our results do not address the issue of whether or not such associations

among αβ heterodimers have significant modifying effects on the enzyme activity.

The oligomeric state of the Na,K-ATPase has been the subject of ongoing controversy

within the field for over 20 years. In 1983, Brotherus et al demonstrated by analytical

ultracentrifugation that the C12E8 solubilized αβ heterodimer was a single unit that could be

reconstituted in phospholipids to regain Na,K-ATPase activity (33). The same group later

showed the soluble αβ heterodimer retained the ion occlusion properties of the membrane bound

Na,K-ATPase (22). This work established that the αβ heterodimer forms the membrane ion

binding sites and suggested that oligomerization of multiple units is not necessary to transport

ions. This early work, however, could not exclude the possibility that Na,K-ATPase may

spontaneously form higher order associations in vivo or reform them in vitro. Subsequently, a

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significant body of evidence has accumulated which suggests that the Na,K-ATPase can form

higher oligomers. Furthermore, this association of subunits has been claimed to influence Na,K-

ATPase functioning (for review, see (7)). It should be noted that subsequent work with C12E8-

solubilized membranes has demonstrated the existence of (αβ)2 and higher order oligomers in

addition to αβ heterodimers, the relative amounts of which were dependent on buffer ionic

composition (34). More recently, work with a Na,K-ATPase preparation from salt-adapted duck

nasal gland has supplied evidence for the monomeric αβ heterodimer as the functional unit in the

membrane. This preparation which can be fully phosphorylated contains primarily monomeric

αβ heterodimers (35,36). However, in light of mounting evidence from many methods and

systems demonstrating the oligomerization of the Na,K-ATPase, including our current work, it

seems likely that in a native membrane environment, the Na,K-ATPase can and does form higher

order oligomers.

Numerous other studies have been presented in the literature which support the

oligomerization of the Na,K-ATPase. For more extensive reviews, see (7,8). Briefly, physical

interactions between αβ, αα and ββ have been implicated from crosslinking studies

(14,16,24,37-39), co-immunoprecipitations have demonstrated the interaction of

different α−subunit isoforms (19), thermal inactivation studies suggest αα contact (18),

fluorescence resonance energy transfer provides distance measurements consistent with (αβ)2

(40), and HPLC/LALLS (41) as well as single molecule detection (17) indicate the presence of

(αβ), (αβ)2 and higher order oligomers. Numerous studies in which half-site phosphorylation

and half or quarter site ATP-analog binding stoichiometries have been interpreted in a model that

proposes that the Na,K-ATPase co-exits simultaneously in the E1ATP, E2ATP or EPATP forms

(42). The existence of such oligomerization of the Na,K-ATPase in native membranes provides

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a mechanism which can explain the recent observation of dominant-negative α−mutants in

Drosophila (43).

In an earlier study using baculovirus-infected insect cells and immunoprecipitation using

isoform-specific antibodies, Blanco et al provided evidence of stable associations of the αβ-

heterodimers in unfractionated cell membranes (19). They also showed that such

oligomerization did not take place between α−subunits from the Na,K-ATPase and the H,K-

ATPase. The present work confirms and extends those findings, using epitope tags on the wild-

type α1β1 heterodimers and demonstrates that the oligomeric forms occur in membranes from

both the ER and PM. It should be borne in mind that we estimate that the heterologously

expressed Na,K-ATPase represents only about 1-2% of the total membrane protein in the insect

cells (5), so it is unlikely that the observed associations are produced by over-enrichment or

artificially-induced close interactions of the heterodimeric units.

The observation of Na,K-ATPase oligomerization naturally leads to the question of how

heterodimeric units interact to form higher order complexes. Work utilizing Na,K-ATPase and

H,K-ATPase chimeras suggests that the cytoplasmic loop of the Na,K-ATPase is important for

oligomerization (9). This data is in agreement with the observation that the bacterially expressed

major cytoplasmic loop between membrane segments M4 and M5, in isolation, can dimerize in

an ATP-dependent way (21). However, if the M4M5 loop is exclusively responsible for

oligomerization, it is surprising that, in the work presented here, the α−4,5LL mutant, from

which ~65% of the M4M5 cytoplasmic loop had been removed, is able to associate with the

wild-type protein. The formation of oligomers between the wild-type and the α−4,5LL mutant

demonstrates that the full M4-M5 loop is not necessary for oligomeric protein-protein

interactions and suggests that an association interface may extend beyond the cytoplasmic loop,

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possibly including other regions of the α−subunit or the β−subunit. It is interesting that although

stable associations are formed in the ER between wild-type and α−4,5LL α−subunits, this does

not enable these (αβ)(α’β) assemblies to leave the ER in the same way as (αβ)2. As an

extension of our current findings, we are initiating studies on the effects of Na,K-ATPase ligands

on the oligomerization.

Our findings that the α−4,5LL mutant is retained in the ER raises some interesting

questions. Since the deleted region of the α−subunit in the α−4,5LL mutant is on the

cytoplasmic side of the membrane, retention by ER luminal quality control apparatus does not

seem to be a likely explanation for the ER retention unless some segments of the α−4,5LL

mutant are incorrectly inserted in the membrane. However, our observations that (1) the

α−4,5LL is relatively stable, i.e. not readily degraded in the ER, and (2) the β−subunit assembles

with the α−4,5LL mutant support the idea that the α−4,5LL achieves a fairly normal membrane

insertion. ER retention of the α−4,5LL subunit even though it associates with the β−subunit,

also supports our previous conclusion that αβ assembly, while being necessary, is not sufficient

for exit from the ER (4). It should be noted that our present result implies that structural

elements of the α−subunit may also be important to accomplish ER exit,

We do not know yet whether the formation of oligomeric αβ subunit association is

modulated via direct interactions alone or if other components, such as the cytoskeleton play a

role. Based on primary structure, two putative ankyrin binding domains have been identified in

the Na,K-ATPase α−subunit and claimed to be important for Na,K-ATPase trafficking (44,45).

These sites are located in the M2M3 and the M4M5 cytoplasmic loops. The putative ankyrin

binding site in the M4M5 loop, which is deleted in the α−4,5LL mutant, is predicted to be buried

based on alignment of the Na,K-ATPase in the SERCA crystal structure (46). Since the

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α−4,5LL is able to associate with the wild-type Na,K-ATPase, oligomerization cannot occur

only through cytoskeletal associations with the putative ankyrin binding site in the M4M5 loop.

Studies are currently underway to investigate the role of the other putative ankyrin binding site in

the M2M3 loop.

In summary, we have shown that the Na,K-ATPase αβ heterodimers, when

heterologously expressed in insect cells, form stable intermolecular associations leading to at

least bis-heterodimers in both the ER and PM compartments. Our work does not address the

issue of whether or not such associations among αβ heterodimers have significant modulating

effects on the enzyme activity, trafficking, or cellular stability of the Na,K-ATPase. It is likely

that these intermolecular interactions among Na,K-ATPase αβ heterodimers may modulate

interactions between the Na,K-ATPase and other cellular proteins. The components and/or

interactions which mediate the oligomerization of the Na,K-ATPase heterodimers and the

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Acknowledgements.

We thank John Eisses for construction of the 4,5-LL α−subunit mutant, technical guidance,

and helpful discussions.

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FIG. 1 Insect cell expression of Na,K-ATPase containing α−subunits with C-terminal

epitope tags. High Five cells were infected with baculovirus containing the cDNA for the wild-

type β−subunit and an α−subunit with either a (His)6 (α−His,β) or FLAG (α−FLAG,β) epitope

tag. Membrane fractions were separated via a step sucrose gradient and A) 30 µg were resolved

on a 7.5% SDS-PAGE, transferred to nitrocellulose and probed with anti-α1, anti-β1, anti-

(His)6-HRP, or anti-FLAG antibody. B) Na,K-ATPase activity was determined as ouabain-

sensitive ATPase activity and is reported as µMol Pi /mg hr.

FIG. 2 Co-expression of epitope tagged Na,K-ATPases. High Five cells were co-infected with

α-His,β and α-FLAG,β baculovirus particles. Membranes were fractionated followed by A)

Western blot analysis with anti-(His)6-HRP, anti-FLAG, or anti-β1 antibodies and B) ouabain-

sensitive Na,K-ATPase activity determination. C) Total membrane preparations were suspended

in IPB with 2% DDM and the soluble (S) and insoluble (IS) fractions separated by

centrifugation. D) The soluble fraction from C was subjected to immunoprecipitation by the

anti-(His)6 or the anti-FLAG antibody and the associated β−subunit was detected by Western

blot analysis following gel electrophoresis.

FIG. 3 The Na,K-ATPase forms oligomers in the ER and PM. High Five cells were co-

infected with α-His,β and α-FLAG,β baculoviruses and membranes fractionated, as in Fig 2A.

A) ER and PM fractions were suspended in 2% DDM followed by separation of soluble and

insoluble fractions. B) The soluble ER and PM fractions were immunoprecipitated with the anti-

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FLAG antibody and subject to Western blot analysis detecting with the anti-(His)6–HRP

antibody. C & D) High five cells were co-infected with baculovirus containing cDNA for

hCTR1 and either α-His,β or α-FLAG,β baculovirus. C) Western blot analysis of fractionated

membranes detecting with anti-α1 and anti-hCTR1 antibodies. D) Total membrane preparations

were solubilized in IPB with 2% DDM as in A and soluble material (far left lanes) was subjected

to immunoprecipitation by the anti-FLAG or anti-(His)6 antibody, followed by Western blot.

FIG. 4 Schematic diagram of α−4,5LL. The M4M5 loop of the α−subunit composes two

cytoplasmic domains, the phosphorylation domain (P) and the nucleotide binding domain (N).

The α−4,5LL mutant lacks the amino acid residues Asp443 through Val707. Portions of both the

P and N domain are removed, however the phosphorylation site (D369) in the P domain, and the

ATP binding motif (GCGxNDxP) in the N domain are retained in the α−4,5LL subunit.

FIG. 5 Cellular distribution and tryptic sensitivity of α−4,5LLβ, and wild-type αβ. High

Five cells were infected with baculovirus containing α−4,5LL,β or wild-type α,β cDNA. A)

Western blot analysis of fractionated membranes was performed detecting with anti-KETYY,

which recognizes the C-terminus of the α−subunit, with anti-β1, or with anti-α1, which

recognizes an epitope in the M4M5 loop. B) ER membrane preparations of separately expressed

α−4,5LL,β and wild-type α,β were suspended in buffer containing either 2mM ATP-Tris, 20mM

Na+, 20mM K+ or no pump ligands as indicated, followed by trypsin digestion.

FIG. 6 Solubility of co-expressed α−4,5LL,β and wild-type α,β. High Five

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cells were co-infected with the α−4,5LL,β and wild-type α,β baculoviruses followed by

fractionation of membrane compartments. A) Western blot analysis detecting with the anti-

KETYY and anti−β1 antibodies. B) Ouabain sensitive Na,K-ATPase activity of the membrane

fractions expressed as µMol Pi / mg hr. High Five cells were co-infected with α−4,5LL,β and

wild-type α,β baculoviruses. C) Total membrane preparations were suspended in IPB containing

specified detergents followed by centrifugation to separate soluble (S) and insoluble (IS)

fractions. Western blot detection was performed with the anti-KETYY antibody.

FIG. 7 Assembly of the β−subunit with the α−4,5LL and wild type α−subunit. High Five

cells were infected with either the α−4,5LLβ or the wild-type α,β baculovirus. A) ER

membrane preparations were suspended in IPB with 1% Fos-Choline and the soluble (S) and

insoluble (IS) fractions separated. B) Soluble fractions were subjected to immunoprecipitation

by either the anti−α1 or anti-KETYY antibody and the co-immunoprecipitated β−subunit was

detected by Western blot analysis.

FIG. 8 Co-infection and association of α−4,5-LL and wild-type heterodimers. High Five

cells were co-infected with the α−4,5LL,β and wild-type α,β baculoviruses followed by

fractionation of membrane compartments. A) ER membrane fractions of two different

preparations were suspended in IPB with 1% Fos-Choline and soluble (S) and insoluble (IS)

fractions were separated. B) Soluble ER fractions were subject to immunoprecipitation by the

anti-α1 antibody, which does not recognize the α−4,5LL subunit. Western blot analysis was

done with the anti-KETYY antibody, which recognized both the α−4,5LL and wild-type

α−subunit.

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Melissa D. Laughery, Matthew L. Todd and Jack H. KaplanOligomerization of the Na,K-ATPase in cell membranes

published online June 18, 2004J. Biol. Chem. 

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