(Received for publication, December 3, 1996, and in revised form, January 23, 1997)
From the Institute of Pharmacology and Toxicology, University of Lausanne, rue du Bugnon 27, CH-1005 Lausanne, Switzerland
Initial folding is a prerequisite for subunit
assembly in oligomeric proteins. In this study, we have compared the
role of co-translational modifications in the acquisition of an
assembly-competent conformation of the subunit, the assembly of
which is required for the structural and functional maturation of the
catalytic Na,K-ATPase
subunit. Cysteine or asparagine residues
implicated in disulfide bond formation or N-glycosylation,
respectively, in the Xenopus
1 subunit were eliminated
by site-directed mutagenesis, and the assembly efficiency of the
mutants and the functional expression of Na+,K+
pumps were studied after expression in Xenopus oocytes. Our
results show that lack of each one of the two most C-terminal disulfide bonds indeed permits short term but completely abolishes long term
assembly of the
subunit. On the other hand, lack of the most
N-terminal disulfide bonds allows the expression of a small number of
functional Na+,K+ pumps at the cell surface.
Elimination of all three but not of one or two glycosylation
sites produces
subunits that remain stably expressed in the
endoplasmic reticulum, in association with binding protein but not as
irreversible aggregates. The assembly efficiency of nonglycosylated
subunits is decreased but a reduced number of functional
Na+,K+ pumps is expressed at the cell surface.
The lack of sugars does not influence the apparent K+ or
ouabain affinity of the Na+,K+ pumps. Thus,
these data show that disulfide bond formation and N-glycosylation may play important but qualitatively
distinct roles in the initial folding of oligomeric protein subunits.
Moreover, the results suggest that an endoplasmic reticulum degradation pathway exists, which is glycosylation-dependent.
Na,K-ATPase is a ubiquitous plasma membrane transporter that is
responsible for the maintenance of the potassium and sodium homeostasis
in animal cells. The minimal functional enzyme unit is composed of two
subunits. The large subunit has 10 putative transmembrane spans and
carries out the catalytic and transport functions of the enzyme. The
smaller
subunit is a type II glycoprotein with a short cytoplasmic
domain, one transmembrane segment, and a large C-terminal ectodomain
that comprises several N-glycosylation sites and three
highly conserved disulfide bridges. The
subunit plays an essential
role in the structural and functional maturation of the
subunit.
Only
subunits that assemble with
subunits in the
ER1 are stably expressed, acquire their
functional properties, and are transported to the plasma membrane (for
review see Ref. 1). Upon assembly, the
subunit itself also
undergoes a structural maturation that is reflected by an increased
resistance against proteolysis (2).
The molecular mechanisms that guide the important process of assembly
of the subunit with the
subunit are still poorly understood.
The structural requirements for
-
assembly have only been
partially characterized (3-7). Several regions in the
subunit
participate in the interaction with the
subunit. It is possible to
distinguish, on the one hand, structural interaction sites including
the ecto- and the transmembrane domains of the
subunit that mediate
the maturation of the
subunit and, on the other hand, functional
interaction sites such as the cytoplasmic N terminus that participate
in the modulation of the transport activity of the functional enzyme
(2).
Little is known how the subunit acquires a conformation that is
compatible with assembly, e.g. how it folds into a correct tertiary structure that exposes the structurally defined surfaces assuring the specific interaction with the
subunit. Molecular chaperones such as BiP (binding protein) are associated with the newly
synthesized
subunit (8) suggesting that they might be important for
the correct folding and/or in the ER retention of unassembled
subunits.
Because the subunit, as many other membrane and secretory proteins,
acquires N-linked sugars and forms intramolecular disulfide bonds during its synthesis in the ER, it appeared interesting to us to
use this protein as a model protein to compare the role of these
co-translational modifications in protein folding, assembly, and in the
functional expression. The role of N-glycosylation and
disulfide bonds in the folding of proteins has been extensively studied
(for review, see Refs. 9 and 10) but rarely compared in the same
protein. Their importance in the folding process varies. When
N-glycosylation is prevented by site-directed mutagenesis, many but not all glycoproteins misfold, aggregate, and degrade in the
ER (11). Disulfide bonds do or don't directly affect the folding
process, but, in most cases, their formation stabilizes the folded
form. In this study, we used the Xenopus oocyte expression system to study 1) the structural properties of mutant
subunits deficient in glycosylation or disulfide bonds, 2) their interaction with BiP, and 3) their ability to associate with
subunits and to be
transported to the plasma membrane as functional
·
complexes. Our results show that both co-translational modifications play an
important but qualitatively distinct role in the correct folding of the
subunit. Indeed, the interaction of the
subunit with molecular
chaperones, its stability, and its association efficiency are
differently affected after abolition of glycosylation sites or cysteine
bonds.
Site-directed mutagenesis was
performed according to the polymerase chain reaction method of Nelson
and Long (12). A linearized pSD5 vector (13) containing a 1cDNA
(pSD5
1) of Xenopus Na,K-ATPase (14) was used as a
template for the preparation of cysteine mutants (see Fig. 1). First,
single mutants in which Cys149, Cys175, or
Cys276 were substituted by a serine residue were prepared.
The DNAs of the single mutants served as templates to prepare double
mutants in which Cys126/Cys149 (CC1),
Cys159/Cys175 (CC2), or
Cys213/Cys276 (CC3), respectively, were
substituted with serine residues. The mutated DNA fragments of the CC1
and CC2 mutants were introduced into the wild type pSD5
1 by using
unique BamHI and HindIII restriction sites and
that of the CC3 mutant by using HindIII and SmaI
(present in the vector).
A linearized pGEM2 vector or a pSD5 vector containing a
1cDNA (pGEM2
1, pSD5
1) of Xenopus
Na,K-ATPase was used as a template for the preparation of the N2 or the
N1 and the N3 glycosylation mutants, respectively (see Fig. 1). In
single mutants Asn158, Asn193, or
Asn265, respectively, were substituted by a glutamine
residue. The mutated DNA fragments of the N1 and N2 mutants were
introduced into the wild type pSD5
1 by using BamHI and
PstI restriction sites and that of the N3 mutant by using
HindIII and SmaI. For the construction of the
double mutant N2,3, the mutated BamHI-PstI
fragment of the pGEM
1 N193Q mutant was introduced into the pSD5
1
N265Q (N3) mutant. For the preparation of the triple mutant N1,2,3, the
mutated BamHI-HindIII fragment of the N158Q (N1)
mutant was introduced into the pSD5
1 N193Q/N265Q (N2,3) mutant. All
polymerase chain reaction-generated fragments were sequenced by dideoxy
sequencing (15). All mutant
1 subunits were tested for their
translation efficiency in a reticulocyte lysate (6).
cRNAs encoding Xenopus
1 (14),
1 (14), and
mutants were obtained by in
vitro transcription (16). Stage V and VI oocytes were obtained
from Xenopus females (Noerdhoek, Republic of South Africa)
as described (6). Routinely, 5-7 ng of
and/or 0.2-0.6 ng of
cRNA were injected into oocytes. Oocytes were incubated in modified
Barth's medium containing 0.6-2.5 mCi/ml
[35S]methionine (Amersham Corp.) for the times indicated
in the figure legends and eventually subjected to different chase
periods in the presence of 10 mM cold methionine.
Microsomes were prepared as described (2), and the
and
subunits
of Na,K-ATPase or BiP were immunoprecipitated under denaturing or
nondenaturing conditions as described (6, 8) by using
antibodies
produced against the N terminus of the Xenopus
1 subunit
(17),
antibodies against the ectodomain of the Xenopus
1 subunit (17), or BiP antibodies produced against
Xenopus BiP (8). In some instances, immunoprecipitates were
subjected to endoglycosidase (Endo H) (Calbiochem) treatment as
described (18). Dissociated immunocomplexes were separated by SDS-PAGE
in the absence or presence of 2%
-mercaptoethanol and the labeled
proteins revealed by fluorography. Quantifications and determinations
of the molecular mass of the immunoprecipitated bands were performed
with an analytic program for electrophoretic images (Bio-1D) from
Vilbert Lourmat (Marne La Vallée, France).
Wild type and mutant cRNAs
were injected into oocytes together with 3 µCi/oocyte of
[35S]methionine. Oocytes were incubated for 5 h
before homogenization by 12 gentle strokes with a glass Teflon
homogenizer in a solution containing in mM, 250 sucrose, 50 potassium acetate, 5 MgCl2, 1 dithiothreitol, 50 Tris-HCl,
pH 7.5. After addition of 10 mM CaCl2, samples
were incubated in the absence or presence of 200 mg/ml proteinase K
(Merck) and in the absence or presence of 1% Triton X-100 for 1 h
at 4 °C. Proteolysis was stopped with 5 mM phenylmethylsulfonyl fluoride and 3.7% hot SDS before
immunoprecipitation of the samples.
[3H]Ouabain binding was essentially done as described (6). After cRNA injection, oocytes were incubated for 2-4 days. Oocytes were then loaded with Na+ by incubation for 2 h in a solution containing in mM, 110 NaCl, 10 Tris-HCl, pH 7.5. After recovery for 30 min in a solution containing in mM, 90 NaCl, 2 CaCl2, 5 BaCl2, 20 tetraethylammonium chloride, 5 MOPS, pH 7.4, the oocytes were transferred to a solution containing in mM, 50 NaCl, 50 N-methyl-D-glucamine-Hepes, 2 CaCl2, 5 BaCl2, 20 tetraethylammonium chloride, 5 MOPS, pH 7.4, and 1 µM [21,22-3H]ouabain (Amersham Corp.) was added. In some instances, increasing concentrations of KCl were added to study the competition of ouabain binding. In this case, the osmolality of the solution was maintained by adapting the concentration of N-methyl-D-glucamine-Hepes. After 12 min at 19 °C, oocytes were washed with a solution containing in mM, 90 NaCl, 2 CaCl2, 5 BaCl2, 5 MOPS, pH 7.4, before dissolution in 0.5% SDS and counting. Nonspecific ouabain binding determined in the presence of a 1000-fold excess of cold ouabain amounted to about 3-7% of the total binding.
86Rb+ Uptake MeasurementsTo study
the K+ activation of the Na,K-ATPase-mediated
86Rb+ uptake, oocytes were injected with
Xenopus wild type or mutant 1cRNA plus
cRNA coding for
1 subunits of Bufo marinus Na,K-ATPase that is more
ouabain resistant than the Xenopus
1 subunit (19). After
loading of the oocytes with Na+ (see above) and recovery in
a solution containing in mM, 90 NaCl, 1 CaCl2,
5 BaCl2, 10 Hepes, pH 7.4, oocytes were transferred to a
solution containing varying concentrations of KCl, 90 mM
TMA-Cl or 90 mM NaCl, 2 mM CaCl2, 5 mM BaCl2, 1 mM MgCl2,
10 mM Hepes, pH 7.4, and 1 µM ouabain to
inhibit the endogenous, oocyte Na,K-ATPase (19). After addition of 5 µCi/ml 86Rb+ (Amersham Corp.), oocytes were
incubated for 12 min at room temperature before washing in a solution
containing in mM, 90 TMA-Cl, 1 CaCl2, 1 MgCl2, 10 KCl, Hepes, pH 7.4. To determine the ouabain
sensitivity of the Na,K-ATPase-mediated 86Rb+
uptake, oocytes were injected with Xenopus wild type or
mutant
1cRNA plus Xenopus
1cRNA, and
86Rb+ uptake measurements were performed in the
presence of 90 mM NaCl and varying concentrations of
ouabain. The parameters of the Hill equation V = Vmax/[1+(K1/2/CK)nH
were fitted to the data of the 86Rb+ uptake
(V) induced by various concentrations of K+ (CK)
and yielded least-square estimates of the maximal uptake (Vmax), the half-maximal activation constant
(K1/2), and the Hill coefficient
(nH).
To assess the role in the initial folding of the three
disulfide bonds present in the subunit of oligomeric P-type ATPase, we introduced mutations in each S-S bond-forming cysteine pair (CC1,
CC2, CC3, see Fig. 1) of Xenopus
1
subunits. The degree of misfolding of the disulfide bond-deficient
mutants was probed by various tests after expression and metabolic
labeling in Xenopus oocytes. In a first test, the structural
characteristics of the immunoprecipitated, wild type
subunits and
of the cysteine mutants were compared by performing SDS-PAGE under
nonreducing conditions (Fig. 2). The abolition of each
one of the disulfide bonds appears to have a distinct effect on the
structure of the mutants since their migration in the absence of
-mercaptoethanol differed slightly from that of the wild type and
among each other (Fig. 2A, lanes 5-8). In comparison, the
migration in the presence of
-mercaptoethanol of wild type and
mutant
subunits was generally slower and identical (Fig. 2A,
lanes 1-4). Overexposure of a gel from a similar experiment (Fig.
2B) revealed in addition that a small fraction of individual Xenopus
1 subunits, which are retained in the ER (17),
occur as disulfide-bonded multimers. However, aggregate formation seen with nonreduced samples is quantitatively and qualitatively similar to
wild type (Fig. 2B, lane 5) and disulfide bond-deficient
(lanes 6-8)
subunits. This result indicates that the
lack of one cysteine bond does not enhance the tendency of individual
subunits to form inappropriate, intermolecular disulfide bonds and
aggregates.
To further characterize the structural effect of the abolition of
disulfide bonds, homogenates of oocytes expressing wild type or mutant
subunits were subjected to proteolysis in the absence or presence
of Triton X-100. Proteinase K did not attack the wild type
subunit
(Fig. 3A, lanes 1 and 2) but
digested the three mutants by about 40% (lanes 5, 8, and
11). Probably due to partial cleavage of the cytoplasmic N
terminus (2), protease digestion also produced a slight shift in the
molecular mass of the residual mutant proteins (lanes 5, 8,
and 11) and to a lesser extent in that of the wild type
subunits (lane 2). Finally, in the presence of Triton X-100,
which opens closed membrane vesicles and exposes the ectodomain of the
subunit, only the wild type (lane 3) but not the mutant
subunits (lanes 6, 9 and 12) is partially
resistant to proteinase K digestion. Thus, abolition of each one of the
disulfide bonds produces a structural effect on the
subunit that is
reflected by an increased protease sensitivity.
We have previously shown that Xenopus 1 when expressed in
Xenopus oocytes without an
subunit is slowly degraded in
or close to the ER (17). Despite the increased protease sensitivity, the disulfide bond-deficient mutants showed a similar rate of cellular
degradation to the wild type
subunits (Fig. 3B).
Furthermore, the wild type as well as the newly synthesized CC1, CC2,
and CC3 mutants interacted with BiP and the chaperone interaction
correlated with the degradation rate of the
subunits (compare Fig.
3, C with B).
Thus altogether, these data indicate that abolition of only one
disulfide bond in the subunit leads to a significant change in its
tertiary structure which, however, does not influence the cellular
half-life of the proteins or their interaction with BiP.
To assess whether the cysteine
mutants can adopt an assembly-competent conformation, we co-expressed
the mutants with subunits in oocytes and performed
immunoprecipitations with an
-antibody under nondenaturing
conditions that preserve
-
interactions. As expected, wild type
subunits co-precipitated with
subunits either in their
core-glycosylated form after a pulse (Fig. 4A, lane 3) or in their fully glycosylated form after a chase period (lane 4) reflecting the transport of the
·
complexes
from the ER to the plasma membrane (18). Furthermore,
subunits that were expressed together with
subunits became stabilized
(lanes 3 and 4) in contrast to
subunits that
were synthesized alone in the oocyte (lanes 1 and
2). The CC1, CC2, and CC3 mutants associated, although
inefficiently, with co-expressed
subunits during a pulse period
(lanes 5, 7 and 9), but they did not visibly
stabilize them (lanes 6, 8 and 10). As shown for
the CC1 mutant, the cysteine mutants also associated transiently with
the endogenous, oocyte
subunits (lanes 11 and
12) that represent a stable pool of unassembled
subunits
(15). Results obtained on the conformation and on the ability to
assemble with
subunits were similar for mutants affected in only
one of the cysteine residues forming the disulfide bonds (data not
shown).
During the chase period, the CC2 and CC3 mutants co-expressed with subunits were degraded in a pre-Golgi compartment in their
core-glycosylated form, similar to when they are expressed alone
(compare Fig. 4B, lanes 7-10 to lanes 11 and
12 and to Fig. 3B). On the other hand, a small
portion of the CC1 mutants became fully glycosylated when they were
co-expressed with
subunits (Fig. 4B, lane 6) but not
when they were expressed alone (lane 12). This result
suggested that in contrast to CC2 and CC3 mutants, the CC1 mutant is
still able, although inefficiently, to form
·
complexes that
can leave the ER. To verify this observation, we tested the expression
of functional pumps at the cell surface by measuring
[3H]ouabain binding and Na,K-ATPase-mediated
86Rb+ uptake in oocytes co-expressing
and
wild type
or one of the
mutants. Oocytes expressing wild type
·
complexes produced about three or four times more functional
Na+,K+ pumps at the cell surface than oocytes
expressing
subunits alone (Fig. 5, A and
B). On the other hand, a similar number of Na+,K+ pumps to
-expressing oocytes was
found in oocytes co-expressing
subunits and the CC2 or the CC3
mutant. In agreement with the biochemical data shown in Fig. 4, the CC1
mutant, however, produced a small but significant increase in the
number of functional pumps at the cell surface. This increase was only
observed when the CC1 mutant was co-expressed with
subunits (Fig.
5).
In conclusion, despite the considerable perturbation of the structural
integrity of the ectodomain of the subunit by the abolition of only
one disulfide bond, all the mutants are capable to associate
transiently with
subunits. However, only the CC1 mutant affected in
the most N-terminal disulfide bond is partially able to form stable and
functional
·
complexes. This result indicates that the
C-terminal domain containing the last two disulfide bonds is
particularly important to ensure the acquisition of a conformation
compatible with an efficient association of the
subunit with the
subunit.
All isoforms of the Na,K-ATPase as well as the
subunits of H,K-ATPases are subjected to N-glycosylation
during their synthesis. To assess the role of the sugar moieties in the
initial folding of
subunits, we prepared mutants in which one (N1,
N2, or N3), two (N2, 3) or three (N1,2,3) of the potential
glycosylation sites in the
1 subunit of Xenopus
Na,K-ATPase were abolished by replacement of the consensus asparagine
residues with glutamine (see Fig. 1). Expression of these mutants in
Xenopus oocytes revealed that all three potential
glycosylation sites are used. Single, double, and triple mutants
exhibited a progressively decreasing molecular mass (Fig.
6). For so far undefined reasons, but perhaps due to differential trimming, the core-glycosylated, single mutants N1, N2,
and N3, which each lacks one of the glycosylation sites, exhibited slightly different molecular masses (compare lanes 3, 5, and
7). However, after deglycosylation with Endo H, all mutants
as well as the wild type
subunit showed the same molecular mass
that was identical to that of the triple mutant lacking all
glycosylation sites (compare lanes 2, 4, 6, 8, and
10, to lanes 11 and 12) or to that of
the
subunit synthesized in a reticulocyte lysate (data not
shown).
To assess the effect of glycosylation on the structural stability of
the newly synthesized subunit, we subjected the triple N1,2,3
mutant synthesized in oocytes to proteolysis in the presence or absence
of Triton X-100. In contrast to the wild type
subunit (Fig.
7A, lanes 1-3), the triple glycosylation
mutant was completely degraded by proteinase K in the presence of
Triton X-100 (lanes 4-6) indicating that the sugar chains
either influence the folding of the
subunit or directly protect it
from proteolytic attack.
With the exception of the N1,2,3 mutant, all glycosylation mutants
expressed without subunits in the oocyte were degraded during a
chase period of 3 days similar to the wild type
subunit (Fig.
7B). Interestingly, the nonglycosylated N1,2,3 mutant
remained stable over the whole chase period (Fig. 7C).
During its stable expression in the ER, the N1,2,3 mutant remained
associated with BiP (Fig. 7D). This result provides further
support for the previously raised hypothesis (8) that interaction with
BiP might be important for the ER retention of partially misfolded or
overexpressed Na,K-ATPase
subunits until they are recognized by a
pre-Golgi degradation system. On the other hand, a comparison of the
gel migration of nonreduced samples (Fig. 7E) reveals no
increase in the formation of disulfide-bonded aggregates by
nonglycosylated
subunits that could explain their high
stability.
The ability of the
glycosylation mutants to form stable ·
complexes was assessed
by performing immunoprecipitations under nondenaturing conditions on
microsomes of pulse/chase-labeled oocytes expressing
and
mutants. After the pulse period, all glycosylation mutants
co-precipitated with an
antibody indicating that they were able to
associate with the
subunit (Fig. 8A). The
mutants with one or two residual glycosylation sites were in their
core-glycosylated (Endo H-sensitive) form (Fig. 8, A and
B). After a chase of 3 days, the
mutants remained
associated with the
subunit (Fig. 8C), and with the
exception of the N1,2,3 mutant that is nonglycosylated, the other
mutants with residual glycosylation were now mainly processed to the
fully glycosylated (Endo H-resistant) form (Fig. 8, C and
D) indicating that the
·
complexes had been
transported through a distal Golgi compartment. In comparison to the
residually glycosylated
mutants that stabilized the
subunit
similarly to the wild type
subunit (Fig. 8C, compare lanes 2-5 to lane 1), the nonglycosylated
mutant only achieved about 50% (average of two experiments)
stabilization of the
subunit (Fig. 8C, compare
lane 6 to lane 1). Thus, these data indicate that
the acquisition of an assembly-competent conformation of the
subunit depends on the presence of the sugars. However, the presence of
only one sugar chain is necessary and sufficient to ensure an efficient
association with the
subunit.
The cell surface expression of ·
complexes containing the
N1,2,3 mutant was estimated by [3H]ouabain binding on
intact oocytes combined with pulse/chase experiments. Denaturing
immunoprecipitations of the labeled, wild type
subunit expressed
without the
subunit showed that a small fraction of the
subunit
population became fully glycosylated during the chase (Fig.
9A, lanes 3 and 4) due to its
association with endogenous, oocyte
subunits (15). The fraction of
fully glycosylated
subunits was more important when the
subunit was expressed together with
subunits (lanes 7 and
8). In contrast to the wild type
subunit, the
nonglycosylated
subunit showed, as expected, no change in its
molecular mass after the chase when it was expressed alone (lanes
5 and 6) or together with
subunits (lanes
9 and 10). The N1,2,3 mutant co-precipitated with the
subunit during the pulse period similarly to the wild type
subunit but, as shown above, it was not able to stabilize the
subunit to a similar extent during the chase period (Fig.
9B, compare lanes 5 and 6 to
lanes 3 and 4).
Expressed alone, the wild type subunit and the N1,2,3 mutant
increased the expression of the number of
Na+,K+ pumps at the cell surface about 2-fold
compared with noninjected oocytes due to association with the
endogenous
subunit (Fig. 9C). When co-expressed with
subunits, the wild type
subunit allowed for about a 4-fold increase
and the N1,2,3 mutant for about a 3.5-fold increase in the number of
Na+,K+ pumps (Fig. 9C). A comparison
between the stabilization of the
subunit by the wild type or mutant
subunit and the expression of Na+,K+ pumps
at the cell surface consistently showed that the expression of
·N1,2,3 mutant complexes was more important than expected from the
ability of the
mutant to stabilize the
subunit. While the
expression of
·N1.2,3 mutant complexes, assessed by ouabain binding, was only about 20% less than that of
·wild type
complexes (Fig. 9C), the stabilization of
·N1,2,3
complexes was about 50% lower (average of two experiments) than that
of
·wild type
complexes (Fig. 9B, lanes 3-6). This
discrepancy might be explained by a regulated expression of
Na+,K+ pumps in Xenopus oocytes that
only permits the expression of a limited number of pumps at the cell
surface and that would establish an intracellular pool of pumps
(20).
Another possibility to explain the differences between the expression
and the stabilization of the ·N1,2,3 complexes might be that the
mutant complexes have a different ouabain sensitivity than the
·wild type
complexes. This latter possibility incited us to
study the effect of the sugars in the
subunit on several functional
properties of the cell surface-expressed
·
complexes. The
apparent K+ affinity (K1/2) of
Bufo Na,K-ATPase, measured as the half-maximal K+ activation of the Na+,K+
pump-mediated 86Rb+ flux, was about 300 µM in the absence of extracellular Na+ and 1 mM in the presence of extracellular Na+ for
wild type as well as for mutant, nonglycosylated
Na+,K+ pumps (Fig. 10,
A and B) These values are similar
to those previously measured by electrophysiological means (21).
Furthermore, the affinity for ouabain of the Xenopus
Na+,K+ pumps containing the nonglycosylated
mutant
subunit was similar to that of the glycosylated wild type
pumps. The inhibition constant (Ki) measured as the
half-maximal concentration needed to inhibit the Na,K-ATPase-mediated
86Rb+ uptake was close to 50 nM for
both types of pumps (Fig. 10C). Finally, no significant
differences could be observed between the wild type and the
nonglycosylated Xenopus pumps in the K+
concentrations needed to compete with ouabain binding (Fig.
10D).
Thus, these data demonstrate that the presence of at least one sugar
moiety is necessary for the correct folding of the 1 subunit of
Na,K-ATPase permitting its efficient assembly with the
subunit. On
the other hand, our functional tests did not permit us to reveal a role
of the sugars in the functional properties of the mature enzyme
expressed at the cell surface.
The present study reveals the importance of co-translational
modifications for the initial folding of the subunit of
Na,K-ATPase. We show that disulfide bond formation and to a lesser
extent core glycosylation during the synthesis of the
subunit are
needed to render it competent for the assembly with the catalytic
subunit.
Six cysteine residues in the ectodomain of the subunits of the
Na,K-ATPase and the H,K-ATPase, the positions of which are highly
conserved, form disulfide bonds in a sequential pattern (22-24).
Cleavage of these disulfide bonds by reducing agents leads to
structural and functional changes in purified enzyme preparations (25-28) suggesting that they are important to maintain the
subunit in a conformation that is compatible with a correct association with
the
subunit. In this study, we show that prevention of the
formation of only one of the three disulfide bonds in the
subunit
of Na,K-ATPase indeed is sufficient to abolish its ability to correctly
assemble with
subunits and as a consequence to impede the
maturation and efficient expression of functional pumps.
Acquisition of disulfide bridges is catalyzed by protein disulfide
isomerase in the ER, and it is thought to limit the number of folding
states a protein can adopt during or after synthesis, to guide the
folding toward a final state and ultimately to contribute to the
stability of the correctly folded state (for reviews see Refs. 10 and
29). Thus, the observed inability of an efficient assembly of the subunit of Na,K-ATPase or of oligomeric, viral proteins (for review see
Ref. 9) in which cysteine residues involved in disulfide bond formation
are changed by site-directed mutagenesis is probably due to
destabilization and increased misfolding of the newly synthesized
proteins. However, in the cysteine-deficient
subunit, misfolding is
not accompanied, as in many other proteins, by the formation of
intermolecular disulfide-bonded aggregates or by a more pronounced
interaction with the molecular chaperone BiP (for review, see Ref. 10).
Possibly, the lack of only one cysteine residue in
subunits is not
enough for the formation of covalent aggregates or for a global
misfolding but leads to local structural perturbations that impede the
acquisition of a completely folded association domain. Such partial
misfolding might explain why cysteine bond-deficient
subunits can
transiently but not permanently associate with
subunits.
Our data indicate that the relative importance of the three disulfide
bonds in the subunit on the acquisition of an assembly-competent form is different. Indeed, while
mutants, affected in the formation of disulfide bonds Cys159-Cys175 (numbering
refers to the
1 subunit of Xenopus laevis) or
Cys213-Cys276, completely lose their ability to
assemble permanently with the
subunit, the
mutant affected in
the formation of the most N-terminal disulfide bond
Cys126-Cys149 permits the formation of a small
number of functional
·
complexes at the cell surface. Thus, the
domain comprised between the two most C-terminal disulfide bonds is
able to adopt a structure that partially permits correct subunit
interaction. Probably this domain in the
subunit is more compact as
suggested by its lower sensitivity to reduction by
-mercaptoethanol
that first breaks the least stable disulfide bond
Cys126-Cys149 in purified enzyme preparations
(26). On the basis of these results, it has been speculated that the
most N-terminal disulfide bond might function as a kind of hinge that
allows establishment of close contact between the hydrophobic,
membrane-anchored domain and the hydrophilic C-terminal part of the
subunit that contains the sugar chains.
Our data showing the structural importance of the two most C-terminal
disulfide bonds in the subunit are in agreement with similar
studies performed with cysteine mutants of Torpedo
subunits. However, our results obtained with the
mutant lacking the
first N-terminal disulfide bond differ from those of Noguchi et
al. (30) who reported that Torpedo
subunits lacking
this disulfide bond are able to assemble and stabilize
subunits but
cannot form functional Na+,K+ pumps. Although
we cannot completely rule out that species differences are responsible
for the discrepancies in the results, it is more likely that they are
due to differences in the experimental approach. Indeed, Noguchi
et al. (30) performed continuous pulse labeling protocols to
analyze
-
interaction. In contrast, in this study, we performed
pulse/chase protocols that are the only means to distinguish between a
short and a long term association of
subunits. Furthermore, it is
likely that, in contrast to the highly sensitive 86Rb+ uptake measurements used in our study,
the Na,K-ATPase assay used by Noguchi et al. (30) was not
sensitive enough to detect a small fraction of functional pumps formed
with mutant
subunits and/or that sodium thiocyanate used during
sample preparation led to dissociation and inactivation of the loosely
assembled
·
mutant complexes.
As with most cell surface proteins, all isoforms of Na,K- or
H,K-ATPase are glycoproteins and possess 3-7 N-linked sugar chains (for review see Ref. 31). The functional role of the glyco-moieties is poorly understood. In this study, we confirm previous
reports (32-34) that nonglycosylated
subunits are able to assemble
with
subunits and permit the expression of functional Na+,K+ pumps at the cell surface. Nevertheless,
our data show that in the
subunit of Na,K-ATPase, as in many other
glycoproteins (for review see Ref. 11), inhibition of glycosylation
using tunicamycin (35) or elimination of the consensus sequences for
N-linked glycosylation by site-directed mutagenesis leads to
folding problems of the newly synthesized protein. These are reflected
by an increased sensitivity of the
subunit against proteolysis and
a decreased efficiency in the assembly with
subunits. As reported
for vesicular stomatitis virus G-glycoprotein or influenza
hemagglutinin, the effects of elimination of carbohydrate sites in the
subunit is additive (for references, see Ref. 9). No single
oligosaccharide is essential for the correct folding, but all sugars
must be eliminated to significantly affect the assembly competence.
Addition of N-linked sugar chains occurs as soon as the nascent polypeptide emerges in the ER lumen and thus occurs, in most cases, before folding and assembly. The addition of the large, hydrophilic carbohydrate chains facilitates the ongoing folding process of the nascent chain by favoring the recruitment of discrete polypeptide domains to the surface of the protein (for review and references see Ref. 36). In addition to local effects on folding, it has been proposed that core sugars have a global effect on the solubility of newly synthesized proteins that counteracts the tendency to form irreversible aggregates (37).
Although the degradation rates of wild type and sugar-deficient
glycoproteins have not rigorously been compared, proteins in which the
sugar moieties have been eliminated can be degraded in the ER (38). The
nonglycosylated subunit, however, is very resistant to ER
degradation. The high stability of the nonglycosylated mutant compared
with that of the wild type, glycosylated
subunit is surprising,
since the presence rather than the absence of sugars would be expected
to protect from proteolytic attack. In contrast to the prediction
mentioned above, the nonglycosylated
subunit does not form
disulfide-bonded aggregates that could explain its resistance to
cellular degradation. On the other hand, it is permanently associated
with BiP, perhaps in aggregates formed by hydrophobic interactions that
could impede the recognition of the nonglycosylated
subunit by the
pre-Golgi degradation pathway. Alternatively, the
subunit of
Na,K-ATPase might be an example of a protein whose quality control and
degradation depends on glycosylation. A similar hypothesis has recently
been raised for carboxypeptidase Y whose unglycosylated species is
stably retained in the ER (39). An important player in the quality
control of glycoproteins is calnexin that binds to newly synthesized
monoglucosylated N-linked oligosaccharides. A role for
glycoprotein trimming and/or calnexin interaction is suggested by a
recent study that reports that a mutant, secretory
1-antitrypsin Z is degraded by the proteasome by first
digesting the cytoplasmic tail of calnexin molecules that are
associated with
1-antitrypsin Z (40).
In contrast to disulfide bonds, complex-type sugar chains are often not
important for overall stability and function of mature glycoproteins
(for references see Ref. 11). In agreement, enzymatic deglycosylation
of the subunit in purified Na,K-ATPase has little effect on the
enzyme activity or ouabain binding (41). Our studies using
subunits
in which the glycosylation sites had been abolished by site-directed
mutagenesis also do not reveal significant effects of the sugars in the
1 subunit on either the K+ activation or the ouabain
binding of functional Na+,K+ pumps expressed at
the cell surface. The question remains open whether complex-type
oligosaccharides in the
1 subunit of Na,K-ATPase might represent
specific recognition sites for interaction with external ligands, as
suggested for many glycoproteins (42).
We thank Jean-Daniel Horisberger and Dmitri Firsov for a critical reading of the manuscript.