(Received for publication, October 2, 1995; and in revised form, November 20, 1995)
From the
Studies of structure-function relationships in Na,K-ATPase
require high yield expression of inactive mutations in cells without
endogenous Na,K-ATPase activity. In this work we developed a
host/vector system for expression of fully active pig Na,K-ATPase as
well as the inactive mutations D369N and D807N at high levels in Saccharomyces cerevisiae. The 1- and
1-subunit cDNAs
were inserted into a single 2-µm-based plasmid with a high and
regulatable copy number and strong galactose-inducible promoters
allowing for stoichiometric alterations of gene dosage. The
protease-deficient host strain was engineered to express high levels of
GAL4 transactivating protein, thereby causing a 10-fold increase in
expression to 32,500 ± 3,000 [
H]ouabain
sites/cell. In one bioreactor run 150-200 g of yeast were
produced with 54 ± 5 µg of Na,K-pump protein/g of cells.
Through purification in membrane bound form the activity of the
recombinant Na,K-ATPase was increased to 42-50 pmol/mg of
protein. The Na,K dependence of ATP hydrolysis and the molar activity
(4,500-7,000 min
) were close to those of
native pig kidney Na,K-ATPase. Mutations to the phosphorylation site
(D369N) or presumptive cation sites (D807N), both devoid of Na,K-ATPase
activity, were expressed in the yeast membrane at the same
-subunit concentration and [
H]ouabain
binding capacity as the wild type Na,K-ATPase. The high yield and
absence of endogenous activity allowed assay of
[
H]ATP binding at equilibrium, demonstrating a
remarkable 18-fold increase in affinity for ATP in consequence of
reducing the negative charge at the phosphorylation site (D369N).
Na,K-ATPase is responsible for the generation and maintenance of
transmembrane Na/K
ion gradients
required for generation of action potentials, control of cell volume,
and secondary active transport. The functional Na,K-pump is a
heterodimer consisting of one
-subunit and one
-subunit(2) . At least three isoforms of each subunit are
expressed in a tissue- and development-specific way. The
-subunit
isoforms consist of 1,016-1,023 amino acids and traverse the
membrane eight to ten times. The
-subunit is an N-glycosylated protein, consisting of 302 residues, with a
single transmembrane domain and three disulfide
bonds(3, 4) .
The production of recombinant
Na,K-pumps is potentially a powerful approach for study of
structure-function relationships, but heterologous expression of
membrane proteins like the heterodimeric -unit of
Na,K-ATPase, with multiple transmembrane segments, has proven to pose
special problems. The Na,K-pump has been expressed in the yeast Saccharomyces cerevisiae(1) , COS cells(5) ,
HeLa cells(6) , NIH 3T3 cells(7) , Xenopus
oocytes(8) , and in insect cells(9) . The
properties of isozymes of Na,K-ATPase have been analyzed (10, 11) and important structure-function correlates
have been established by work on
mutants(5, 7, 12, 13) . However, it
has been difficult to express Na,K-ATPase in large quantities and
higher eucaryotic cell lines like HeLa (6) and COS cells (5) and Xenopus oocytes (8, 14) express endogenous Na,K-ATPase of almost the
same magnitude as the transfected activity. The ouabain selection
methodology (5, 6) does not allow analysis of the
interesting mutants that are blocked in the reaction cycle. The
expression level in baculovirus infected insect cells is high, but only
a small fraction of the recombinant pumps are enzymatically active, and
insect cells also possess endogenous Na,K-ATPase
activity(9, 11) . A major advantage of yeast cells for
expression studies is their lack of endogenous Na,K-ATPase activity.
The work of Farley and co-workers(1, 15, 16) demonstrated that yeast cells are capable of expressing
fully active Na,K-ATPase at the cell surface, but the expression is
limited to levels of 2-4 pmol/mg of protein as determined by
[
H]ouabain binding.
In the present work the
capacity of S. cerevisiae for the production of enzymatically
active Na,K-pumps was characterized with respect to the dependence upon
gene copy number, promoter strength, and growth medium composition. The
1- and
1-subunit gene dosage was altered in parallel by
insertion of their cDNAs into a single plasmid with a particularly high
and regulatable copy number. The promoter activity was increased by the
combination of a strong galactose regulated promoter and a host strain
modified to express high levels of GAL4-transactivating
protein(17) . The plasmid was constructed to allow separation
of the growth of the host cells from the phase of Na,K-ATPase
expression. Computer-controlled bioreactors were used to increase the
yield and growth experiments were performed to examine the influence of
medium composition and induction time using
[
H]ouabain binding to assess the expression
levels. Established methods were modified for partial purification of
Na,K-ATPase from the yeast plasma membranes, in conditions where the
endogenous H-ATPase was removed. The quantity, ligand binding, and
enzymatic properties of the recombinant Na,K-ATPase were determined.
The versatility of the expression system was examined by
characterization of mutations to two side chains of the
-subunit,
the D369N mutation of the phosphorylated side
chain(7, 18) , and D807N at a presumptive site for
cation binding(19) . These mutants were devoid of Na,K-ATPase
and potassium-dependent para-nitrophenyl phosphatase
activities, but they could be expressed in the yeast membranes at the
same
-subunit concentration and [
H]ouabain
binding capacity as the wild type Na,K-ATPase. The high yield of
Na,K-ATPase from yeast and the absence of endogenous activity allowed
assays not previously achieved for recombinant enzyme, such as
[
H]ATP binding at equilibrium.
The Klenow polymerase-treated
941-bp NcoI-1-DraI fragment from pNK
31 was
ligated into XhoI-digested, Klenow polymerase-treated RS421.
The resulting plasmid carries the NcoI-
1-DraI
fragment between the PMA1 (yeast H-ATPase) promoter and terminator
region. The promoter-
1-terminator region present on a HindIII fragment was cloned into the unique HindIII
site in pEMBLyex4 in the correct orientation with respect to the
CYC-GAL promoter. A 1,637-bp NruI-HpaI fragment was
excised from the plasmid to eliminate one of three KpnI sites.
The resulting plasmid was digested with KpnI and religated to
remove the PMA1 promoter from the plasmid. The resulting
1
expression plasmid contains the pig
1 cDNA under control of the
CYC-GAL promoter. The final
1
1 expression plasmid, pPAP1466,
carries the 5.0-kilobase NruI-CYC-GAL-
1-HindIII
fragment inserted into the NruI-HindIII-digested
1 expression plasmid.
The plasmid pPAP1647 contains the NheI-URA3-Amp-BglI fragment from pYES2.0 and the NheI-P15A ori-Amp-BglI fragment from pACYC177 (34) . The ScaI-P15A ori-ScaI fragment from
pPAP1647 was cloned into ScaI-digested pEMBLyex4. The
resulting plasmid pPAP1657 has a 100-fold lower copy number in E.
coli compared with pEMBLyex4. The 1
1 expression plasmid
pPAP1666 carries the ApaI-P15A ori-AatII fragment
from pPAP1657, the ApaI-
1-AatII fragment, and
the AatII-
1-AatII fragment from pPAP1466. The
resulting
1
1 expression plasmid has a 100-fold lower copy
number in E. coli compared to pPAP1466.
The plasmid pPAP1485 bears the 3.4-kilobase pair EcoRI-GAL10-GAL4-HindIII fragment from pKHint-C inserted into EcoRI and HindIII-digested pUC19. pPAP1488 was constructed by inserting the 850-bp EcoRI-TRP1-EcoRI fragment from pEMBLYr25 into EcoRIdigested pPAP1485.
For large scale production, 1000 ml of preculture was used to inoculate 10 liters of minimal medium in an Applicon® fermentor equipped with an ADI 1030 Bio Controller. The culture was agitated at 158 rpm, and air was supplied through a 0.2-µm filter. The carbon source was 5% glucose and 2% lactate, and the medium was supplemented with all amino acids except leucine, tryptophan, and histidine. During growth at 30 °C in glucose, the pH of the medium was kept at 6.0 by computer-controlled addition of 1 M NaOH. The shift from growth on glucose to growth on lactate was monitored as an increase in pH of the growth medium and a decrease in growth rate. At this point galactose was added to a final concentration of 2%. Cells were harvested after 48 h.
For ATP binding experiments, gradient membranes were incubated at 2 mg of protein/ml in lysis buffer with 0.3 mg/ml SDS and proteolysis inhibitors for 30 min at 20 °C. The mixture was centrifuged for 30 min at 70.000 rpm in the Beckman 100 A ultracentrifuge and resuspended in lysis buffer.
For assay of vanadate sensitive H-ATPase
activity(33) , 10-µl portions were transferred to test
tubes containing 1 ml of 50 mM MES adjusted to pH 6.5 with
Tris, 5 mM MgSO, 50 mM KNO
(to inhibit vacuolar ATPase), 5 mM sodium azide (to
inhibit mitochondrial ATPase) 0.2 mM ammonium molybdate (to
inhibit acid phosphatase), and 2 mM Na
ATP without
and with 1 mM NaVO
. After 10 min at 37 °C, the
reaction was stopped, and inorganic phosphate was measured as described
above for Na,K-ATPase.
Molecular sieve high performance liquid
chromatography was performed with a TSK 3000 SW (7.5 300 mm)
Toyo Soda gel filtration column with a TSK SW guard column (7.5
75 mm) operated at flow rates of 0.2 ml/min using a Pharmacia Biotech
Inc. solvent delivery system as before (40) . Prior to
solubilization, the membrane-bound Na,K-ATPase was sedimented at
100,000 rpm for 10 min in the Beckman Airfuge and resuspended in 300
mM potassium acetate, pH 6.0, 2 mM dithiothreitol, 2
mM EDTA-Tris. This suspension was mixed with equal volumes of
C
E
, 10 mg/ml. The insoluble residue was
removed by centrifugation for 10 min at 100,000 rpm in a Beckman
Airfuge(40) .
Figure 1:
Structural map of the 15.7-kilobase
yeast 1
1 expression plasmids, pPAP1466 and pPAP1666.
Abbreviations used:
1(pig), the
1-subunit cDNA from
the pig kidney Na,K-ATPase;
1(pig), the
1-subunit
cDNA from pig kidney Na,K-ATPase; CYC-GAL P, a hybrid promoter
containing the GAL10 upstream activating sequence fused to the
5`-nontranslated leader of the cytochrome-1 gene; 2µ, the
2-µm origin of replication; URA3, the orotitin-5`-P
decarboxylase gene; leu2-d, a poorly expressed allel of the
-isopropylmalate dehydrogenase gene; pMB1, the pMB1
origin of replication; P15A, the P15A origin of replication. Amp
, a
-lactamase gene; T,
the PMA1 (H-ATPase from yeast) terminator region. Arrows indicate the direction of
transcription.
The two expression plasmids differ in their E.
coli origin of replication in that pPAP1466 is a pUC derivative (31) with a very high copy number, while pPAP1666 is a
P15A-derived plasmid (34) with a 100-fold reduced copy number
in E. coli compared with pPAP1466. The use of pPAP1666 with a
low copy number in E. coli was necessary, as we were unable to
clone various 1-subunit mutations into pPAP1466.
The yeast
strain BJ5457(20) , lacking the PEP4 and PRB1 protease
activities, was chosen as the basic host strain for the production of
recombinant Na,K-ATPase in order to reduce the possibility of
proteolytic degradation of the enzyme during synthesis and
purification. The transactivating GAL4 protein is known to be limiting
for expression controlled by galactose-regulated
promoters(17) . The GAL4 protein level can be increased by
integrating a GAL10-GAL4 transcriptional fusion into the yeast
chromosome(17) . Therefore, we constructed the
yeast-integrating plasmid pPAP1488 (Fig. 2), which was targeted
to the trp1 locus of BJ5457 by homologous recombination
generating strain PAP1500. Addition of galactose to PAP1500 transformed
with pPAP1466 or pPAP1666 should initiate a cascade reaction leading to
expression of GAL4 protein and Na,K-ATPase 1- and
1-subunits.
Figure 2:
Structural map of the 6.6-kilobase yeast
integrating plasmid pPAP1488. Abbreviations used: GAL10 P, the
GAL10 promoter; GAL4, the GAL4 transactivator gene; TRP1, the PR-antranilate isomerase gene; pMB1, the
pMB1 origin of replication; Amp, a
-lactamase gene. The arrow indicates the direction of
transcription.
Figure 3:
Separation of the yeast growth phase and
the Na,K-pump production phase: without galactose induction ( and
with addition of galactose 2% (
. Growth of PAP1500(pPAP1466) was
in minimal medium at 30 °C with 0.5% glucose or 2% lactate as
carbon source. Yeast cells were growing exponentially with glucose as
carbon source between A
= 0.04 and A
= 0.5 and exponentially with lactate as
carbon source between A
= 0.5 and A
= 1.0. Induction of Na,K-pump
biosynthesis with 2% galactose at A
= 1.0
and higher is seen to arrest cell growth. Abbreviations: GLU,
growth on glucose; LAC, growth on lactate; GAL,
addition of galactose to a final concentration of
2%.
Integration of a GAL10-GAL4
transcriptional fusion in the yeast chromosome had a dramatic effect on
the level of expression of Na,K-ATPase, in media selecting for the high
plasmid copy number. Fig. 4shows that induction of Na,K-ATPase
was much faster and that the maximal number of
[H]ouabain binding sites was about 10-fold higher
in cells with the chromosomal fusion than in the yeast strain, with
only a few copies of GAL4 protein/cell.
Figure 4:
The
accumulation of [H]ouabain sites in the membranes
of yeast strains without (
) or with (
) a transcriptional
GAL10-GAL4 fusion. At the indicated times 80-ml aliquots of the growth
medium were removed and crude cell membranes were prepared. Aliquots
containing 200 µg of protein were assayed for binding at 10 nM [
H]ouabain as described under
``Experimental Procedures.''
A number of experiments were
performed to determine the influence of composition of the growth
medium and induction time on the time course of appearance of high
affinity [H]ouabain sites measured in crude yeast
membranes. Fig. 5shows that the highest density of
[
H]ouabain sites is seen in yeast cells, with a
high copy number of the expression plasmid, and growing in synthetic
minimal medium supplemented with all amino acids except leucine,
tryptophan, and histidine. In medium without the supplement of amino
acids, accumulation of Na,K-ATPase ceased after 48 h, and then the
level decreased, suggesting that low levels of amino acids may be
rate-limiting for synthesis of Na,K-ATPase.
Figure 5:
Effect of composition of growth medium on
accumulation of ouabain sites. Crude yeast membranes were isolated from
strain PAP1500(pPAP1466) after induction with 2% galactose at time 0.
Procedures for membrane preparation and
[H]ouabain binding were as in Fig. 4.
Cells were grown with 0.5% glucose and 2% lactate as carbon source
prior to induction, as illustrated in Fig. 3. Abbreviations
used: - leu - aa, growth in the absence of leucine
and the presence of lysine; + leu - aa, growth in
the presence of leucine and lysine; -leu + aa,
growth medium supplemented with all amino acids except leucine,
tryptophan, and histidine.
In
crude yeast membranes, the binding capacity for
[H]ouabain was 10-15 pmol/mg of protein.
For characterization of the recombinant enzyme, it was essential to
reduce the endogenous H-ATPase activity and to purify the enzyme by
removal of extraneous protein. Fig. 6shows the SDS curve
required for application of the purification scheme developed
previously for purification of the Na,K-ATPase from
kidney(38, 39) . In contrast to the 3-5-fold
activation of Na,K-ATPase in kidney membranes after incubation with
SDS, due to demasking of closed right-side-out vesicles, the activity
of Na,K-ATPase or H-ATPase in yeast cell membranes increased only about
20%. At the membrane protein concentration of 2 mg/ml, an optimum
concentration range for SDS was found (0.5-0.7 mg/ml) where most
of the H-ATPase of the yeast cell membranes was inactivated, while
[
H]ouabain binding and Na,K-ATPase activity of
the recombinant enzyme were preserved. The peak of
[
H]ouabain binding in crude yeast cell membranes
was found at equilibrium densities in the range 1.15-1.2
g/ml(42) . However, after incubation with 0.64 mg/ml SDS, it
was difficult to recover the recombinant Na,K-ATPase after
centrifugation in discontinuous sucrose gradients, because the activity
was distributed in a broad band at relatively low densities around 1.10
g/ml. The [
H]ouabain binding data in Fig. 7show a 3-5-fold enrichment of binding capacity from
10-15 pmol/mg [
H]ouabain binding in crude
yeast membranes to 42-50 pmol/mg of protein in the partially
purified preparation. Note that the dissociation constant was reduced
from 21 ± 2 nM to 11 ± 2 nM upon
incubation with SDS and fractionation, presumably because the detergent
promotes the access of the ligands, Mg
and vanadate,
required for facilitation of [
H]ouabain binding.
Figure 6:
Demasking and inactivation of H-ATPase
() and [
H]-ouabain binding (
) of
crude yeast cell membranes during incubation with SDS in presence of
ATP. Membrane protein at 4 mg/ml was incubated at 20 °C in 200
µl with SDS at the indicated concentrations in a medium containing
1 mM EDTA, 3 mM Na
ATP, 10 mM MES-Tris, pH 7.0. For assay of vanadate-sensitive H-ATPase
activity, 10-µl portions were transferred to test tubes and
analyzed as described under ``Materials and Methods.'' For
assay of [
H]ouabain binding, portions of 25
µl containing 100 µg of membrane protein were transferred to
centrifuge tubes containing 1 ml of binding medium with 10 nM [
H]ouabain without or with 1 mM unlabeled ouabain. Other components and separation of free and
bound ligand were as described under ``Materials and
Methods.''
Figure 7:
Scatchard plots of
[H]ouabain binding to crude yeast cell membranes
isolated from strain PAP1500(pPAP1466) (
) and partially purified
recombinant Na,K-ATPase (
). Aliquots of the preparations
containing 100-200 µg of protein were incubated with
[
H]ouabain and unlabeled ouabain at final
concentrations ranging from 10.8 to 161 nM. The data were
fitted by nonlinear least squares regression (line). The K
and binding capacities were 21 ±
2 nM and 10.0 ± 0.5 pmol/mg of protein for the crude
membranes and 11 ± 2 nM and 44 ± 2 pmol/mg of
protein for the purified Na,K-ATPase.
A higher yield, but a lower binding capacity (15-25 pmol/mg of protein, cf. Fig. 12), were obtained when the crude membranes were fractionated on sucrose gradients prior to SDS incubation. The gradient membranes at the interphase between 15 and 40% (w/v) sucrose were collected by centrifugation and subsequently incubated at 2 mg of protein/ml with SDS in a lower concentration, 0.5 mg/ml SDS in the presence of 3 mM ATP, or at 0.3 mg/ml SDS in the absence of ATP.
Figure 12:
Scatchard plot of
[H]ATP binding to SDS-treated gradient membranes
of wild type and mutant D369N recombinant Na,K-ATPase. Procedures for
preparation of gradient membranes, incubation with SDS, and assay of
[
H]ATP in NaCl or KCl medium were as described
under ``Materials and Methods.'' The data were fitted by
nonlinear least squares regression to the lines with K
-109 ± 11 nM and
a binding capacity of 14.1 ± 0.8 pmol/mg of protein for wild
type (WT) and K
-5.9
± 0.4 nM and a binding capacity of 21 ± 1
pmol/mg of protein for D369N.
Fig. 8shows a comparison of Western
blots of the recombinant Na,K-ATPase from this preparation and renal
Na,K-ATPase. From this comparison, the purity in terms of -subunit
protein was 0.71% (7.1 µg of
-subunit protein/mg of
SDS-extracted yeast membrane protein). The purity in terms of
[
H]ouabain sites was 0.65%, corresponding to 44
pmol
mg
protein/6.8 nmol
mg
protein
100. The similarity of the two values suggests
that the unit binding one molecule of [
H]ouabain
contains one recombinant
-subunit. The Na- and K-ion dependence of
ATP splitting in Fig. 9is identical to that observed before for
preparations from pig kidney. The molecular activity of Na,K-ATPase in
the preparation was 4,500-7,000
P
min
per
[
H]ouabain site. This value is in the lower range
of the molecular activity of the native pig kidney Na,K-ATPase
(7,000-8,000 P
min
).
Figure 8:
Western blot of SDS-extracted membranes
from yeast cells. Recombinant wild type -units (lanes
a-e), recombinant D369N
-units (lanes j-m),
recombinant D807N
-units (lanes r-u) partially
purified pig kidney Na,K-ATPase (lanes f-i, n-q, and v-z) after separation by SDS-polyacrylamide gel
electrophoresis. The amount of pig kidney Na,K-ATPase is calculated as
µg
-units assuming one
-unit per
[
H]ouabain binding site ((35) ) as
determined from Scatchard analysis, see ''Materials and
Methods.`` Protein in lanes a, j, and r, 0.48
µg; in lanes b, k, and s, 1.0 µg; in lanes c, l, and t, 5.0 µg; in lanes d,
n, and u, 10.0 µg; and in lane e, 20.0
µg. Na,K-ATPase
-unit in lanes f, n, and v, 0.016 µg; in lanes g, o, and x 0.031
µg; in lanes h, p, and y, 0.078 µg; and in lanes i, q, and z, 0.156 µg. Arrows indicate the position of the
-subunit. From this
standardization, the purity in terms of
-subunit protein was
estimated from scans of the blots to be 0.71% for wild type, 0.68% for
D369N, and 0.54% for D807N.
Figure 9:
Na,K-ATPase activity of partially purified
recombinant Na,K-ATPase as a function of Na and
K
concentrations. The reaction mixture contained 3
mM ATP at pH 7.5 and 37 °C. Isotonicity was maintained by
exchanging NaCl and KCl in the medium for assay of Na,K-ATPase
described under ``Materials and
Methods.''
Chromatography of soluble Na,K-ATPase in CE
on TSK 3000 SW size exclusion columns was shown to be a sensitive
procedure for detecting the aggregation of
-units
accompanying thermal denaturation of Na,K-ATPase(40) . In the
experiment in Fig. 10, it is seen that the
[
H]ouabain complex of soluble, wild type,
recombinant Na,K-ATPase is stable during chromatography in
C
E
at 2-4 °C and that it elutes
quantitatively at a volume (9.0 ml) corresponding to the elution volume
(8.5 ml) of the [
H]ouabain complex with the
-unit of kidney Na,K-ATPase. The elution volume of the D369N
mutant
-unit was in the same range (9.0 ml). This provides
evidence for organization of the recombinant, wild type, and mutant
D369N, Na,K-ATPase in
-units with hydrodynamic properties
similar to those of native renal Na,K-ATPase. There was no tendency for
aggregation of the soluble protein to oligomers similar to the
(
-
) aggregates demonstrated in solubilized preparations of
the recombinant
-subunit from SF9 cells infected with baculovirus (43) .
Figure 10:
Size-exclusion chromatography of
recombinant Na,K-ATPase. The method was as described in (40) using 7.5 300-mm plus 7.5
75-mm TSK-gel G
3000 SW column equilibrated with C
E
, 5 mg/ml,
in 150 mM potassium acetate, pH 6.0, and operated at 0.2
ml/min at 4 °C. Prior to solubilization in C
E
((40) ), Na,K-ATPase was incubated with 100 nM [
H]ouabain, 3 mM MgSO
,
and 1 mM NaTris
VO
as in (39) .
Figure 11:
Scatchard plots of
[H]ouabain binding to wild type Na,K-ATPase
(
) and the mutants (
) D369N and D807N in medium containing
MgSO
plus vanadate. Aliquots of the preparations containing
100-200 µg of protein were incubated for 60 min at 37 °C
with [
H]ouabain as described under
``Materials and Methods.'' The data were fitted by nonlinear
least squares regression (lines) to give the following
capacities and dissociation constants (K
): D807N, 10.5 ± 0.4 pmol/mg of
protein and 13 ± 2 nM; wild type (WT), 10.0
± 0.5 pmol/mg of protein and 21 ± 2 nM; D369N,
10.9 ± 0.3 pmol/mg of protein and 279 ± 13
nM.
The data in Fig. 12show that linear Scatchard
plots were obtained for both wild type and the mutant D369N and that
the mutation D369N led to a remarkable change in dissociation constant
of the protein-ATP complex. The capacities for binding were in the same
range as the capacities for [H]ouabain binding
and the concentrations of
-subunit protein (cf. Fig. 8), suggesting that there was one high affinity binding
site for ATP or ouabain per
-unit (cf. (39) ). However, the dissociation constant for wild type (109
± 11 nM) was 18-fold higher than for the D369N mutant
(5.9 ± 0.4 nM) (Fig. 12) and 32-fold higher than
for the D369A mutant (3.4 ± 0.3 nM, data not shown).
The concept behind the host/vector system for Na,K-ATPase
expression in this study was to separate the phase of yeast cell growth
with low basal expression from the expression phase, where the
expression level of Na,K-ATPase in the yeast cell membranes increases
to a maximum. In the latter phase, the expression system allowed for a
high gene copy number and a high transcriptional activity due to the
strong inducible promoter. Precise alteration of the :
gene
dose required that the
and
cDNAs be present on the same
plasmid and be expressed under control of identical promoters. A
regulated increase in plasmid copy number in the Na,K-ATPase production
phase was possible, due to the presence of a URA3 gene and the poorly
expressed leu2-d gene on the expression plasmid. Selection for
leucine autotrophy increased the plasmid copy number and simultaneously
elevated the pump concentration 2-3-fold (Fig. 5). This
change is smaller than the 8-fold increase in expression of
schistosomal antigen P28-I observed upon changing from selecting for
uracil to leucine autotrophy(45) . However, these data are not
directly comparable as the schistosomal antigen is a soluble protein,
while Na,K-ATPase is inserted into the membrane. Transcriptional
activity was increased by engineering the yeast host strain to express
elevated concentrations of the GAL4-transactivating protein, causing a
10-fold increase in the expression level (Fig. 4). This is
consistent with the finding of Schultz et al.(46) who
reported a 10-fold increase in the expression level of the membrane
bound Epstein-Barr virus gp350 protein after integration of a
GAL10-GAL4 fusion into the genome of the yeast host strain. The high
transcriptional activity may cause subsequent steps in the synthesis
process to become rate-limiting and account for the 2-fold increase in
expression level upon supplementing the yeast growth medium with amino
acids (Fig. 5).
The combined effect of engineering these
parameters was to increase the density of Na,K-pumps to 32,500 ±
3,000 sites/cell or 54 ± 5 µg of Na,K-pump protein/g of
yeast cell. In the crude membrane fraction from the yeast (10-15
pmol/mg of protein) and in the SDS-extracted membranes (42-50
pmol/mg of protein), the pump density was higher than achieved
previously in eucaryotic cells, cf. Table 1. The
recombinant enzyme is fully active, with one site for binding of ATP
and ouabain per -subunit and a range of molecular activities close
to those of the native Na,K-ATPase of pig kidney. The relatively high
activity and the lack of endogenous background allowed for analyses not
previously achieved for recombinant Na,K-ATPase, such as ATP binding at
equilibrium.
An important reason for separating the growth phase
from the phase of Na,K-ATPase biosynthesis is the apparent toxicity of
Na,K-ATPase protein synthesis. The evidence for this is the immediate
arrest of cell growth and division following the galactose induction of
Na,K-ATPase biosynthesis. This could be due to the activity of the
Na,K-pump, but arrest of cell growth was independent of the enzymatic
activity of the expressed pump as yeast strains expressing wild type or
inactive mutations behaved identically. Also, the expression levels
obtained in this work do not, in general, prohibit yeast cell growth.
Much higher levels of constitutive heterologous expression has been
described for soluble proteins(47) . Also the endogenous GAL1
protein accumulates to 0.8% of total cell protein after galactose
induction without affecting cell viability(48) . It is
therefore reasonable to propose that the toxicity of Na,K-ATPase
protein synthesis is due to perturbation of the yeast membranes
following the insertion of protein with hydrophobic membrane-embedded
sequences. Previous experiments with membrane proteins, OmpA (49) and -hemagglutinin(50) , demonstrated
selection against the production of hydrophobic intramembrane segments
in response to the poor tolerance by the host cell. Selection for low
expressing variants has been observed in several cases, where
constitutive promoters were used to drive expression of membrane
proteins(51, 52) . In the present expression system,
the selection pressure was avoided through the use of a strong
inducible promoter, but the toxicity of the expression suggests that
the capacity of the yeast membrane system may set the limit for the
concentration of Na,K-ATPase that can be achieved in these cells.
The highest expression level of an ATPase in yeast has been
described for isoforms of the Arabidopsis thaliana H-ATPase
using a multicopy plasmid and the strong constitutive yeast PMA-1
(plasma membrane H-ATPase) promoter(33) . This expression
system directs the plant proton pump to the endoplasmic reticulum
membranes, where it constitutes 45% of the endoplasmic reticulum
proteins, but the plant proton pump may also complement the proton pump
in the yeast plasma membrane(53) , like the Neurospora H-ATPase(54) . In our experience, insertion of
-unit cDNA in this plasmid did not lead to expression of
significant amounts of Na,K-ATPase. (
)The constitutive PMA-1
host/vector system (33) also proved inefficient for expression
of the SR-Ca-ATPase(55) . The Na,K-ATPase expression system in
yeast described by Horowitz et al.(1) also used
constitutive promoters and achieved only low expression levels, 0.05%
of the plasma membrane protein, cf. Table 1. It seems
that the high expression level obtained by constitutive expression of
the plant H-ATPase is the exception, while a high expression level for
Na,K-ATPase and SR-Ca-ATPase is incompatible with constitutive
promoters. A probable explanation could be that insertion of H-ATPase
into yeast membranes is less prone to eliciting selection for low
expressing variants than is expression of the Na,K- or Ca-ATPases. In
agreement with these notions, the SR-Ca-ATPase has recently been
expressed in a relatively high concentration, 0.3% in the yeast
membranes, under control of a galactose-regulated
promoter(55) .
For the purification of the recombinant
Na,K-ATPase, it was important to establish conditions where the plasma
membrane H-ATPase could be removed. This enzyme constitutes background
activity in the enzyme assay and [H]ATP binding
experiments. Fortunately, the H-ATPase turned out to have a relatively
high sensitivity to denaturation by incubation with SDS. Our data show
that Na and K dependence of ATP hydrolysis, and ATP binding, of the
recombinant enzyme are similar to the kinetics of the native renal
Na,K-ATPase. In the best of our preparations, the molar activity was
close to 7,000 P
/min, but the number varied in the range
4,500-7,000 P
/min for reasons that are not yet fully
understood. A variation in molecular activity is also apparent among
the previous preparations from yeast, in the range from 792 to 8212
P
/min(1, 15, 56, 57) , cf. Table 1. The relatively large scatter of the
reported molecular activity among preparations from different host
systems may reflect difficulties in determining site concentrations in
preparations with relatively low activity. The highest expression in
terms of
-subunit protein is achieved in baculovirus-infected Sf-9
cells(9) , but only about 3% of the protein is active in terms
of Na,K-ATPase activity and ligand binding, Table 1. The insect
cells(9, 43) , as well as the COS-1 (5) and
HeLa (10, 20, 58) cells, also express
endogenous Na,K-ATPase activity as denoted in Table 1.
The
yeast expression system clearly does not distinguish active Na,K-ATPase
from inactive mutant -units with respect to biosynthesis and
translocation to the cell membranes. The D369N and D807N mutations were
expressed in yeast membranes at concentrations of
-subunit and
[
H]ouabain binding sites that are comparable with
those of the wild type enzyme. For the D369N mutant it was further
demonstrated that the concentration of protein expressed in the yeast
membranes was equal to the concentration of
[
H]ATP sites. The
-units of the D369N
mutant and recombinant Na,K-ATPase have the same hydrodynamic
properties as purified Na,K-ATPase from kidney.
Mutation at the
phosphorylation site of the negatively charged aspartic acid 369 for an
asparagine residue caused a remarkable decrease in dissociation
constant of the protein-ATP complex. This is most likely due to a true
change in the binding constant of the E conformation for
ATP and not to a shift in conformational equilibrium. Our ATP binding
data for D369N demonstrate that Na
and K
stabilize the E
Na and E
K conformations
with widely different affinities to ATP. In addition, the D369N mutant
has the same affinity for [
H]ouabain as the wild
type in the presence of Mg
. Previous data also show
that the [
H]ouabain complex of the mutant
responds to Na
and K
in the same
manner as the wild type Na,K-ATPase(7) . In the Ca-ATPase from
SR, modification of either Asp
or the neighboring
Lys
abolish phosphorylation(59) . Our data show
that reduction of the negative charge of Asp
greatly
increases the affinity for ATP. Although the negatively charged Asp
residue is essential for enzymatic turnover, it may thus reduce the
affinity for ATP through electrostatic repulsion of the negatively
charged
-phosphate present on the ATP molecule.
The
extraordinary high affinities of D369N or D369A for ATP turn these
mutants into powerful tools for future identification of residues
involved in ATP binding to Na,K-ATPase and for studying the influence
of Mg upon ATP binding. Given the high degree of
homology of the amino acid sequence around the phosphorylation site
(ICS D KTGTLT), it can be expected that the large increase in
affinity for ATP upon reducing the negative charge on the
phosphorylated side chain will be a general phenomenon for the P-type
cation pumps, H,K-ATPase, Ca-ATPase, and H-ATPase.