©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A Cytoplasmic Region of the Na,K-ATPase -Subunit Is Necessary for Specific / Association (*)

Joseph C. Koster , Gustavo Blanco , Robert W. Mercer (§)

From the (1)Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

While most structural studies of the Na,K-ATPase support a subunit stoichiometry of one -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-ATPaseNa,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.


INTRODUCTION

The Na,K-ATPase, or sodium pump, is a ubiquitous, multi-spanning membrane protein that actively maintains a high internal K 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.

Although several lines of evidence seem to support an 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.

Using the baculovirus expression system, we have recently demonstrated the ability of the -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.


MATERIALS AND METHODS

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.

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 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) .


RESULTS

Recombinant baculoviruses containing the cDNAs coding for the Na,K-ATPase 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.

To delineate the domains necessary for Na,K / 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 (polyA). 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.

Recently, a naturally occurring truncated 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).

In conclusion, the data presented here imply that a peptide segment from Gly 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.


DISCUSSION

While most structural studies of the Na,K-ATPase support an 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.

The interaction between Na,K-ATPase -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.

Preliminary reports of cross-linking studies on the proteolytic fragments of the Na,K-ATPase also indicate / 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.

To confirm the results of the cross-linking studies in the baculovirus expression system, an 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.

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 / 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.

The role of the Na,K-ATPase midregion segment in mediating protein interactions does not appear to be restricted to / 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.

The results presented here provide strong evidence that a cytoplasmic region of the Na,K-ATPase 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.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant GM 39746 and the George M. O'Brien Kidney and Urological Diseases Center at Washington University School of Medicine. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Dept. of Cell Biology and Physiology, Washington University School of Medicine, 660 S. Euclid Avenue, Box 8228, St. Louis, MO 63110. Tel.: 314-362-6924; Fax: 314-362-7463.

The abbreviations used are: CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; PAGE, polyacrylamide gel electrophoresis; IAP, integrin-associated protein.


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