From the Laboratory of Membrane Biology, Neuroscience Center, Massachusetts General Hospital, Charlestown, Massachusetts 02129
Received for publication, October 5, 2000, and in revised form, November 16, 2000
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ABSTRACT |
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Thermal denaturation can help elucidate protein
domain substructure. We previously showed that the Na,K-ATPase
partially unfolded when heated to 55 °C (Arystarkhova, E., Gibbons,
D. L., and Sweadner, K. J. (1995) J. Biol.
Chem. 270, 8785-8796). The The Na,K-ATPase has two obligatory subunits, Thermal denaturation of the Na,K-ATPase has produced some surprising
results. From the literature, thermal denaturation of membrane proteins
differs from that of soluble proteins in one important respect. Those
segments of a membrane protein that are embedded in the lipid bilayer,
whether Enzyme Preparations
Pig and dog renal medulla Na,K-ATPase were purified by
extraction of contaminating proteins with low concentrations of SDS followed by centrifugation on sucrose gradients by the method of
Jørgensen (13). The purified enzyme, which remains membrane-embedded throughout the procedure, was stored at Heating
Purified enzyme or crude microsomes were diluted to 0.5 mg of
protein/ml in buffer containing 30 mM histidine, 1 mM EDTA, and 250 mM sucrose (pH 7.2 at room
temperature) and incubated for 30 min at 55 °C in a thermal block
incubator. Subsequently samples were kept on ice.
ATPase Activity Assay
Na,K-ATPase activity was measured at 37 °C in media
containing 3 mM Tris-ATP, 3 mM
MgCl2, 140 mM NaCl, 20 mM KCl, 30 mM histidine (pH 7.2 at room temperature). Na,K-ATPase
activity is defined as the ouabain-sensitive difference in
Pi released per mg of protein per hour. Pi
release was measured colorimetrically (14).
Gel Electrophoresis
Gel electrophoresis was normally carried out with the buffer
system of Laemmli on either 5 or 10% polyacrylamide gels (except for
gradient 5-15% gels which were used to visualize the Electrophoretic transfer to nitrocellulose was performed in a buffer
containing 25 mM Tris, 170 mM glycine, 20%
methanol, and 5% SDS at 100 mA overnight at 4 °C. Nitrocellulose
was quenched with Tris-buffered saline, 0.5% Tween 20, and stained in
the same buffer with primary antibodies and peroxidase-conjugated
secondary antibodies (Sigma). Detection was by chemiluminescence, using luminol-based reagent (Pierce Chemical Co.).
Antibodies
Mouse antibody 6F, which binds near the N terminus of the
Proteolysis
V8 Protease--
Purified pig or dog enzyme (0.5 µg) was
incubated with V8 protease (Sigma) (0.1 µg) in a buffer consisting of
30 mM histidine, 1 mM EDTA, and 250 mM sucrose, for 30 min at 37 °C. Digestion was stopped
by diluting with 1 volume of 2 × electrophoresis sample buffer
acidified with 0.3% trichloroacetic acid, which was effective at preventing digestion during gel electrophoresis. The acidification of the sample lasts only until it penetrates the gel, but it appears to
promote irreversible denaturation of the protease.
Trypsin--
Limited trypsinolysis in K+ at the T1
and T2 sites was carried out based on established protocols (17).
Purified Na,K-ATPase (100 µg of protein) was incubated with 0.5 µg
of TPCK-treated trypsin (Sigma) in a buffer containing 25 mM imidazole, 1 mM EDTA-Tris, and 15 mM KCl (pH 7.5), for 10 min at 37 °C. Proteolysis was
stopped by addition of a 10-fold excess of soybean trypsin inhibitor
(Sigma), and 1 volume of 2 × electrophoresis sample buffer with
0.4% trichloroacetic acid was added 5 min after the trypsin inhibitor.
As shown previously, acidified sample buffer is more effective at
preventing digestion by trypsin during gel electrophoresis than
addition of soybean trypsin inhibitor alone because the inhibitor
paradoxically stabilizes the activity of the protease in the detergent
(18).
Trypsin Treatment of Right-side-out Vesicles--
Right-side-out
pig renal medulla vesicles were prepared by centrifugation on
metrizamide gradients as described (11). The extent of sealing was
assessed by determining how much the Na,K-ATPase activity was
stimulated by addition of detergent to open the vesicles. In the
vesicles used here the stimulation was 5.5-fold, indicating that the
vesicles were ~82% sealed. Vesicles were incubated with TPCK-trypsin
at a ratio of 1:3, trypsin:vesicle protein, for 30 min. The buffer was
250 mM sucrose, 25 mM MOPS (pH 7.4), 1 mM Tris-EDTA. In some experiments, vesicles were
pre-equilibrated with 10 mM RbCl to verify the
stabilization of structure by this K+ analog (data not
shown). Proteolysis was at 37 °C, and the reaction was stopped by
addition of acidified sample buffer. Samples were then directly loaded
on SDS gels. Enzyme in open vesicles is extensively digested and does
not contribute to the results (11).
Heat Denaturation of the Na,K-ATPase--
Fig.
1A shows the predicted
topology of the Na,K-ATPase
Some issues remained unaddressed by the experiments summarized in Fig.
1. One was whether the cytoplasmic domains were really as unperturbed
as implied in the figure, and another was the fate of the Proteolytic Sensitivity of Heated Enzyme--
The experiments
described first were performed with purified enzyme in open membrane
fragments instead of right-side-out medullary vesicles so that we could
assess the extent to which protease sensitivity increased in the
cytoplasmic domains of the
Thermal denaturation does not necessarily mean the complete unfolding
of the polypeptide chain. For many proteins it has been shown that a
"looser" conformational state known as a molten globule forms as an
intermediate. The molten globule state is thought to retain much
secondary structure but to lack tertiary structure, and it can be
quasi-stable. Fig. 3 shows evidence that
the Irreversible Aggregation of
Fig. 4 illustrates the basic observation
that Na,K-ATPase heated in the absence of KCl forms a series of
SDS-resistant aggregates that can be resolved on 5% polyacrylamide. It
was frequently observed that a very small amount of similar bands was
found in unheated preparations, which suggests that heating was
accelerating a process that occurred to a small but detectable extent
during normal sample manipulation. In this experiment 4-5 extra bands
were resolved, but the extent of aggregation varied between
experiments, as will be seen in other figures. Two different
It is difficult to accurately estimate the molecular weights of large
proteins, and large cross-linked proteins are known to behave
anomalously on Laemmli gels, so the Weber-Osborn buffer system was used
on slabs of 3.5% polyacrylamide. Fig. 5
shows the migration of
The next figure makes two important points. First, heating produced the
same aggregates in crude kidney microsome preparations (Fig.
6B) as it did in purified
enzyme (Fig. 6A), again detected with two different
Na,K-ATPase-specific antibodies. The microsomes were isolated by
differential centrifugation alone, and were not treated with SDS like
the purified enzyme. This makes it much less likely that the
aggregation is a trivial consequence of removing other
membrane-associated proteins. Second, the SDS-resistant aggregates
contained no detectable Fate of the
Aggregation of the vesicle
In parallel we examined the trypsin sensitivity of the A Framework for Thinking About the Na,K-ATPase Transmembrane
Domain--
Fig. 9A uses the
Ca2+-ATPase structure to illustrate the probable
organization of the Na,K-ATPase membrane domain. The spans that are
extruded during heat denaturation, M8, M9, and M10, are grouped in the
Ca2+-ATPase at the edge of the membrane domain, and
highlighted in yellow. The view is from the extracellular surface. The
Na,K-ATPase has been known to be homologous to the
sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA) ever
since the cDNAs were sequenced, but whether they share the same
topology in the C-terminal membrane domain has been controversial. Now
that the Ca2+-ATPase structure is known (2), it is clear
that there are many polar amino acids in the membrane, and
discrepancies between hydropathy plots are no longer a strong argument
that the Ca2+-ATPase and Na,K-ATPase structures should be
different. In a gapped-BLAST alignment of the Na,K-ATPase Insights from Thermal Denaturation--
Heat denaturation of the
intact Na,K-ATPase probably occurs in stages. According to the data of
Goldshleger et al. (12), heating at 45 °C with 1-butanol
and
Instability of the organization of the C-terminal part of the
transmembrane domain of P-type ATPases has now been observed in several
contexts. Exposure of the L9-10 loop on the cytoplasmic side of the
membrane was observed in Na,K-ATPase (12), H,K-ATPase (27), and
Ca2+-ATPase (28) after various experimental manipulations,
although it is unambiguously luminal in the SERCA1a crystal structure. Exposure of the L7-8 loop on the cytoplasmic side of the membrane has
been observed in Ca2+-ATPase after low concentrations of a
nondenaturing detergent-like C12E8 and protease
K treatment (29), and it too is luminal in the crystal. M7, M8, M9, and
M10 apparently leave the membrane to the cytoplasmic side principally
after proteolytic cleavage of cytoplasmic loops (12, 27, 28).
Association with the Na,K-ATPase
Calorimetry has been performed on H,K-ATPase and Na,K-ATPase, but the
thermal transitions that have been observed are not easy to reconcile
with the structural changes that were observed here with protease
sensitivity and changes in the exposure of defined sites. Gasset
et al. (30) observed two peaks of heat capacity at 53.9 and
61.8 °C in gastric H,K-ATPase. We would have predicted that the heat
transition at 53.9 °C corresponds to the physical changes we
observed at 55 °C, but protection by K+ confounds this
interpretation. It is well established that K+ stabilizes
the Na,K-ATPase to heat (31), and it specifically increases the
protease resistance of the C-terminal end of H,K-ATPase as well as
Na,K-ATPase (32, 33). Of the two peaks of heat capacity detected in
intact gastric H,K-ATPase, the lower temperature transition
(53.9 °C) occurred in the presence or absence of K+,
while the higher temperature transition (61.8 °C) disappeared in
K+. This would suggest that the 61.8 °C transition
represents the denaturation event described here at a significantly
lower temperature. Gasset et al. (30) also observed no
transitions in enzyme digested at the cytoplasmic surface with
proteinase K, up to a temperature of 80 °C. The interpretation was
that the observed heat capacity signals were derived from denaturation
events occurring in the cytoplasmic loops of the
Somewhat different results were reported for thermal transitions in
dogfish shark Na,K-ATPase (34). They saw denaturation as measured by a
shift in amide I spectrum in Fourier transform infrared spectroscopy
commencing at 58 °C in intact enzyme. Enzyme that had been
extensively trypsinized in RbCl (19-kDa membranes) had a transition at
57 °C, while enzyme trypsinized in NaCl had a biphasic response at
57 and 84 °C. Again, no unique K+-protected transition
was seen near 55 °C.
Protection by K+ probably has its basis in large
conformation changes. K+ induces the E2 conformation in the
Na,K-ATPase. In the Neurospora proton pump, a highly
homologous P-type ATPase, it has been shown that about 175 residues are
shielded from solvent proton exchange in the E2 conformation (35). This
is physical evidence for a more compact, less exposed structure in E2.
Comparison of two SERCA structures and a Neurospora enzyme
structure gives shape to this concept, in that the 8-Å
Ca2+-ATPase structure crystallized in decavanadate has a
compact "head" for the cytoplasmic domain, while the 2.6-Å
structure, crystallized in Ca2+, has three spread-out A-,
P-, and N-domains (2, 36). The Neurospora structure appears
to be similar (37). We infer that an "open" conformation is more
labile to heat and more susceptible to aggregation.
Oligomers of Na,K-ATPase--
It has been demonstrated that
Prior physical evidence for association of Na,K-ATPase into dimers or
tetramers has been received cautiously because of the possibility that
the association was secondary to experimental manipulation: partial
inactivity, nonspecific aggregation in detergents, or the slow capture
of freely diffusing units by chemical cross-linking. The heat
denaturation of membrane proteins can result in aggregation into
complexes that are stable to SDS, and thus migrate at higher apparent
molecular weights in SDS gels (53). With this approach we found
evidence that the Na,K-ATPase can associate as a tetramer through
specific interactions between its
Stable aggregation presumably followed more conventional denaturation.
Reversible aggregation of Na,K-ATPase
The aggregates were stable to SDS, even at pH 2. Spontaneous
disulfide-mediated cross-linking of Na,K-ATPase Predicted Transmembrane Organization of Na,K-ATPase Protomers
(
The picture that emerges is of a Na,K-ATPase complex of subunit unfolded without leaving
the membrane, but three transmembrane spans (M8-M10) and the C terminus
of the
subunit were extruded, while the rest of
retained its
normal topology with respect to the lipid bilayer. Here we investigated
thermal denaturation further, with several salient results. First,
trypsin sensitivity at both surfaces of
was increased, but not
sensitivity to V8 protease, suggesting that the cytoplasmic domains and
extruded domain were less tightly packed but still retained secondary
structure. Second, thermal denaturation was accompanied by
SDS-resistant aggregation of
subunits as dimers, trimers, and
tetramers without
or
subunits. This implies specific
-
contact. Third, the
subunit, like the C-terminal spans of
, was
selectively lost from the membrane. This suggests its association with
M8-M10 rather than the more firmly anchored transmembrane spans. The
picture that emerges is of a Na,K-ATPase complex of
,
, and
subunits in which
can associate in assemblies as large as
tetramers via its cytoplasmic domain, while
and
subunits
associate with
primarily in its C-terminal portion, which has a
unique structure and thermal instability.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and
(reviewed
in Ref. 1). The
subunit (112 kDa) is a polytopic membrane protein
homologous in structure to the Ca2+-ATPase of sarcoplasmic
reticulum, which was recently determined to 2.6 Å (2). The
subunit
(34 kDa) is a single-span membrane protein with a large extracellular
portion that is glycosylated and stabilized by disulfide bonds
(reviewed in Ref. 3). Both
and
are required for the assembly
and function of the enzyme, and once assembled, it has not been
possible to separate them without irreversible loss of activity. In the
renal medulla the Na,K-ATPase is also associated with a 64-70-amino
acid single span membrane protein known as the
subunit (reviewed in
Ref. 4). This protein is not present in all Na,K-ATPase preparations, and in fact it is not even present in all segments of the mammalian nephron (5). It has been demonstrated to act as a regulator of
Na,K-ATPase properties, however, influencing affinity for both Na+ and K+ (5-7) and affecting conformation
(reviewed in Ref. 4). Its association with
and
, when it occurs,
is very stable, surviving extensive proteolytic degradation and
detergent solubilization along with the ability to occlude
K+ (8). Whether the Na,K-ATPase is normally a protomer (one
copy of each subunit) or a dimer or tetramer of
units in the
membrane remains a controversial matter.
helices or
sheets such as in porins, are normally much
more stable to thermal denaturation than the extramembranous portions
(reviewed in Ref. 9). This is not because the lipid bilayer provides
superior stabilization or a greater heat sink, but because of the
absence of water, which provides the competing H-bond donor and
acceptor groups that are instrumental for unfolding these internally
bonded structures (10). Consequently much higher temperatures are
required to denature the membrane domain. In this context, the
observation that relatively mild heating (50 °C in
-mercaptoethanol or 55 °C without) caused a radical
reorganization of part of the transmembrane domain of the Na,K-ATPase
subunit while unfolding the
subunit ectodomain was surprising
(11, 12). Here we have investigated further the consequences of heat
denaturation of the Na,K-ATPase. Our observations on the degree of
denaturation of other portions of the Na,K-ATPase
subunit are in
line with the literature, but some unexpected observations were made
that are pertinent to the tertiary and quaternary structure of the enzyme.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
70 °C in 250 mM sucrose, 30 mM histidine, 1 mM
EDTA (pH 7.2). Final specific activities were 600-2,000 µmol/h/mg of
protein. Pig and dog
1 differ by only 21 amino acid
substitutions; the
subunit antibody used works better on dog,
necessitating the use of that species in a few experiments.
subunit of
the Na,K-ATPase). Samples were reduced with 5%
-mercaptoethanol, and sample preparation was at room temperature. For determination of
the molecular weights of the aggregates, a 3.5% polyacrylamide gel
with 0.2% SDS was used with the Weber and Osborn buffer system (15).
This was necessary because the high molecular weight standards (cross-linked phosphorylase B, Sigma) run anomalously on Laemmli gels.
A Tricine1 peptide gel system
was used in some experiments for resolution of the
subunit
(16).
1 subunit isoform, is available from the Developmental
Biology Hybridoma Bank, University of Iowa (Iowa City, IA). Rat
antibody 2F12, which recognizes
1,
2, and
3 and binds in the intracellular L2-3
loop,2 was obtained from Dr.
Melitta Schachner, (ETH Zurich, Switzerland). Mouse antibody 9A7, which
recognizes
1,
2, and
3 and
binds in the intracellular L4-5 loop near M5,2 was
obtained from Dr. Maureen McEnery, Case Western Reserve University (Cleveland, OH). Peptide-directed antibody ETYY against the
subunit
C terminus was the gift of Dr. Jack Kyte, University of California, San
Diego. Antibody 8A, which is specific for the
1 subunit,
was obtained from Dr. Michael Caplan, Yale University Medical School
(New Haven, CT). We thank all our colleagues for generous gifts of
antibodies. Antibody RCT-G1, which recognizes the C terminus of the
subunit, was described earlier (5).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
,
, and
subunits based on the
literature, and indicates the locations of the epitopes of each of the
antibodies used in this study. Fig. 1B diagrams the extent
of denaturation of the rat Na,K-ATPase
subunit after heating to
55 °C that we previously deduced from investigating the sensitivity
to proteolysis at the extracellular surface in sealed right-side-out
rat renal medulla vesicles (11). Normally the Na,K-ATPase is highly
resistant to proteolysis at the extracellular surface except for two
sites on the
subunit, which nonetheless stays intact because the
fragments are connected and stabilized by disulfide bonds. After
heating, the last three transmembrane spans of the
subunit, and the
associated cytoplasmic segments, became accessible to extravesicular
trypsin (arrows). The protein kinase C phosphorylation site
and several tryptic cleavage sites (T1, T2, T3, T4, and T19) remained
inaccessible from the outside of the vesicles, indicating that the
integrity of the vesicles was unaffected. Simultaneously, the
subunit extracellular domain became very sensitive to trypsin, but its cytoplasmic N terminus and transmembrane span remained protected by the
vesicles. For simplicity, the diagram shows only the truncated portion
of
that remained after proteolysis. After heating (and without
digestion) the C terminus of the
subunit became accessible to an
antibody (ETYY). Heating also caused the protein kinase A
phosphorylation site, which is on the cytoplasmic surface of the
subunit between M8 and M9, to become accessible to kinase from the
extracellular side. The presence of RbCl or KCl protected against the
partial denaturation of both
and
subunits. Similar observations
of K+ protection and the extrusion of M8 and M9 were made
by others with pig kidney Na,K-ATPase (12).
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Fig. 1.
Diagram of the native and partially denatured
state of the Na,K-ATPase. A, cartoon
representing the native structure of Na,K-ATPase and
subunits.
The predicted transmembrane spans are shown as cylinders.
The locations of the epitopes for antibodies 6F, 2F12, 9A7, ETYY, and
8A are shown. B, consequences of heating Na,K-ATPase to
55 °C in K+-free buffer for 30 min, taken from
observations reported earlier (11). The experiments were performed in
sealed rat medullary vesicles so that protein probes (protease,
antibody, and kinase) had access only to the extracellular surface.
M1-M7 remain anchored to the membrane, but M8-M10 unfolded into the
extracellular space, exposing the C-terminal epitope (ETYY), the
cytoplasmic protein kinase A site (PKA), and new tryptic
cleavage sites (arrows). The protein kinase C site
(PKC) and other known tryptic cleavage sites, T1, T2, T3
(17), T4 (64), and T19 (65), remained inaccessible in the
vesicle.
subunit.
In addition, we fortuitously observed SDS-resistant aggregation or
cross-linking of the
subunits as a consequence of the heat
denaturation event.
subunit. An increase in sensitivity to
trypsin after heating was readily demonstrated (Fig.
2). Normally only a few tryptic cleavage
sites are exposed in the Na,K-ATPase, and the accessibility of the
sites is influenced by whether NaCl or KCl is present, causing the
enzyme to adopt E1 or E2 conformations. In E2 (KCl), cleavage occurs first at T1, then at T2 and T4 and secondarily at some other sites near
those (Fig. 1). A concentration of trypsin and length of time were
chosen that permit digestion at T1, T2, and T4, but not at T19. For
these experiments the enzyme was heated in the absence of KCl (because
KCl prevents the denaturation), but KCl was added prior to addition of
the protease. Digestion produced the usual set of fragments from the
unheated control, stained with antibodies against the N terminus (6F)
and C terminus (ETYY). An antibody that we have mapped to the
C-terminal half of the L4-5 loop, 9A7, stained the same set of
fragments that were stained by ETYY. After heating, trypsin completely
digested both the N-terminal and C-terminal epitopes, leaving blank
lanes. Stain with the 9A7 antibody, however, revealed that there was a
small amount of almost intact
subunit (which must have been cleaved
at both ends) and some residual fragments of the same size as in the
control. More than 90% of the stain was gone, however. The conclusion
is that multiple additional tryptic cleavage sites were exposed.
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Fig. 2.
Heating increased sensitivity to trypsin
digestion. Pig kidney Na,K-ATPase was first heated at 55 °C for
30 min or not, and then samples were subjected to trypsinization under
conditions that normally give a limited number of cleavages. Trypsin
was quenched prior to gel electrophoresis. Each panel shows stain by a
different antibody, 6F against the N terminus, ETYY against the C
terminus, and 9A7 against the C-terminal half of the P domain of the
large intracellular loop. After heating, both and its aggregates
were more sensitive. The N- and C-terminal epitopes were completely
removed, and an internal epitope was substantially digested. The gels
were of 10% polyacrylamide. All data shown is representative of
replicate experiments.
subunit heated to 55 °C was not completely or irreversibly
unfolded: although sensitive to trypsin, it did not acquire sensitivity
to Staphylococcus V8 protease (Glu-C). Normally the
Na,K-ATPase is completely resistant to this protease unless it is
treated with low concentrations of SDS. We previously showed that
0.04% SDS (50 times less than required to fully denature the enzyme)
permitted limited digestion of pig kidney enzyme (19). V8 protease can
also be used to generate proteolytic fingerprints of the
subunit by
adding it to enzyme after dissolving it in SDS-containing Laemmli
sample buffer (20, 21), but as we show here, this can be prevented by
inactivating the protease with trichloroacetic acid before
running the gel. Fig. 3 shows first, that V8 protease did generate
fragment fingerprints when digestion was allowed to occur during gel
electrophoresis (when no trichloroacetic acid was added to
inactivate the protease). Both
and
were digested in the gels,
and the antibody against the
C terminus, ETYY, stained a different
set of fragments than the antibody against the L2-3 loop, 2F12.
Second, the figure shows that there was negligible digestion if 1.5%
trichloroacetic acid was added after the incubation but before
starting electrophoresis, even when using heated enzyme. This means
that V8 protease cleavage sites were not exposed by the heating. It is
quite surprising that even the extruded C terminus of heated
was
largely resistant to the protease, as indicated by the lack of loss of
ETYY binding. This implies that although denatured, the protein
retained some compact secondary structure.
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Fig. 3.
Heating does not increase sensitivity to V8
protease. Purified dog kidney enzyme was heated as described in
the legend to Fig. 2. Control and heated samples were incubated with
protease for 30 min where indicated, and then prepared for
electrophoresis either in the usual way, or with addition of
trichloroacetic acid to acidify the samples just before
electrophoresis. Each panel shows stain by a different antibody, ETYY
against the C terminus, 2F12 against the L2-3 loop, and 8A against the
subunit. There was no detectable digestion of
or aggregates by
V8 protease, although digestion of
was enhanced by heating. The
gels were of 10% polyacrylamide.
but Not
or
--
It can be
seen in Figs. 2 and 3 that some high molecular weight aggregates of the
subunit were present in the heated samples. There was also some
aggregation seen in the trichloroacetic
acid-acidified controls in Fig. 3, and it is notable
that the aggregates were stable to such harsh acidic detergent
conditions. In the next experiments, we used more porous polyacrylamide
gels to investigate the structure and composition of the heat-induced aggregates.
-subunit-specific antibodies were used to identify the high
molecular weight bands, one that binds in the N-terminal half and the
other at the C terminus. The presence of 15 mM KCl (or
RbCl, not shown) abolished the aggregation, just as it prevented the
physical changes illustrated in Fig. 1B.
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Fig. 4.
Aggregates of Na,K-ATPase subunit stable to gel electrophoresis. Purified pig kidney
Na,K-ATPase was heated for 30 min at 55 °C with or without KCl, and
prepared for electrophoresis on Laemmli gels of 5% polyacrylamide.
Higher molecular weight bands were seen that stained with two different
Na,K-ATPase-specific antibodies, and these bands were much more
pronounced when heating was performed in the absence of KCl.
as monomers, dimers, trimers, and tetramers after heating, as identified by mobility calibrated with cross-linked phosphorylase b (Sigma) in the high molecular weight range.
In this gel system, larger aggregates were not observed, suggesting that they might have been Laemmli gel artifacts.
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Fig. 5.
Formation of dimers, trimers, and
tetramers. Pig kidney Na,K-ATPase was heated without KCl as
described in the legend to Fig. 4, but electrophoresed on Weber-Osborn
gels with three kinds of molecular weight markers. A shows a
blot stained with anti-ETYY antibody against , and B the
plot of electrophoretic mobility versus molecular weight of
the standards. The standards were Bio-Rad prestained, high molecular
weight range; Amersha Pharmacia Biotech rainbow prestained, high
molecular weight range, and Sigma cross-linked phosphorylase
b. The latter is unreliable on Laemmli gels. The mobilities
of the Na,K-ATPase bands are consistent with being multiples of 110 kDa.
subunit. Since
and
are normally
inseparable, this indicates that the aggregation was the result of
specific interaction between
subunits, not simply the production of
a physical state like cooked egg. Nor did
subunits aggregate with
one another in an SDS-resistant form. The same was true for the
subunit, as illustrated in Fig. 7. In
this case the samples were electrophoresed in parallel on 5% polyacrylamide gels to resolve the aggregates of
(Fig.
7A) and on 5-15% gradient gels to resolve the
doublet,
which is only 7.5 kDa (Fig. 7B). It can be seen, however,
that no higher molecular weight forms of
were detected in either
type of gel. The same result was obtained with Tricine gels (not
shown). In summary, the SDS-resistant aggregation or
cross-linking event is clearly very selective, since it creates
multimers of the
subunit and excludes the two proteins that have
the most intimate association with
.
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Fig. 6.
Aggregation in microsomes; no
subunits aggregate. Dog kidney-purified
enzyme (panel A) and crude microsomes (panel B)
were treated with heat identically, as described above. The gel was of
5% polyacrylamide. It can be seen that heat induced the same
aggregates in microsomes as in purified enzyme. It can also be seen
that there was no detectable aggregation of
with
or with
itself.
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Fig. 7.
The aggregates do not contain
subunit. Pig kidney-purified Na,K-ATPase was
heated as above, and samples were run on both a 5% polyacrylamide gel
(A) and a 5-15% gradient gel (B), and the blots
were each stained with the appropriate antibody, ETYY to detect
and
RCT-G1 to detect
. The
subunit is actually a doublet contributed
by two splice variants,
-a and
-b (57).
Subunit in Right-side-out Vesicles--
Our
previous work on heat denaturation in right-side-out rat renal vesicles
indicated that a portion of the
subunit from M8 to the C terminus
was extruded from the membrane into the extracellular space (Fig.
1B). The N terminus of the
subunit, which is on the
cytoplasmic surface, did not become accessible to protease. The
subunit (in all the species that have been sequenced) has potential
tryptic cleavage sites on both sides of the membrane. The extracellular
site, which removes the alternatively spliced N terminus and a few more
amino acids, is accessible in right-side-out vesicles (6, 22). We too
previously detected cleavage in right-side-out vesicles by a shift in
the gel mobility of the
subunit (5). Our antibody to
, RCT-G1,
binds to an epitope at the C terminus, and there are several potential
tryptic cleavage sites that would destroy or remove the epitope if
trypsin had access to the C-terminal end. This sets the stage to test
the hypothesis that during heating the
subunit remains anchored in
the membrane (like the
subunit) or comes out (like M8, M9, and M10
of the
subunit).
subunit was promoted by heating just as
it was in purified enzyme and crude microsomes, and the ETYY epitope on
aggregated
was very sensitive to digestion from the outside of the
vesicles (Fig. 8A). In the
present experiments, some Na,K-ATPase
subunit remained completely
undigested (20-30% as determined by densitometry), but we did not
detect any ETYY-stained fragments, consistent with extensive digestion
of the majority of the C terminus that was extruded to the
extracellular surface (Fig. 8A). Goldshleger et
al. (12) reported that while M8 and M9 left the membrane, at least
some of M10 and the C terminus retained their normal topology because
proteolytic fragments stained with ETYY could be detected after
digestion from the extracellular surface. Minor differences in protocol
probably account for the difference between laboratories, since we
performed the digestion at 37 °C instead of room temperature, and
this may have resulted in better exposure of tryptic sites near ETYY in
a molten globule-like physical state in the extracellular space.
View larger version (45K):
[in a new window]
Fig. 8.
The subunit is
extruded from the membrane upon heating. Pig kidney right-side-out
vesicles were heated, and then treated with trypsin from the outside.
The gel was made of 12% polyacrylamide, and it used the Tricine buffer
system (16) to obtain better resolution of
. For this reason the
aggregate bands are crowded at the top of the gel.
A, the blot was stained with ETYY antibody. No small
fragments were obtained that stained with ETYY, but a substantial
fraction of the ETYY stain was digested in heated vesicles.
B, the bottom half of the gel was restained with
RCT-G1 to stain the
subunit. It was cleaved to a single
faster-migrating band from the outside of both control and heated
vesicles, but the recovery of the fragment was much lower in the heated
sample, indicating accessibility of the RCT-G1 epitope at the outside
surface after heating. Losses of
and
were quantified by
densitometry.
subunit
after heating right-side-out pig kidney vesicles (Fig. 8B). First, it can be seen that the
doublet (
-a and
-b) was
reduced to a single band in the control (unheated) vesicles, consistent with digestion at a lysine in the extracellular space, (K)GDVD for pig
(23). In our previous work we used 16% polyacrylamide gels and the
faster-migrating digested product was better resolved (5); here on a
12% gel it ran only slightly faster than the
-b band. In the
experiment shown, a 74% loss of the subunits
C-terminal epitope
(ETYY) was accompanied by a loss of 68% of the
subunits C-terminal
stain, as determined by densitometry (compare the 2nd and 4th lanes).
This indicates that
, like M8-M10, is extruded from the membrane,
allowing the epitope at the C terminus to be exposed to trypsin in the
extravesicular space. In experiments performed with rat renal medulla
right-side-out vesicles, almost all of the
subunit epitope was
digested (data not shown). We also confirmed with both pig and rat
vesicles that the cytoplasmic N terminus of the
subunit, and its
transmembrane span, were not digested and remained with the vesicles
(data not shown).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
subunit
with SERCA1a, there is significant homology in all major domains (other
than a few deletions and insertions), including in the C-terminal end
and the connecting loops and tail.3 Thus the gapped-BLAST
alignment is consistent with the adoption of the same fold. Homologous
C-terminal topology for the individual transmembrane hairpins of
Na,K-ATPase is now supported by biochemical evidence (12, 24).
Cross-linking data suggests proximity of M1-M2 to M8-10 in the
Na,K-ATPase (25, 26), and this can be accommodated in the model by the
fact that M2 is adjacent to M9, and both have cysteine residues that
could be the cause of the observed cross-link. (The appearance in Fig.
9A that M4b separates M2 from M9 is misleading, because that
portion of M4 is actually in the cytoplasm, below the plane of the
membrane-embedded segments.) In addition, there is a cross-link between
M9 and M10 in the Na,K-ATPase that could also be accommodated
by this model. The figure shows predicted positions of
the
and
subunit transmembrane spans, based on data discussed
below.
View larger version (47K):
[in a new window]
Fig. 9.
Predicted Na,K-ATPase subunit
association. A, model for the packing of ,
, and
transmembrane spans. The actual transmembrane spans of the SERCA1a
enzyme are shown in blue, as seen from the luminal surface
with the longest luminal loops cut away. M8, M9, and M10, and the
L9-10 loop are highlighted in yellow. Although it appears
that M4b lies between M2, M6, and M9, in fact M4b is in the cytoplasm
below the other helices. The predicted position of the
span
(purple) is adjacent to M7, and close to the L7-8 loop at
the extracellular surface. The predicted position of the
span
(red) is in association with M8, M9, and M10. In this
position it would be under the N domain on the cytoplasmic surface.
B, conceptual diagram of the proposed tetraprotomeric
organization of the Na,K-ATPase. The outline of the
subunit roughly
corresponds to the projection outline of the Ca2+-ATPase as
seen from the luminal surface. The smallest lobe, conjectured here as
the point of contact, is the location of the Ca2+-ATPase A
domain, but of course other parts of
could be in contact instead.
The
and
subunits are shown at the periphery (and in the
positions shown above) because of their failure to aggregate with
or each other.
-mercaptoethanol exposes the extracellular L7-8 and L9-10
loops to digestion. Heating at 50 °C with dithiothreitol or at
55 °C without it next exposes the intracellular L8-9 loop at the
extracellular surface (11, 12). We also observed (here and earlier)
that the C terminus was exposed to the extracellular side, and since
Goldshleger et al. (12) did not, the extrusion of M10
and the C terminus might be the last step in the denaturation of the
C-terminal portion of the protein. In this study we obtained evidence
that the cytoplasmic domains of the
subunit are also perturbed to
the extent that they become much more sensitive to digestion by
trypsin. The denaturation is unlikely to take the form of complete
unfolding, however, because of the continued resistance of the
subunit to digestion by Staphylococcus V8 protease on both
sides of the membrane.
subunit may favor loss to the
extracellular surface when proteolytic cleavage is not a factor (11,
12).
subunit, which is
consistent with the concept that secondary and tertiary structures that
are embedded in the membrane have very high stability (9). It was not
ruled out, however, that portions of the membrane domain became so
unstable after protease K that they were already denatured at a lower
temperature, or even digested away. It is also puzzling that no
transition was seen for the
subunit.
particles (protomers) are capable of hydrolyzing ATP and of carrying
out active ion transport (38-40), but there is much evidence that
suggests that the enzyme normally associates as dimers
(
)2 or as tetramers (
)4. This was
initially proposed as a result of cross-linking experiments (41-43),
but is supported with many other kinds of evidence as well: by
saturation transfer EPR (44) and fluorescence energy transfer between
subunits (45, 46), gel filtration (47), co-precipitation (48), kinetic
evidence that could be best explained by the interaction of active
sites (49), by measurements of the binding of different nucleotide
analogs that suggest the existence of as many as four nonidentical
sites (50), and by electron microscopy of solubilized particles (50).
With electron microscopy of two-dimensional membrane crystals,
Na,K-ATPase units (
) have been observed in both monomeric and
dimeric associations (51, 52).
subunits. While
and
subunits may participate in quaternary interactions and even form
SDS-sensitive aggregates, they did not participate in formation of
SDS-resistant multimers (Fig. 9B).
subunits as a consequence of
heating has been reported previously, when enzyme was first heated and
then solubilized in the detergent C12E8 and sedimented in an analytical centrifuge (31). Those aggregates dissociated in SDS for gel electrophoresis, however. It is possible that such aggregation occurred here as an intermediate step in the
formation of SDS-resistant aggregates, and we observed that enzyme
activity was lost significantly faster than SDS-resistant aggregates
were formed (data not shown). That the aggregates proved to be more
trypsin-sensitive than unaggregated material in the same samples also
implied that denaturation preceded aggregation and that not all of the
was equally denatured at the time point used. Aggregation of
detergent-solubilized Na,K-ATPase units has also been reported (31,
54), but is unlikely to be related to the events observed here. The
formation of anomalously-migrating
subunit forms after heating
Na,K-ATPase in SDS sample buffer (55) is also not related to the
observations reported here.
subunit and fragments of
has been observed (26), but the aggregates studied here were resistant to reducing agents and unlikely to be due to
disulfide bonds. Slower migrating species eventually formed, but it is
not clear whether they were higher-order aggregates or internally
aggregated forms with reduced electrophoretic mobility. We did not
observe aggregates so large that they failed to enter the gel.
)--
In Fig. 9A we show a model of the
predicted organization of
,
, and
transmembrane spans based
on the known structure of the SERCA1a Ca2+-ATPase (2) and
on the behavior of the
and
transmembrane spans during heat
denaturation. In the figure,
's span is shown associated with
M8-M10. The
subunit is closely enough associated with
to be
labeled by cardiac glycoside derivatives with reactive groups (reviewed
in Ref. 56). The fact that the
subunit, like the C-terminal segment
of
, was selectively lost from the membrane upon heating could be
interpreted two ways. As an optional regulatory subunit,
might not
be tightly bound to the Na,K-ATPase, and it could be hypothesized that
its membrane association is spontaneously labile to heat. The highly
hydrophobic nature of
's membrane span (57), however, makes it
unlikely that it would spontaneously leave the membrane in mild heat
unless it were bound to something more soluble. The
subunit has
been observed to dissociate from extensively digested Na,K-ATPase
destabilized by calcium (8), but the centrally located M5-M6 hairpin of
also dissociated under those and similar conditions (58, 59), and
conditions have been observed in which the M5-M6 hairpin was lost but
not the
subunit (60). Such disruption must reflect major
disintegration of the complex of membrane spans. Further evidence that
the loss of
is linked to
structure is that it is prevented by
K+, just as
and
denaturation is prevented. The data
thus suggest that
is associated with M8-M10 rather than other more
firmly anchored transmembrane spans.
's span is shown associated with M7 because M7 is close to the
known extracellular
-
association site in the L7-8 loop (61,
62), and because
and M7 remain anchored during heat denaturation.
Since the
subunits extracellular domain denatures but its
transmembrane and cytoplasmic domains do not leave the membrane with
M8-M10, it must be associated with transmembrane spans that remain in
the membrane or with a cytoplasmic domain of
. M7 does not have a
cysteine residue available for oxidative cross-linking, but
has
been shown to form a disulfide cross-link to M8 (25, 26, 63). As
pointed out by the authors, however, this occurred preferentially after
detergent treatment or extensive digestion, not in native enzyme. In
the Ca2+-ATPase structure, M8 is more buried than M7.
Consequently it appears likely that
's proximity is primarily to M7
and secondarily to M8 in the Na,K-ATPase structure. This is a
refinement of the position of
proposed by Or et al. (26)
and Ivanov et al. (63) based on cross-linking studies alone.
,
, and
subunits in which
can associate in assemblies as large as
tetramers (Fig. 9B), probably via an cytoplasmic domain,
while
and
subunits associate with
primarily in its
C-terminal portion, which has a unique structure and thermal
instability. In the high resolution Ca2+-ATPase structure,
transmembrane spans 1-6 are associated with the large A and P domains,
while spans 7-10 are associated only with smaller extramembranous
loops which themselves are not part of self-contained,
-helix, or
-strand-containing secondary structures. This C-terminal structure,
an assembly of contributing segments, helps to explain the relative
ease with which the C-terminal end of the Na,K-ATPase undergoes radical
reorganization. We conclude that
subunits associate upon heating,
but that the
and
subunits are either too distant or too easily
dissociated to participate in aggregation.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grant R01 HL36271 (to K. J. S.).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.
To whom correspondence should be addressed. Tel.: 617-726-8579;
Fax: 617-726-7526; E-mail: sweadner@helix.mgh.harvard.edu.
Published, JBC Papers in Press, November 30, 2000, DOI 10.1074/jbc.M009131200
2 C. Donnet and K. J. Sweadner, unpublished observations.
3 K. J. Sweadner and C. Donnet, unpublished observations.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; MOPS, 4-morpholinepropanesulfonic acid; TPCK, L-1-tosylamido-2-phenylethyl chloromethyl ketone; SERCA, sarcoplasmic/endoplasmic reticulum Ca2+-ATPase.
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