Heterologous expression of Na+-K+-ATPase in insect cells: intracellular distribution of pump subunits

Craig Gatto1,2, Scott M. McLoud1, and Jack H. Kaplan1

1 Department of Biochemistry and Molecular Biology, Oregon Health Sciences University, Portland, Oregon 97201-3098; and 2 Department of Biological Sciences, Illinois State University, Normal, Illinois 61790-4120


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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The Na+-K+-ATPase is a heterodimeric plasma membrane protein responsible for cellular ionic homeostasis in nearly all animal cells. It has been shown that some insect cells (e.g., High Five cells) have no (or extremely low) Na+-K+-ATPase activity. We expressed sheep kidney Na+-K+-ATPase alpha - and beta -subunits individually and together in High Five cells via the baculovirus expression system. We used quantitative slot-blot analyses to determine that the expressed Na+-K+-ATPase comprises between 0.5% and 2% of the total membrane protein in these cells. Using a five-step sucrose gradient (0.8-2.0 M) to separate the endoplasmic reticulum, Golgi apparatus, and plasma membrane fractions, we observed functional Na+ pump molecules in each membrane pool and characterized their properties. Nearly all of the expressed protein functions normally, similar to that found in purified dog kidney enzyme preparations. Consequently, the measurements described here were not complicated by an abundance of nonfunctional heterologously expressed enzyme. Specifically, ouabain-sensitive ATPase activity, [3H]ouabain binding, and cation dependencies were measured for each fraction. The functional properties of the Na+-K+-ATPase were essentially unaltered after assembly in the endoplasmic reticulum. In addition, we measured ouabain-sensitive 86Rb+ uptake in whole cells as a means to specifically evaluate Na+-K+-ATPase molecules that were properly folded and delivered to the plasma membrane. We could not measure any ouabain-sensitive activities when either the alpha -subunit or beta -subunit were expressed individually. Immunostaining of the separate membrane fractions indicates that the alpha -subunit, when expressed alone, is degraded early in the protein maturation pathway (i.e., the endoplasmic reticulum) but that the beta -subunit is processed normally and delivered to the plasma membrane. Thus it appears that only the alpha -subunit has an oligomeric requirement for maturation and trafficking to the plasma membrane. Furthermore, assembly of the alpha -beta heterodimer within the endoplasmic reticulum apparently does not require a Na+ pump-specific chaperone.

sodium-potassium-adenosinetriphosphatase; High Five cells


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INTRODUCTION
MATERIALS AND METHODS
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MAINTENANCE OF LOW INTRACELLULAR Na+ and high intracellular K+ in most eukaryotic cells is achieved by the active export of three Na+ with the concomitant import of two K+ powered by the hydrolysis of one ATP molecule. The Na+-K+-ATPase (EC 3.6.1.37) mediates this coupled transport via a reaction mechanism that resembles that of other P2-type ATPases involving the participation of an acylphosphate intermediate (35). This class of cation transporters is widely spread throughout both plant and animal kingdoms and includes such members as the gastric H+-K+-ATPase, the sarcoplasmic reticulum Ca2+-ATPase, the plasma membrane Ca2+-ATPase, and the Neurospora H+-ATPase (for reviews see Refs. 30 and 35). The Na+-K+-ATPase has a catalytic alpha -subunit of ~100 kDa with 10 transmembrane-spanning domains (25) and an additional 55 kDa beta -subunit. The beta -subunit spans the membrane only once, with the majority of the protein protruding into the extracellular space, including three glycosylation sites.

The role that the beta -subunit plays in the active translocation of Na+ and K+ remains unclear, although some data are starting to emerge. For example, ligand-induced conformational changes in the alpha -subunit produced distinctly different trypsin proteolysis patterns of the beta -subunit (34). Similarly, loss of Na+-K+-ATPase activity after reduction of the beta -subunit S-S bridges was prevented when K+ were occluded by the enzyme (33). These data indicate that the beta -subunit changes conformation concomitantly with the alpha -subunit in response to physiological ligands. Until recently, much of the information pertaining to the biochemical and biophysical properties of the Na+-K+-ATPase have been gathered from studies of highly purified enzyme preparations. However, in the last decade, several isoforms of the alpha - and beta -subunits have been cloned from a number of species, and functional expression has been achieved in several systems (30). Consequently, researchers have been able to use a variety of mutagenesis approaches toward elucidating the functional role of the beta -subunit as well as the part it plays in the maturation of the alpha -beta complex and targeting the complex to the plasma membrane.

Many protein complexes en route to the plasma membrane take a common pathway by which assembly is required to overcome endoplasmic reticulum retention and degradation. Indeed, in many cases where individual subunits are expressed in the absence of their partners, the expressed proteins are retained in the endoplasmic reticulum and degraded (see Ref. 7). For example, the Na+-K+-ATPase beta -subunit has been reported to play an essential role in the maturation of the alpha -subunit (18). Specifically, when the alpha -subunit was expressed alone in Xenopus oocytes, it was rapidly degraded in the vicinity of the endoplasmic reticulum, and only coexpression and assembly with the beta -subunit facilitated alpha -subunit maturation (18). While subunit assembly is a widespread phenomenon, the molecular mechanisms involved in the process of subunit assembly of heterooligomeric proteins are still poorly understood. Some of the approaches used to gain information on alpha -beta assembly have included immunoprecipitation experiments involving coexpression of truncated beta -subunits (21, 42), chimeras between the Na+-K+-ATPase and H+-K+-ATPase alpha -subunits (and between respective beta -subunits) (20), and in vivo translation/insertion experiments (2, 4). In general, these studies conclude that the beta -subunit is required for the membrane insertion and plasma membrane targeting of the alpha -subunit. However, these studies were performed in tissues (e.g., Xenopus oocytes) with endogenous Na+-K+-ATPase expression, which makes interpretation of experiments using heterologously expressed enzyme in these systems difficult.

In contrast, it has been suggested that, in some systems, the beta -subunit is not required to target the alpha -subunit to the plasma membrane. Specifically, DeTomaso and colleagues (13) detected the Na+-K+-ATPase alpha -subunit on the plasma membrane surface of Sf9 cells via immunocytochemistry when it was expressed in the absence of the beta -subunit. Even more striking was the claim that Sf9 cells expressing the alpha -subunit alone had ouabain-sensitive ATPase activity independent of the presence of the beta -subunit (5). The properties of the ATPase activity, not surprisingly, bore little resemblance to normal Na+-K+-ATPase activity. In other studies, pulse-chase and immunoprecipitation experiments suggested that newly synthesized alpha -subunits were delivered to the plasma membrane of A6 epithelia in the absence of the beta -subunit (12). Thus the issue of assembly of the Na+-K+-ATPase alpha -beta complex and its delivery to target membranes remains unresolved.

In the present study, we use cultured insect cells [i.e., Trichoplusia ni (High Five) cells] infected with baculovirus containing the cDNA for the Na+-K+-ATPase subunits to examine further alpha - and beta -subunit assembly as well as the functional distribution of the enzyme. High Five cells, like Sf9 cells, have little or no endogenous Na+-K+-ATPase activity. Thus, in this system, assembly and trafficking of the alpha -beta complex and separately expressed subunits may be characterized and studied in the absence of any endogenous polypeptides. Also, insect cells have the ability to posttranslationally modify and assemble multisubunit proteins in a manner similar to that found in mammalian expression systems (1, 28, 44). These characteristics make the baculovirus system attractive for studying cellular processing of heterodimeric proteins such as the Na+-K+-ATPase. We isolated the endoplasmic reticulum, Golgi apparatus, and plasma membrane fractions of infected High Five cells to specifically determine the structural and functional characteristics of the alpha - and beta -subunits along the protein maturation pathway. We found that when the alpha -subunit was expressed in the absence of the beta -subunit it was degraded in the endoplasmic reticulum, similar to the findings in Xenopus oocytes (18). Furthermore, this degradation was completely prevented by coexpression with the beta -subunit. In contrast, expression of the beta -subunit alone resulted in proper folding and trafficking of the solitary protein through the Golgi to the plasma membrane (in contrast to findings in oocytes). In addition, we measured protein function at the various stages of protein maturation. Interestingly, the alpha -beta dimer was fully functional even in the endoplasmic reticulum, indicating that, after subunit assembly, no further processing is necessary.


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Materials. [3H]ouabain and rainbow high- and low-molecular-weight markers were from Amersham. Acrylamide, ammonium persulfate, Coomassie brilliant blue R-250, N,N,N',N'-tetramethylethylenediamine, and SDS were purchased from Bio-Rad. Ammonium molybdate, hydrochloric acid, and sodium phosphate were from Fisher. Cupric sulfate, potassium chloride, sucrose, and urea were from Mallinckrodt. Polyvinylidene difluoride (PVDF) electroblotting membrane was from Millipore. Dog kidneys were from Pelfreeze. Ascorbic acid, beta -mercaptoethanol, EDTA, Folin's reagent, imidazole, magnesium chloride, Na2ATP, sodium bicarbonate, sodium chloride, antipain, pepstatin, leupeptin, phenylmethylsulfonyl fluoride, HEPES, and Trizma base were from Sigma. A full-length cDNA clone encoding the wild-type alpha 1- and beta 1-subunits of sheep Na+-K+-ATPase was a gift of Dr. Elmer Price (Department of Veterinary Biomedical Sciences, University of Missouri-Columbia). The Bac-To-Bac baculovirus expression system was from Life Technologies, High Five cells were from Invitrogen, and the Ex-Cell-405 insect cell growth medium was from JRH Biosciences.

Na+-K+-ATPase purification from dog kidney. Na+-K+-ATPase was purified from dog kidney as described by Jorgensen (26) with the modifications reported earlier (16). Specifically, the dog kidney enzyme was purified through a continuous sucrose gradient (15-45% sucrose) achieved with a zonal rotor. The enzyme was judged >95% pure through SDS-PAGE. Protein concentration was determined by the method of Lowry et al. (32).

Baculovirus production and viral infections of High Five cells. Recombinant baculovirus was produced following the Bac-To-Bac system protocols as described previously (25). Briefly, the donor plasmid pFASTBACDUALalpha beta (pFBDalpha beta ) was constructed by subcloning the Na+ pump beta - and alpha -subunit cDNAs into the multiple cloning sites I and II of the pFASTBACDUAL vector, respectively. The cDNA used contained two silent mutations to allow for future cassette mutagenesis (see Ref. 25). The beta -subunit cDNA was introduced into the vector as an EcoRI/SpeI fragment and the alpha -subunit cDNA as a SmaI/StuI fragment. DH10BAC cells were transformed with pFBDalpha beta to obtain recombinant baculovirus shuttle vectors (i.e., bacmids), which were used subsequently for High Five cell transfections to generate recombinant baculovirus.

High Five cells were maintained at 27°C in 250-ml suspension cultures and split every 3 days with fresh media to maintain cell density between 0.5 and 4 × 106 cells/ml. Viral amplification was carried out by infecting log-phase high-viability High Five cells (>98%, trypan blue exclusion) with recombinant baculovirus at a multiplicity of infection of 0.1-1. After 5 days, cells were centrifuged (500 g, 10 min), and the resulting supernatants were collected as viral stocks. For protein expression, log-phase high-viability High Five cells were infected with viral stocks at a multiplicity of infection of 10-15. The inclusion of 1% ethanol (vol) in the growth media significantly increases protein production (25). High Five cells were harvested and the membrane fractions separated via a five-step sucrose gradient as originally described by Yang et al. (47) and modified by Hu and Kaplan (25). Membrane fractions were characterized by enzymatic marker activities representative of endoplasmic reticulum, Golgi, and plasma membrane as described by Ma et al. (36).

Briefly, for the alpha -glucosidase assay (endoplasmic reticulum marker), 100 µg of protein from each membrane band were diluted into 100 µl of 1× PBS buffer containing 1% Triton X-100 and 2.5 mM p-nitrophenol-alpha -glucoside. Samples were incubated at 37°C for 5 h, after which the reaction was stopped with 0.2 M Na2CO3. Enzyme activity was determined by measuring the absorbance at 410 nm. For the alpha -mannosidase assay (Golgi marker), 100 µg of membrane protein were diluted to 1 ml with homogenizing buffer. A 0.4-ml volume of a reaction mixture containing 4 mM 4-methylumbelliferyl-alpha -D-mannopyranoside, 1× PBS, and 3% Triton X-100 was added to the protein solution and incubated at 37°C for 30 min. The reaction was stopped with the addition of 1 ml of ice-cold 0.5 M glycine, 0.5 M Na2CO3. Enzyme activity was determined by measuring the fluorescence emission at 448 nm after exciting at 364 nm. For the alkaline phosphodiesterase assay (plasma membrane marker), 50 µg of membrane protein were diluted in a total volume of 600 µl reaction buffer containing 100 mM Tris, 10 mM sodium thymidine 5'-monophosphate, and 1 mM p-nitrophenyl ester. The mixture was incubated for 2 h at 37°C. The reaction was stopped by adding 1 ml of ice-cold stopping solution containing 0.5 M glycine and 0.5 M Na2CO3. Enzyme activity was determined by measuring the absorbance at 410 nm.

After purification, each fraction was analyzed for Na+-K+-ATPase activity and ouabain binding capacity (see [3H]ouabain binding). In addition, quantitative slot-blot analyses were run to determine the amount of heterologously expressed Na+-K+-ATPase with respect to the total membrane protein in each fraction.

Na+-K+-ATPase assay. ATP hydrolysis was measured essentially as reported previously (25). Briefly, the typical Na+-K+-ATPase assay contained 500 µl of assay medium containing (in mM) 0.6 EGTA, 156 NaCl, 24 KCl, 3.6 MgCl2, 3.6 ATP, 60 imidazole (pH 7.2), 10 sodium azide, and 100 µl of cell membrane preparations containing 8 µg of protein. The assay mixture was incubated at 37°C for 30 min, and the phosphate release was determined as reported by Brotherus et al. (9). Na+-K+-ATPase activity was the difference between the ATP hydrolysis measured in the presence and absence of 20 µM ouabain. In the measurements of K+ or Rb+ dependence (see Figs. 3 and 7), the respective cation was omitted from the assay medium, and the concentrations indicated in the figures were added directly to the assay tubes.

86Rb+ uptake into Sf9 cells. Ouabain-sensitive 86Rb+ uptake was measured in Sf9 cells essentially as reported by Minor et al. (37) with minor modifications. Sf9 cells grown attached to six-well plates (35 mm well diameter) were infected with wild-type viral stocks (multiplicity of infection = 10-15). Cells were allowed to grow to confluence (72-84 h) at 27°C. The growth medium was removed and replaced with 0.9 ml of an incubation medium containing (in mM) 149 NaCl, 2.5 MgCl2, and 25 HEPES (pH 7.4). Cells were incubated in this solution at 25-27°C (i.e., room temperature) for 30 min to load them with Na+. A 100 µM final concentration of bumetanide was added to each well, and cells were incubated for an additional 5 min. The 86Rb+ uptake was initiated by adding 0.1 ml of an appropriate RbCl stock to achieve the desired final Rb+ concentration. The specific radioactivity was ~2.5 µCi/ml. The 86Rb+ uptake into Sf9 cells, which was not mediated by the Na+ pump, was determined by performing the same assay in the presence of 20 µM ouabain. At the times indicated, the uptake reaction was stopped by aspirating off the flux medium, and the cells were washed twice with 2.5 ml of ice-cold incubation medium containing 5 mM RbCl. The cells were solubilized with 1.5 ml of 100 mM NaOH, and the radioactivity from aliquots of each sample was determined by liquid scintillation spectroscopy. The total protein concentration from each well was determined by the method of Bradford (8).

[3H]ouabain binding. Specific ouabain binding was measured as described earlier (25). Briefly, High Five cell membrane protein (100 µg) was incubated at 37°C for 1 h in 50 µl of an incubation buffer containing 3 mM MgSO4, 1 mM Na2VO4, 1 mM EGTA, 10 mM MOPS-Tris (pH 7.2) containing 5 µM [3H]ouabain in the presence or absence of 1.4 mM unlabeled ouabain. The reaction mixtures were filtered through Millipore filters (pore size 0.45 µm), washed three times with ice-cold MOPS-Tris buffer, and the amount of radioactivity was determined by liquid scintillation spectroscopy. The difference between samples incubated in the absence and presence of excess ouabain was considered specific ouabain binding.

Quantification of expressed Na+-K+-ATPase. To estimate the relative amounts of Na+-K+-ATPase present in each of the membrane fractions, we performed a quantitative slot-blot analysis strictly according to the methods described by Bio-Rad for their slot-blotting system. Standard curves were generated by plotting the pixel density of chemiluminescent autoradiographs against the corresponding amount of purified dog kidney Na+-K+-ATPase. To control the total amount of protein added to each slot well, BSA was added along with the purified dog Na+-K+-ATPase to bring the total protein content added to 10 µg. Consequently, a 10-µg quantity of the respective High Five cell membrane fractions was added to the slot wells. Duplicate or triplicate determinations were used for the various membrane fractions, and the corresponding pixel densities were converted to Na+-K+-ATPase using the slope generated from the standard curves.


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Isolation of intracellular insect cell membranes. The separate membrane fractions from High Five cells were separated via a five-step sucrose gradient (0.8-2.0 M sucrose). After centrifugation there were three distinct bands at ~1.5 M (band 1), 1.3 M (band 2), and 1 M (band 3) sucrose densities. The three separate fractions were each analyzed for specific membrane marker enzyme activities. Figure 1 shows the results for glucosidase, mannosidase, and alkaline phosphodiesterase activities characteristic of endoplasmic reticulum, Golgi apparatus, and plasma membrane, respectively. These assays clearly show that band 1 and band 3 are extremely pure preparations of endoplasmic reticulum (Fig. 1A) and plasma membrane (Fig. 1C), respectively. In addition, it appears that band 2 is an enriched preparation of the Golgi apparatus (Fig. 1B). These membrane distributions were not significantly altered when High Five cells were expressing the sheep Na+-K+-ATPase alpha -subunit alone, the beta -subunit alone, or both subunits simultaneously (data not shown).


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Fig. 1.   Marker enzymes for membrane fractions. Experimental verification of the respective membrane pools was achieved by performing characteristic enzyme assays on the 3 isolated fractions. Equal amounts of protein were used from the 3 isolated fractions in each of the 3 enzyme activities measured. These were alpha -glucosidase assay (endoplasmic reticulum marker; A), alpha -mannosidase assay (Golgi marker; B), and alkaline phosphodiesterase (plasma membrane marker; C). The membrane bands isolated from the sucrose gradient were numbered 1-3, with 1 corresponding to the most dense fraction. These experiments clearly indicate that band 1 is endoplasmic reticulum, band 2 is Golgi apparatus, and band 3 is plasma membrane. Abs., absorbance.

The amount of functional Na+-K+-ATPase present in each fraction was determined by measuring ouabain-sensitive ATPase activity (Fig. 2). For each fraction, the total ATPase activity was measured in the absence and the presence of 20 µM ouabain. These data indicate that Na+-K+-ATPase activity is present at all stages of the protein trafficking pathway. Specifically, the Na+-K+-ATPase activity present in the endoplasmic reticulum and Golgi was about one-half of the activity measured in the plasma membrane (Fig. 2). Further characterization of the Na+-K+-ATPase activity revealed no differences in the K+ dependence between each of the three membrane fractions (Fig. 3). The similar half-maximal activation concentration (K1/2) for K+ suggests that the Na+-K+-ATPase molecules present in each fraction are coordinating cations in the same fashion, providing additional evidence that the structural and functional properties are not different during the stages of protein maturation. Expression levels of functional enzyme were also assayed by measuring the specific [3H]ouabain binding capacity of each fraction (Table 1). It is important, if we are to describe the properties of the Na+-K+-ATPase, a plasma membrane protein, and its trafficking in High Five cells, to be confident that we are able to separate the plasma membrane from other intracellular membranes. The distribution of the marker enzymes is good support for successfully accomplishing this. It should be noted that contamination of the endoplasmic reticulum and Golgi membranes by the plasma membrane is no higher than 9% and 11%, respectively, based on the degree of alkaline phosphodiesterase activity in these compartments. Alkaline phosphodiesterase is a widely used marker for plasma membranes. Thus, in terms of Na+-K+-ATPase activity, where levels of 12 µmol Pi · mg protein-1 · h-1 are observed in the plasma membrane fractions, a specific activity of maximally 1-1.4 µmol Pi · mg protein-1 · h-1 could be expected to be due to plasma membrane contamination of the endoplasmic reticulum and Golgi fractions. However, activity in the 5 µmol Pi · mg protein-1 · h-1 range are observed. In other experiments, where lower enzyme levels are observed (see Table 1), similar distributions are obtained. Thus the levels of Na+-K+-ATPase activity that we see in the endoplasmic reticulum and Golgi fractions are much higher than could be accounted for by contamination from the plasma membrane compartment.


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Fig. 2.   ATPase activity of membrane fractions. Total ATPase activity (open bars) and the ATPase activity in the presence of 20 µM ouabain (shaded bars) are shown. The specific Na+-K+-ATPase activity is represented as the differences between these 2 measurements (solid bars). Data are means ± SE of triplicate determinations from a single experiment, representative of several such experiments. E.R., endoplasmic reticulum; mem., membrane; ouab. sens., ouabain-sensitive activity.



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Fig. 3.   K+ dependence of Na+-K+-ATPase activity. High Five cells (72 h postinfection) were lysed and the membranes fractionated on a 5-step sucrose gradient (see Fig. 1). Each membrane fraction was assayed for ouabain-sensitive ATPase activity as the K+ concentration was increased as indicated. In each membrane fraction, the Na+-K+-ATPase-mediated ATP hydrolysis showed saturation kinetics with respect to K+. Each plot is a single experiment, representative of at least 3 experiments from different insect cell membrane preparations. A: endoplasmic reticulum. B: Golgi. C: plasma membrane. The half-maximal activation concentration (K1/2) calculated from all such experiments were (in mM) 2.6 ± 0.21, 3.1 ± 0.41, and 3.4 ± 0.66 for the endoplasmic reticulum, Golgi, and plasma membrane, respectively.


                              
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Table 1.   Postinfection time course of Na+-K+-ATPase expression

Quantitation of expressed Na+-K+-ATPase. Immunostaining density comparisons between membrane fractions was used as a means to estimate the amount of Na+-K+-ATPase subunits present in each fraction. For these experiments, we used commercially available antibodies for both the alpha - and beta -subunits, as well as a polyclonal antibody raised against a bacterially produced peptide corresponding to the rat alpha 1 ATP-binding domain (17). With the use of quantitative slot-blot analyses, a standard densitometry curve (Fig. 4B) was created by applying known amounts of purified dog kidney Na+-K+-ATPase to the PVDF membrane (Fig. 4A). The total protein applied to each well was kept constant by mixing purified dog enzyme with an appropriate amount of BSA. Likewise, the amount of protein added from the respective membrane fractions was equal to the total protein applied to the wells in the standard curve. This procedure effectively eliminates any differences, which could arise from saturating the binding capacity of the PVDF membrane. The results from several slot blots are summarized in Table 1. Our observations indicate that there is not overexpression of the renal enzyme producing large quantities of inactive protein. Specifically, in our High Five cell experiments, the heterologously expressed Na+-K+-ATPase comprises between 0.5 and 2% of the total membrane protein in the respective fractions. It is interesting to note that the ratio of Na+-K+-ATPase expression to total protein remains relatively constant over the postinfection times measured (Table 1). Indeed, differences in ouabain-sensitive ATPase activity between experiments always correlated well with differences in the Na+-K+-ATPase expression level seen with immunostaining (data not shown). This confirms that large quantities of inactive protein were not produced in this system.


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Fig. 4.   Quantitative slot-blot analysis. To get an estimate of the relative amounts of Na+-K+-ATPase present in each of the membrane fractions, we performed a quantitative slot-blot analysis according to the methods described by Bio-Rad for their slot-blotting system. A: example of an autoradiography of purified dog kidney Na+-K+-ATPase used to generate a standard curve. Duplicate samples were run in adjacent lanes and total protein was accounted for by adding BSA to a final total protein quantity of 10 µg/slot. The primary antibody used was developed against the ATP-binding domain of the rat alpha 1-subunit (15). The control curve (B) was generated by plotting the pixel density against the known amount of purified dog kidney Na+-K+-ATPase in the corresponding well. In this instance, the 20-ng quantity of Na+-K+-ATPase () was not included within the curve, as it obviously exceeds the resolution of the film. Triplicate determinations were used for the various membrane fractions. The slope of the standard curve was used to convert pixel densities into µg of Na+-K+-ATPase.

Assembly and trafficking of the alpha - and beta -subunits. High Five cells were infected with baculovirus constructs containing either alpha -only, beta -only, or alpha - and beta -cDNA. The separate membrane fractions were then isolated from the various infections and analyzed. Interestingly, we observed normal ouabain-sensitive activity from alpha /beta expressing cells even at the level of the endoplasmic reticulum, suggesting that these subunits are assembled early in the maturation pathway and that further processing is not required for activity (Table 1). Moreover, since the activity is similar in the different membranes, protein processing itself does not modify enzymatic activity. However, when the alpha -subunit was expressed in the absence of the beta -subunit, it was retained within the endoplasmic reticulum (Fig. 5A) and subjected to increased degradation (only detectable on larger gels, data not shown). These observations are consistent with the findings in Xenopus oocytes (18). In contrast, when the beta -subunit was expressed alone, it was processed correctly and sent to the plasma membrane (Fig. 5B); multiple bands in Fig. 5B are indicative of heterogeneous glycosylation of the translated beta -subunit. Ouabain-sensitive ATPase assays were performed on the endoplasmic reticulum, Golgi, and plasma membrane fractions from the various infections. There was no detectable Na+-K+-ATPase activity in any membrane fraction from either alpha -only- or beta -only-expressing High Five cells (data not shown), whereas the alpha /beta infected cells had activity in each fraction (Table 1).


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Fig. 5.   Electroblot analysis of the endoplasmic reticulum (ER), Golgi (G), and plasma membrane (PM) fractions from alpha /beta , alpha -only, and beta -only infected High Five cells. Insect membrane protein (30 µg) was loaded onto the corresponding lanes and run through a 7.5% Laemmli gel and electrotransferred onto a PVDF membrane. All measurements are in molecular weight standards (kDa). A: blots probed with anti-alpha antibody. Left: membrane fractions from alpha /beta infected cells. Right: membrane fractions from alpha -only infected cells. Clearly, when the alpha -subunit is expressed alone it remains within the endoplasmic reticulum fraction. B: blots probed with anti-beta antibody. Left: membrane fractions from alpha /beta infected cells. Right: membrane fractions from beta -only infected cells. The beta -subunit appears to be processed normally in the absence of the alpha -subunit.

Cation transport into Na+-K+-ATPase-infected insect cells. Ouabain-sensitive 86Rb+ uptake was measured on Sf9 cells (Rb+ is a congener for K+); Sf9 cells were used in these experiments because they adhere much better than High Five cells when grown as a confluent monolayer attached to a cell culture flask. 86Rb+ uptake is a powerful tool for determining the functional characteristics of Na+ pump expression in insect cells for several reasons: 1) the assay can be performed quickly and easily on very little material (e.g., 35 mm confluent layer of cells), 2) the signal-to-noise ratio is significant to make clear interpretations about Na+ pump function, and 3) in situations where large amounts of the expressed protein are nonfunctional and retained within the endoplasmic reticulum and Golgi (13), one can use whole cell transport to assess only the functional molecules that have been correctly targeted to the plasma membrane.

Figure 6 shows time-dependent Rb+ transport into noninfected Sf9 cells and cells infected with baculoviruses containing the Na+-K+-ATPase alpha - and beta -subunit cDNA; experiments were performed in the presence or absence of 20 µM ouabain. It is clear that cells producing sheep renal Na+-K+-ATPase have a significantly increased rate of Rb+ uptake that can be completely eliminated by the presence of ouabain. To more critically test the function of the plasma membrane Na+-K+-ATPase, we measured the Rb+ concentration dependence of both 86Rb+ transport and ATPase activity (Fig. 7, A and B, respectively). The Km for Rb+ was 3.2 ± 0.7 and 3.9 ± 1.1 mM for Na+-K+-ATPase and Rb+ uptake, respectively. These values agree well with the values for K+ dependence of ATPase activity shown in Fig. 3.


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Fig. 6.   Time course of 86Rb+ uptake into Sf9 cells. Sf9 cells grown attached to 6-well plates were either noninfected or infected with Na+-K+-ATPase alpha /beta -subunit-containing baculovirus and allowed to grow to confluence at 27°C (i.e., 72-84 h). Each well constituted an individual time point, and 20 µM ouabain was added to the bottom three wells of each plate. Uptake reactions were initiated by the addition of 86RbCl at a final concentration of 1 mM and performed at 25-27°C (i.e., room temperature). At the times indicated, the uptake reaction was stopped and the amount of 86Rb+ incorporation determined as described in MATERIALS AND METHODS; , alpha /beta infected, without ouabain; , alpha /beta infected, with ouabain; black-triangle, noninfected, without ouabain; open circle , noninfected, with ouabain. Data were fit to a linear equation; points represent the 3 corresponding wells within each category from a single experiment, representative of at least 4 separate experiments.



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Fig. 7.   Rb+ dependence of ouabain-sensitive 86Rb+ uptake and Na+-K+-ATPase activity. Sf9 cells were infected with Na+-K+-ATPase-containing baculoviruses and treated as in Fig. 6. However, in these experiments, the flux time was 3 min and the extracellular concentration of RbCl was varied as indicated on the abscissa. A: ouabain-insensitive Rb+ flux was linearly dependent on RbCl concentration ([RbCl]); this linear flux rate was subtracted from the total flux data at each [RbCl] to yield the specific Na+ pump component. Each point represents a single well (3 at each [RbCl]) from a single experiment, representative of at least 4 separate experiments. B: High Five cells were infected with Na+-K+-ATPase-containing baculoviruses and grown in spinner flasks at 27°C for 4 days. The plasma membrane fractions were prepared as described in MATERIALS AND METHODS. The assay medium was identical to that described in MATERIALS AND METHODS, with the exception that the [RbCl] shown replaced the KCl in the assay medium. The ouabain-insensitive ATPase activity was independent of [RbCl] and remained constant over the [RbCl] range used in these experiments. The ouabain-insensitive component was subtracted from the total ATPase activity to obtain the Na+ pump activity displayed. Duplicate determinations from a single experiment are shown, representative of at least 4 separate experiments. Data for both A and B were fit to the Michaelis-Menten equation.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Our results show that the alpha - and beta -subunits of the Na+-K+-ATPase assemble within the endoplasmic reticulum, and only then does the complex proceed to the plasma membrane. In addition, it appears that association of the alpha -beta heterodimer is sufficient for protein function, as the enzyme complex turns over normally within the endoplasmic reticulum and the K+ dependence of enzyme activity is unchanged between endoplasmic reticulum and plasma membrane. These determinations are possible because the endoplasmic reticulum, Golgi, and plasma membrane fractions from baculovirus-infected High Five cells can be separated via sucrose gradient centrifugation. This membrane separation also allowed us to follow protein processing as well as function throughout the trafficking pathway. In particular, we demonstrate that expression of the alpha -subunit alone resulted in endoplasmic reticulum retention, whereas the beta -subunit was processed and transported to the plasma membrane in the absence of the alpha -subunit. Quantitative immunostaining suggests that the heterologously expressed protein comprises between 0.5 and 2% of the total membrane protein from the various fractions and that the majority of the expressed Na+-K+-ATPase is functional.

The P2-type ATPase family is comprised of several enzymes, several of which exist in eukaryotic cells [e.g., Na+-K+-, gastric H+-K+-, yeast H+-, sarcoplasmic/endoplasmic reticulum Ca2+- (SERCA)-, and plasma membrane Ca2+ (PMCA)-ATPases] (for review see Ref. 35). Of these enzymes, all but SERCA are processed and delivered to the plasma membrane. Na+ pump trafficking is further complicated, as it requires assembly of an alpha -subunit (containing 10 transmembrane segments) with a beta -subunit (containing a single transmembrane segment), followed by delivery of this alpha -beta complex to the plasma membrane. In addition, during the assembly and trafficking process, the first five amino acids are cleaved from the alpha -subunit and the beta -subunit is glycoslylated. Our data suggest that all essential processing of the Na+-K+-ATPase occurs within the endoplasmic reticulum, since the characteristics of activity of the endoplasmic reticulum protein are the same as the protein residing within the plasma membrane. Consequently, it appears that any improperly folded or assembled Na+-K+-ATPase is rapidly degraded within the endoplasmic reticulum fraction in High Five cells. Indeed, it is becoming more apparent that the endoplasmic reticulum is not only a site for protein synthesis and folding, but also an organelle that performs protein editing (6, 27, 42). Endoplasmic reticulum editing appears to be the case in High Five cells infected with the alpha -subunit alone, since it is predominantly degraded within this organelle (Fig. 5A). However, there are conflicting reports in the literature concerning whether or not the Na+-K+-ATPase beta -subunit is essential for proper folding and trafficking of the alpha -subunit. For example, observations of alpha -only expression and processing to the plasma membrane have been initially reported in Sf9 cells (13) and A6 epithelia (12), but more recent studies in Sf9 cells found only intracellular retention (31); in contrast, endoplasmic reticulum degradation of alpha -only expression was reported in Xenopus oocytes (18) and in the present study on High Five cells. The reason for these differences remains unclear. Of these systems, Sf9 and High Five cells are the only ones in which trafficking of heterologously expressed alpha -subunits or alpha -beta complexes are not susceptible to influences from high levels of endogenous alpha - and beta -polypeptides. However, as yet there are no published reports of detailed analyses of trafficking in these cells to compare with such studies in oocytes (18). It is clear that there are different elements that control protein folding and assembly compared with those that regulate function. It has been shown that Na+-K+-ATPase with all 23 cysteines substituted in the alpha -subunit is more susceptible to degradation within the endoplasmic reticulum and thus has probably affected folding or assembly kinetics but is fully functional when delivered to the plasma membrane (24).

Activity level of expressed Na+-K+-ATPase. The initial application of the baculovirus system to Na+-K+-ATPase expression in insect cells concluded that the major portion of the enzyme was inactive (13). However, in the present study, it appears that most of the expressed enzyme is functional. Interestingly, our expression yields roughly the same amount of functional enzyme per milligram of total membrane protein as reported by DeTomaso and co-workers (13) in the absence of significant quantities of nonfunctional enzyme. They report similar activity and ligand-binding numbers (see Table 1 and Ref. 13) to the values reported here; however, they estimate their Na+-K+-ATPase content to be 5-10% of the membrane protein (compared to ~1% reported here). Indeed, they report a clearly discernible Coomassie blue-stained alpha -subunit band on SDS-PAGE, whereas we are unable to routinely observe discernible bands attributable to Na+-K+-ATPase subunits. The reason for this difference may reflect a difference between infecting cells with separate viruses for alpha  and beta  (13) and infecting with a single virus containing both alpha  and beta  (the present study).

In a more recent paper by Liu and Guidotti (31), using a baculovirus containing both alpha - and beta -cDNA, Na+-K+-ATPase expression in Sf9 cells was reported and cell fractionation was attempted. These authors were unable to obtain clearly separated membrane fractions, and several of their findings differ significantly from those reported here. They found that the majority of the expressed polypeptides were greatly aggregated and not functional, and thus they concluded that only ~15% of the expressed protein was functional.

Endogenous Na+-K+-ATPase activity in insect cells. A great advantage in the use of Sf9 and High Five cells is that the uninfected cells have zero or very low endogenous Na+-K+-ATPase levels. This raises several interesting questions: How low is low? Do contaminating levels affect our observations? Since the Na+-K+-ATPase is known to be expressed in insects, What is special about the cellular physiology of these cells? Although it is clear that insect cells have the potential to express the Na+-K+-ATPase, it has long been know that many insect cells in epithelia lack a ouabain-sensitive cation-activated ATPase or ouabain-sensitive cation pathway. It is likely that in these cells nutritional uptake is driven by proton gradients (rather than Na+ gradients), which are maintained by a plasma membrane V-type ATPase (45). We are unaware of direct studies on Sf9 or High Five cells, and their cellular physiology awaits such an examination.

We have made considerable efforts to document an endogenous level of Na+-K+-ATPase. Reports in the literature describe low levels of activity of Sf9 cells (5, 13). We have been unable to obtain reproducible measurements of activity in uninfected High five cells that reliably differ from zero. To examine whether or not infection with baculovirus per se affects this situation, we recently characterized the properties of High Five cells expressing a D369A mutant Na+-K+-ATPase. This mutation removes the catalytically essential phosphorylation site. The resulting membranes were devoid of ouabain-sensitive ATPase activity (Stephenson DA and Kaplan JH, unpublished observations). We are also unable to detect the presence of alpha - or beta -subunit polypeptides in uninfected cells using an array of antibodies to each subunit at very high titers. In another earlier paper on the expression of Na+-K+-ATPase in Sf9 cells, the issue of endogenous activity was also addressed (46). These workers concluded that their experiments create serious doubts about the existence of an endogenous enzyme in Sf9 cells with properties similar to any of the known Na+-K+-ATPase isoforms (46).

We believe that the infected cells are also very appropriate for transport studies, since our Rb+ uptake measurements indicate that ouabain can reduce the Rb+ flux to the level seen in uninfected cells. Furthermore, a ouabain-sensitive Rb+ uptake is not observable in uninfected cells. The issue of which transporters are responsible for the residual ouabain-resistant activity cannot yet be completely answered. However, if the flux measurements are performed in the absence of bumetanide, the ouabain-insensitive Rb+ uptake rates are enhanced, implying that there is a significant endogenous bumetanide-sensitive cotransport activity (Gatto and Kaplan, unpublished observations). There may also be K+ channels in the plasma membrane, which may mediate part of the residual uptake.

beta -Subunit role in Na+ pump maturation. It has been known for about a decade that expression of both the alpha - and beta -subunits is necessary for functional Na+-K+-ATPase activity (Refs. 40 and 23, respectively). Furthermore, there appears to be a consensus emerging that the beta -subunit may play a role in the maturation of the alpha -subunit (19) and in the trafficking of assembled Na+ pump molecules to the plasma membrane (3, 18, 22, 42). Indeed, our experiments support such a hypothesis, since the alpha -subunit was retained within the endoplasmic reticulum and was degraded in the absence of the beta -subunit. Conversely, we observed that the beta -subunit was processed, glycosylated, and delivered to the plasma membrane without any requirement for its partner. The observation that only the alpha -beta complex and beta -only are correctly delivered to the plasma membrane is consistent with the beta -subunit acting as a chaperone capable of preventing incorrect folding or degradation of the alpha -subunit. Our observation that the beta -subunit alone is trafficked in a normal fashion and appears in the endoplasmic reticulum, Golgi, and plasma membrane fractions in the absence of alpha -subunit is apparently different from a previous finding in oocytes (3). These authors found that beta -subunit expression without alpha -subunit resulted in predominantly endoplasmic reticulum retention. In contrast, H+-K+-ATPase beta -subunit was not retained. We do not yet know whether these kinds of effects are cell specific. However, in the insect cell system, we have the advantage that, when we express beta -only, we do not have to consider interactions with endogenous alpha -subunits; this is not the case in oocytes. Such considerations do not apply to the previous observations in Sf9 cells (31), which report that beta -only expression produces high-molecular-weight aggregates of beta , which are insoluble in Triton X-100 and formed via disulfide linkages. In earlier work with Sf9 cells (13), like our work, trafficking of beta -only to the plasma membrane was found; this was shown by immunocytochemical imaging of infected cells.

Consequently, it seems clear that the Na+-K+-ATPase does not have any other specific chaperone requirements, because it is processed correctly by High Five cells that lack an endogenous Na+-K+-ATPase, i.e., High Five cells would not likely produce a specific chaperone for a membrane protein that they do not normally express, and yet that have the ability to process this foreign protein when infected with the cDNA. Therefore, either the Na+-K+-ATPase contains chaperone-like qualities itself (e.g., the beta -subunit) or its assembly utilizes a general chaperone within the insect cell itself. Our conclusion that no specific chaperone seems to be required for Na+-K+-ATPase assembly and trafficking in insect cells may seem superfluous or redundant, since protein-specific chaperones have not been widely described. However, for another P2-type ATPase, such activities have been reported in yeast. From studies of PMA1p (plasma membrane H+-ATPase), the elements important in this ATPase trafficking pathway have been identified; these are reviewed extensively by Morsomme et al. (38). Briefly, MOP2p influences the amount of ATPase in the plasma membrane (39): AST1p, which targets mutated ATPase to the plasma membrane rather than the vacuole (11), and LST1p, which affects the selective transport of PMA1p from the endoplasmic reticulum to the Golgi (43).

Is the Na+-K+-ATPase functional in organelles? Until now, there have been no reports identifying at what stage during the assembly and trafficking process the Na+ pump becomes functional. Previous reports using indirect methods have suggested that fully processed and functional Na+-K+-ATPase is produced before its insertion in the plasma membrane of polarized epithelial cells from kidney (10) and toad bladder (48). In the present study, we directly confirm these suggestions, demonstrating that Na+-K+-ATPase expressed in insect cells is fully functional within the endoplasmic reticulum. This raises the question of whether the enzyme is working in the endoplasmic reticulum in vivo, and, if so, what physiological purpose might be served by this function. For example, it is well known that the gastric H+-K+-ATPase is functional while sequestered within intracellular vesicles and these vesicles are direct descendents of the trans-Golgi. Indeed, it is the extremely acidic environment of these vesicles that is exploited pharmacologically by thiophilic sulfenamides to specifically inhibit the H+-K+-ATPase (29, 44). The Na+ pump may also function at the organellar level and play an as yet undetermined role in cellular physiology.

Cation transport into Sf9 cells. For these whole cell flux experiments, we chose to use Sf9 cells instead of High Five cells, because baculovirus-infected High Five cells do not adhere strongly to the culture flask and are dislodged during the washing steps of the transport experiment (Gatto and Kaplan, unpublished observations). Because ouabain-sensitive Rb+ uptake is a very sensitive assay that only requires small amounts of cells per experiment, this protocol can be used to quickly screen mutant pumps to see if they are processed and delivered to the plasma membrane as well as to determine whether or not a specific mutant alters pump activity. However, the sensitivity of ouabain-sensitive Rb+ uptake is severely compromised by the existence of endogenous Na+-K+-ATPase. To date, the only Na+ pump expression systems not encumbered by an endogenous enzyme are baculovirus-infected insect cells and yeast (23, 41). The feasibility of measuring ouabain-sensitive Rb+ uptake in yeast remains unclear.

In conclusion, this report demonstrates that baculovirus-delivered Na+-K+-ATPase alpha - and beta -subunits are assembled and trafficked appropriately by High Five cells. More specifically, alpha -subunit processing has an obligatory requirement for simultaneous beta -subunit expression, whereas beta -subunit processing is self-sufficient. When both subunits are expressed, they appear to be assembled within the endoplasmic reticulum. In addition, we demonstrate that the alpha -beta complex is completely functional while still in the endoplasmic reticulum, which suggests that no further processing is necessary and which may imply that the enzyme is performing physiologically relevant transport in intracellular organelles.


    ACKNOWLEDGEMENTS

We thank Yi-Kang Hu and John Eisses for valuable input on the experimental design and protocols used in this study. In addition, we thank Dr. Charles Costa (Eastern Illinois University) for helpful comments on the manuscript and Dr. Elmer Price (Univ. of Missouri-Columbia) for the initial gift of sheep renal Na+-K+-ATPase alpha - and beta -cDNA. We also thank the anonymous reviewers of this paper for several helpful suggestions, which greatly improved its quality.


    FOOTNOTES

This work was supported by National Heart, Lung, and Blood Institute Grant HL-30315 to J. H. Kaplan.

Preliminary reports of this work were presented to the Biophysical Society and the Society of General Physiology (14, 15).

Address for reprint requests and other correspondence: J. H. Kaplan, Dept. of Biochemistry and Molecular Biology, L224, Oregon Health Sciences Univ., 3181 SW Sam Jackson Park Rd., Portland, OR 97201-3098 (E-mail: kaplanj{at}ohsu.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 29 August 2000; accepted in final form 9 April 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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