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
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ABSTRACT |
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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 - and
-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
-subunit or
-subunit
were expressed individually. Immunostaining of the separate membrane
fractions indicates that the
-subunit, when expressed alone, is
degraded early in the protein maturation pathway (i.e., the endoplasmic
reticulum) but that the
-subunit is processed normally and delivered
to the plasma membrane. Thus it appears that only the
-subunit has
an oligomeric requirement for maturation and trafficking to the plasma membrane. Furthermore, assembly of the
-
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 |
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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 -subunit of ~100 kDa with 10 transmembrane-spanning domains (25) and an additional 55 kDa
-subunit. The
-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 -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
-subunit produced distinctly different trypsin
proteolysis patterns of the
-subunit (34). Similarly,
loss of Na+-K+-ATPase activity after
reduction of the
-subunit S-S bridges was prevented when
K+ were occluded by the enzyme (33). These
data indicate that the
-subunit changes conformation concomitantly
with the
-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
- and
-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
-subunit as well as the part it plays in the maturation of the
-
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 -subunit has been reported to
play an essential role in the maturation of the
-subunit
(18). Specifically, when the
-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
-subunit facilitated
-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
-
assembly have included
immunoprecipitation experiments involving coexpression of truncated
-subunits (21, 42), chimeras between the
Na+-K+-ATPase and
H+-K+-ATPase
-subunits (and between
respective
-subunits) (20), and in vivo
translation/insertion experiments (2, 4). In general,
these studies conclude that the
-subunit is required for the
membrane insertion and plasma membrane targeting of the
-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
-subunit is not required to target the
-subunit to the plasma membrane. Specifically, DeTomaso and colleagues (13)
detected the Na+-K+-ATPase
-subunit on the
plasma membrane surface of Sf9 cells via immunocytochemistry
when it was expressed in the absence of the
-subunit. Even more
striking was the claim that Sf9 cells expressing the
-subunit alone had ouabain-sensitive ATPase activity independent of
the presence of the
-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
-subunits were delivered to the plasma membrane of A6
epithelia in the absence of the
-subunit (12). Thus the
issue of assembly of the Na+-K+-ATPase
-
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
- and
-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
-
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
- and
-subunits along the protein
maturation pathway. We found that when the
-subunit was expressed in
the absence of the
-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
-subunit. In contrast, expression of the
-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
-
dimer was fully functional even in the endoplasmic
reticulum, indicating that, after subunit assembly, no further
processing is necessary.
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MATERIALS AND METHODS |
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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, -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
1- and
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 pFASTBACDUAL (pFBD
) was constructed by subcloning the Na+ pump
- and
-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
-subunit cDNA was
introduced into the vector as an EcoRI/SpeI
fragment and the
-subunit cDNA as a SmaI/StuI fragment. DH10BAC cells were transformed with pFBD
to obtain recombinant baculovirus shuttle vectors (i.e., bacmids), which were
used subsequently for High Five cell transfections to generate recombinant baculovirus.
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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RESULTS |
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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
-subunit alone, the
-subunit alone, or both subunits simultaneously (data not shown).
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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 - and
-subunits, as well as a polyclonal
antibody raised against a bacterially produced peptide corresponding to
the rat
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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Assembly and trafficking of the - and
-subunits.
High Five cells were infected with baculovirus constructs containing
either
-only,
-only, or
- and
-cDNA. The separate membrane
fractions were then isolated from the various infections and analyzed.
Interestingly, we observed normal ouabain-sensitive activity from
/
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
-subunit was expressed in the
absence of the
-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
-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
-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
-only- or
-only-expressing High Five cells
(data not shown), whereas the
/
infected cells had activity in
each fraction (Table 1).
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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
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DISCUSSION |
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Our results show that the - and
-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
-
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
-subunit alone resulted in endoplasmic reticulum retention,
whereas the
-subunit was processed and transported to the plasma
membrane in the absence of the
-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 -subunit
(containing 10 transmembrane segments) with a
-subunit (containing a
single transmembrane segment), followed by delivery of this
-
complex to the plasma membrane. In addition, during the assembly and
trafficking process, the first five amino acids are cleaved from the
-subunit and the
-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
-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
-subunit is essential for
proper folding and trafficking of the
-subunit. For example,
observations of
-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
-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
-subunits or
-
complexes are not susceptible to influences from high levels of endogenous
- and
-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
-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 -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
and
(13) and
infecting with a single virus containing both
and
(the present study).
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-Subunit role in Na+ pump
maturation.
It has been known for about a decade that expression of both the
-
and
-subunits is necessary for functional
Na+-K+-ATPase activity (Refs. 40
and 23, respectively). Furthermore, there appears to be a
consensus emerging that the
-subunit may play a role in the
maturation of the
-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
-subunit was retained within the endoplasmic
reticulum and was degraded in the absence of the
-subunit.
Conversely, we observed that the
-subunit was processed,
glycosylated, and delivered to the plasma membrane without any
requirement for its partner. The observation that only the
-
complex and
-only are correctly delivered to the plasma membrane is
consistent with the
-subunit acting as a chaperone capable of
preventing incorrect folding or degradation of the
-subunit. Our
observation that the
-subunit alone is trafficked in a normal
fashion and appears in the endoplasmic reticulum, Golgi, and plasma
membrane fractions in the absence of
-subunit is apparently
different from a previous finding in oocytes (3). These
authors found that
-subunit expression without
-subunit resulted
in predominantly endoplasmic reticulum retention. In contrast,
H+-K+-ATPase
-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
-only, we do not have to consider interactions with
endogenous
-subunits; this is not the case in oocytes. Such
considerations do not apply to the previous observations in Sf9
cells (31), which report that
-only expression produces
high-molecular-weight aggregates of
, which are insoluble in Triton
X-100 and formed via disulfide linkages. In earlier work with
Sf9 cells (13), like our work, trafficking of
-only to the plasma membrane was found; this was shown by
immunocytochemical imaging of infected cells.
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 ![]() |
ACKNOWLEDGEMENTS |
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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 - and
-cDNA. We also thank
the anonymous reviewers of this paper for several helpful suggestions,
which greatly improved its quality.
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FOOTNOTES |
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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.
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