From the
The topological organization of the Na,K-ATPase
The Na,K-ATPase
To study the spatial organization of the
Na,K-ATPase and the sidedness of events of membrane transport, sealed
vesicles have been extensively employed
(11, 14, 15, 16, 17, 18, 19, 20, 21) .
Right-side-out vesicles, i.e. those in which Na,K-ATPase has
the same orientation as in the cell, are formed during homogenization
of renal medulla by the resealing of involuted basolateral membrane
fragments. Vesicles are typically 80-90% sealed, as determined by
measuring how much latent ATPase activity can be detected by assay with
and without mild detergent treatment to open them. Chemical
modification of right-side-out vesicles revealed a 3:1 relative
distribution of reactive amino groups on the
In this paper we report that exposure of a certain stretch
of the
The renal medulla preparation used here
contains the
Fig. 2
also
shows the pattern of
The results verify the accessibility of
the
Fig. 3
illustrates the result of trypsinolysis of rat
kidney vesicles after incubation with 200 m
M DTT at 37 °C
( lanes 1-4) and 50 °C ( lanes 5-8) for
15 min. A distinct doublet of the
Taken
together, the data of Figs. 3, 4, and 5 and the loss of ATPase activity
suggest that harsh treatment caused rearrangements of the structure of
the Na,K-ATPase. The most quantitative digestion of
Prior to examining the
phosphorylation of right-side-out kidney vesicles, we used purified
Na,K-ATPase before and after heating and reduction. Fig. 7 A confirms that Na,K-ATPase was normally phosphorylated only in the
presence of Triton X-100 (compare lanes 9 and 10). In
contrast, samples that were pretreated with 200 m
M DTT at 50
°C for 15 min were phosphorylated even without detergent, although
addition of Triton enhanced it. Heating to 55 °C for 30 min
resulted in the highest level of 32P incorporation, and
addition of Triton had little additional effect.
Phosphorylation by protein kinase A catalytic subunit
was performed in rat kidney vesicles after heating but prior to
digestion by trypsin, to see if the smaller cleaved fragment(s) could
be detected (Fig. 9). In the same experiment, we employed an antibody
(anti-ETYY) capable of detecting fragments containing the
The predicted structure of
the C-terminal third of the Na,K-ATPase has been controversial, and the
10-span model in Fig. 12 A is provisional. Attempts to predict
transmembrane
Heating and reduction, of course, have the potential to bring about
radical structural change. The extensive digestion of the
In the model shown in Fig. 12 A,
the sites at 906-907 are predicted to be on the extracellular
surface, whereas those at 933-936 are predicted to be on the
cytoplasmic surface. If cleavage is really at 933-936, and if the
model is correct, the exposure of this region to proteolysis from the
extracellular side of sealed right-side-out vesicles implies that it
moved (``popped out'') as an early step of denaturation.
The normal location of
the C terminus has been controversial. The weight of evidence suggests
that it is normally inaccessible from either side. Two groups have
produced antibodies against the peptide sequences of the C terminus
that failed to recognize native enzyme
(26, 28) ,
providing no support for binding to exposed sites on either surface.
Another antibody against the C terminus was initially thought to bind
to the extracellular surface based on fluorescence-activated cell
sorting, but the epitope was readily removed by trypsin
(58) ,
suggesting in retrospect that the fluorescence was due to a small
fraction of denatured enzyme or to antibody contaminants binding to
unrelated surface proteins. Two additional antibodies against the C
terminus were thought to bind on the cytoplasmic surface because
binding to right-side-out kidney vesicles was seen only when they had
been opened by treatment with SDS
(17) . However, those vesicles
showed a maximum of 6.7-fold increase in ATPase activity, while the
antibody binding increased more than 1000-fold. The conclusion that the
site is on the intracellular surface is less likely than that the site
was buried and exposed by detergent. Further evidence for the normal
inaccessibility of the C terminus is that access of the sequence ETYY
to carboxypeptidase Y was restricted in trypsin-digested membranes
which have intact Rb+occlusion capacity, but greatly
increased in digested membranes treated with Ca2+,
which destroys Rb+occlusion capacity
(9) . It
was concluded that digestion occurred only in the inactivated sample.
Whether the Ca2+treatment caused externalization of a
large portion of the
The data
presented here throw no light on the normal location of the C terminus.
The observation that there was no loss of ETYY binding (on blots) to
When enzyme
was digested after heating and reduction or heating at 55 °C, an
antibody against the first 12 amino acids of the
Since the
These observations are
consistent with the present report that heating and reduction disrupt
The
effect of Rb+in preserving activity and ouabain
binding and in protecting against digestion is consistent with the
hypothesis that the Rb+-occluded form of the enzyme is
a conformation with maximum compactness and stability
(19, 22, 42) . We can also point out an inverse
correlation between Rb+(K+) binding
and the accessibility of the protein kinase A catalytic subunit
phosphorylation site in unheated, but Triton-treated enzyme: optimal
phosphorylation is seen in the absence of monovalent cations and in the
presence of Mg2+/P
The
conclusion that emerges is that both K+(Rb+) and ouabain stabilize a conformation in
which the protein kinase A catalytic subunit site is buried and other
sites within the 19-kDa C-terminal portion are protected from
proteolysis. The stabilizing effect of Rb+against a
variety of insults (heat, reduction, and proteases), combined with the
fact that both subunits are protected, implies that
Rat kidney vesicles were
presoaked in media containing various ligands for the indicated periods
of time. The latency of the vesicles was determined by the stimulation
of Na,K-ATPase activity by mild detergent treatment. The latency of
untreated vesicles (kept on ice after thawing) in a medium containing
no external ligands was taken as 100%.
Digestion of right-side-out vesicles with
various proteases reveals two ``hot spots'' in the structure
of
Rat kidney vesicles were pre-equilibrated for 1 h
at 4 °C in different media prior to proteolysis. Digestion was
performed for 30 min at 37 °C. Blots were stained with
peptide-directed antibody 757 to detect any fragments containing the N
terminus of the
Effect of heating and reduction on
proteolysis of
Rat kidney vesicles were treated with
various concentrations of DTT for the indicated periods of time prior
to trypsinolysis. No external ligands were added to the incubation
media. Detection of digestion fragments was performed with K1 antiserum
(
Protective effect of Rb+ions
against thermal denaturation
Pig kidney Na,K-ATPase (specific
activity 20 µmol of P
We thank W. J. Ball, Jr., Alicia McDonough, and J.
Kyte for their generosity with antibodies and S. J. D. Karlish for
early and stimulating sharing of research results.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
subunit is
controversial. Detection of extracellular proteolytic cleavage sites
would help define the topology, and so attempts were made to find
conditions and proteases that would permit digestion of Na,K-ATPase in
sealed right-side-out vesicles from renal medulla. The
subunit is
predominantly extracellular and could mask the surface of the
subunit. Most of the tested proteases cleaved
, and some digested
it extensively. However, without further disruption of structure, there
was still no digestion of the
subunit. Reduction (at 50 °C)
of disulfide bonds that might stabilize the
subunit fragments, or
heating alone at 55 °C, permitted tryptic digestion of
at a
site close to the C terminus, while simultaneously increasing digestion
of
. A 90-kDa N-terminal fragment of
was recovered, but the
C-terminal fragment was further digested. Heating and reduction
resulted in the extracellular exposure of a protein kinase A
phosphorylation site, Ser-938, and the C terminus, both of which have
been proposed to be located on the intracellular surface. At the same
time, access to a distant protein kinase C phosphorylation site was not
increased. The data suggest that the harsh treatment simultaneously
resulted in alteration of the
subunit and the extrusion of a
segment of
that normally spans the membrane, without causing
complete denaturation or opening the sealed vesicles. Preincubation
with Rb+was protective, consistent with prior evidence
that it stabilizes the protein segments in the C-terminal third of
. We conclude that this portion of the
subunit contains a
transmembrane structure with unique lability to heating.
(
)
catalyzes the active
transport of ions across the plasma membrane. It has a catalytic
subunit,
, with a molecular mass of 110 kDa, and a smaller
glycoprotein subunit,
, with an apoprotein molecular mass of 32
kDa. A clear role for the
subunit has not been determined, but
there is evidence for its importance in
-
assembly during
biogenesis
(1) , in cell-cell communication
(2) , and in
modulating the affinity for K+(3, 4, 5) . Both subunits have been cloned
(6) , and topological models have been based on deduced primary
structure, hydropathy plot predictions, selective proteolysis, chemical
modification, and electron microscopy
(7, 8, 9, 10) . According to current
models, the N and C termini of the
subunit are located inside the
cell, and there is a large central intracellular portion, anchored by
6-10 transmembrane segments. The models predict that very little
of
is exposed on the extracellular surface, mostly in the
C-terminal third where the structure is much debated. In contrast, the
majority of the
subunit is exposed outside the cell
(11) .
Its extracellular domain has three asparagine-linked carbohydrate
groups and three disulfide bridges
(12, 13) . Only a
small portion, including the N terminus, is intracellular, connected by
one transmembrane span.
subunit between the
intra- and extracellular surfaces
(11) , which predicts a
respectable number of potential tryptic cleavage sites. Attempts to get
tryptic cleavage of the
subunit at the extracellular surface have
been unsuccessful, however
(14, 15, 17, 18) . To explain the
discrepancy, one hypothesis is that the
subunit masks the
subunit.
subunit at the extracellular surface can be achieved in
right-side-out vesicles, and the data implicate the close interaction
of the
and
subunits. Independent work by Karlish et al. (22, 23) has produced evidence for the exposure of
sites at the extracellular surface of
as a consequence of
heating. Evidence for substantial reorganization of the C-terminal
domain is described here within the framework of topological models of
Na,K-ATPase structure.
Materials
Antiserum K1
(24) was raised
against the purified subunit of rat kidney Na,K-ATPase.
Antipeptide directed antisera 757
(25) and 845
(26) were the gift of Dr. William J. Ball, Jr., University of
Cincinnati. Polyclonal antiserum FP
1 was provided by Dr. Alicia
McDonough, University of Southern California; polyclonal antibody UBI
1 was purchased from United Biotechnology Inc. (Lake Placid, NY);
these two antibodies were prepared by the same procedure
(27) .
Monoclonal antibody McK1 was raised against rat kidney Na,K-ATPase
(16) . Peptide-directed antibody ETYY
(28) was the gift
of Dr. Jack Kyte, University of California at San Diego. All proteases
and secondary antibodies used for staining blots were obtained from
Sigma.
General Procedures
Purification of Na,K-ATPase
from pig or rat kidney outer medulla microsomes, determination of
protein concentration, ATPase activity assay, and
[H]ouabain binding were performed as described
elsewhere
(29) .
Right-side-out Vesicles
Right-side-out vesicles
from rat kidney outer medulla were prepared by modification of
published methods
(11) . Briefly, freshly prepared crude
microsomes were loaded onto step gradients consisting of 20 and 5%
metrizamide (Sigma, grade I) in 0.25
M sucrose, 25 m
M MOPS, pH 7.4, 1 m
M Tris-EDTA. Centrifugation was at
27,000 rpm for 4 h at 4 °C in the SW 50.1 rotor of a Beckman
ultracentrifuge. The vesicle fraction was collected from the interface
between the two metrizamide solutions and stored without further
manipulation at -70 °C after the addition of dimethyl
sulfoxide to 5% as a cryoprotectant. The extent of vesicle sealing was
assessed in a latency test by measuring the hydrolysis of ATP before
and after permeabilization, which was with 0.016% sodium dodecyl
sulfate in the presence of 3 m
M ATP to protect activity. For
the preparations used here, activity was stimulated 5.5-7.7-fold,
indicating that vesicles were 82-88% sealed. With cryoprotection,
vesicle sealing was changed very little by storage for weeks or months
at -70 °C.
Proteolytic Digestion
Right-side-out vesicles
(0.5-0.6 mg/ml) were incubated with different proteases (in all
cases, 1:3 w/w with respect to vesicle protein) for periods of time
from 1 min to 1 h as indicated, in medium containing 250 m
M sucrose, 25 m
M MOPS, pH 7.4, 1 m
M Tris EDTA. For
some experiments, different ligands were added as specified in the text
and tables; for others, digestion was preceded by treatment with
reducing agent (dithiothreitol or -mercaptoethanol) at different
temperatures, or by heating at 55 °C for 30 min (the latter
condition according to Ref. 23). Proteolysis was performed at 37
°C, and the reaction was stopped either with the addition of
acidified electrophoresis sample buffer (125 m
M Tris, 4% SDS,
20% glycerol, and 5%
-mercaptoethanol, pH < 2.0) or by addition
of 3 m
M diisopropylfluorophosphate (Sigma), a covalent
inhibitor of serine proteases. As shown previously, acidified sample
buffer is more effective at preventing digestion by trypsin during gel
electrophoresis than addition of soybean trypsin inhibitor
(30) , and it was successful with other proteases as well. The
acidification of the sample lasts only until it penetrates the gel, but
it appears to cause irreversible denaturation of the protease.
Gel Electrophoresis
For most experiments, gel
electrophoresis was performed according to Laemmli
(31) , using
7.5%, 10%, or gradient 5-15% polyacrylamide for separating gels.
To resolve short peptides, the Tricine-SDS buffer system of Schagger
and von Jagow
(32) was employed with 16% polyacrylamide gels.
Electrophoretic transfer was performed in a buffer containing 20 m
M Tris, 150 m
M glycine, and 20% methanol, at 100 mA
overnight at 4 °C. Nitrocellulose was quenched with Tris-buffered
saline, 0.5% Tween 20, and stained with antibodies as described
previously
(16) , with visualization with ECL (enhanced
chemiluminescence) (Amersham). For autoradiography of 32P,
nitrocellulose blots, stained with 0.1% Amido Black, were exposed to
Kodak XAR-5 film. Densitometry was accomplished with a Molecular
Dynamics laser densitometer.
1 and
1 isoforms of the Na,K-ATPase. Without
digestion, both subunits migrate on SDS gels at anomalous positions.
The
subunit migrates as if it had a molecular mass of 95-99
kDa, slightly faster than the 112-kDa mass deduced from the cDNA. This
is thought to be due to peculiarities in the interaction of very
hydrophobic domains with the detergent
(30) . The
subunit,
with its protein molecular mass of 32 kDa, migrates as a fuzzy band of
roughly 60 kDa because of its carbohydrate groups. The extra band of 70
kDa which stained with an antiserum against
1 (K1) seen in
Fig. 1
was absent from Na,K-ATPase purified from renal medulla
microsomes by SDS extraction (data not shown), and it was not
investigated further as it was not digested.
Figure 1:
Digestion of vesicles under mild
conditions. Right-side-out vesicles (36 µg) were equilibrated with
either 15 m
M NaCl ( lanes 1-4) or 15 m
M KCl ( lanes 5-8) overnight at 4 °C. Lanes 1 and 5 were controls with no trypsin. Samples were
digested with trypsin at 37 °C, and aliquots were taken at 1 min
( lanes 2 and 6), 10 min ( lanes 3 and
7), and 30 min ( lanes 4 and 8) of
proteolysis and loaded on a 5-15% gradient gel. Blots were
stained first with antibody K1 to detect ( A) and
reprobed with a polyclonal antibody, UBI
1 ( B). The
positions of undigested
subunit,
subunit, and the 50-kDa
fragment of the
subunit are denoted with arrows. The
undigested band at approximately 70 kDa, indicated by an
asterisk, has been seen consistently in vesicle and microsome
preparations when the K1 antiserum is used, but appears not to be
digested or to affect the results. Its identity is uncertain; it does
not stain with Na,K-ATPase
1-specific monoclonal antibody McK1
(data not shown). Double asterisks indicate the position of
trypsin, at 27 kDa, which is autodigested during the incubation. We do
not know why ECL reagents detect trypsin, but its identity was
confirmed on gels loaded with trypsin alone (not
shown).
Competition Enzyme-linked Immunosorbent
Assay
Heated (55 °C, 30 min) or unheated vesicles were used
directly or permeabilized by treatment with 0.016% SDS in the presence
of 3 m
M ATP for 15 min at room temperature. Various
concentrations of vesicles were preincubated with fixed concentrations
of antibody in a solution containing 250 m
M sucrose, 25 m
M MOPS, 1 m
M EDTA, 0.1% bovine serum albumin for 1 h at 37
°C. Vesicle-bound antibody was removed by centrifugation in an
Airfuge for 15 min. Unbound antibody was measured on microtiter plates
(Nunc Maxi-Sorp immunoplates) coated with rat kidney microsomes (0.7
µg/well in phosphate-buffered saline) and quenched with 1% bovine
serum albumin in phosphate-buffered saline. Detection was performed
with biotinylated goat anti-rabbit or anti-mouse IgG (Life
Technologies, Inc.), and color was developed with horseradish
peroxidase-conjugated streptavidin and o-phenylenediamine as a
substrate. The wells were read on a EIA plate reader (Bio-TEK
Instruments, Inc.).
Phosphorylation by Protein Kinase A
Pretreated or
untreated right-side-out vesicles (10 µg) or purified Na,K-ATPase
from rat kidney (1 µg) were preincubated in a buffer containing 20
m
M HEPES, pH 7.4, 1 m
M EGTA, 5-10 m
M MgClin a total volume 10-20 µl. After 15
min, 0.05% Triton X-100 (if specified) and 100 ng of protein kinase A
catalytic subunit (Sigma) were added. After 5 min, the reaction was
started by addition of 200 pmol of [
-32P]ATP
(DuPont NEN) (specific activity 10,000 cpm/pmol). Phosphorylation was
allowed to proceed for 15 min at 30 °C and was stopped with an
equal volume of electrophoresis sample buffer containing 125 m
M Tris-Cl, pH 6.8, 4% SDS, 20% glycerol, and 5%
-mercaptoethanol. When trypsinolysis of phosphorylated Na,K-ATPase
was performed, vesicles were first heated at 55 °C for 30 min, next
phosphorylated by protein kinase A catalytic subunit without Triton as
described above, and then trypsin was added at 1:3 ratio with respect
to vesicle protein. Proteolysis was for 1 h at 30 °C and was
stopped with acidified electrophoresis sample buffer.
Phosphorylation by Protein Kinase C
An aliquot of
right-side-out vesicles containing 3 m
M ATP (sodium salt) was
first treated with 0.016% SDS for 15 min at room temperature for
permeabilization. Then opened and sealed vesicles were subjected to
treatment with 200 m
M dithiothreitol (DTT) at 50 °C for 15
min or heated at 55 °C for 30 min. For optimal phosphorylation by
protein kinase C (PKC), samples (10 µg) were preincubated in the
presence of 10 m
M Tris phosphate, pH 7.4, 5 m
M magnesium acetate, 500 µ
M CaCl, 100
n
M phorbol 12-myristate 13-acetate (Sigma), and 50 ng of
protein kinase C (Calbiochem). The reaction was started with 200 pmol
of [
-32P]ATP (specific activity 1,000
cpm/pmol) and stopped after 15 min by the addition of an equal volume
of electrophoresis sample buffer.
Trypsinolysis in the Presence of Different
Ligands
Ion-induced conformation changes are well-known to alter
the digestion of cytoplasmic loops of the subunit
(33) .
To evaluate whether such changes would also affect the extracellular
portion of either subunit, trypsinolysis of right-side-out vesicles was
performed after overnight incubation in either 15 m
M Na+or 15 m
M K+, to
induce the E1 or E2 conformation, respectively
(Fig. 1). Digestion from the extracellular surface was unaffected
by the cations. Staining with a polyclonal antibody against
(K1)
revealed no digestion even at 30 min (Fig. 1 A). In
contrast, the
subunit was readily cleaved at 1 min
(Fig. 1 B; the blot shown in A was reprobed with
a second antibody, and the
stain is the difference between the
two images). Prior work
(34) has established that
is
preferentially cleaved at Arg-142, dividing the 302-amino acid protein
into roughly equal pieces. Only the C-terminal half contains
carbohydrate groups, however, and, accordingly, staining of the tryptic
digest with an antibody raised against a fusion protein containing
amino acids 150-302 (UBI
1) revealed a large, smudgy band
with an apparent molecular mass of 50 kDa. The N-terminal 16-kDa
fragment, which contains the cytoplasmic and transmembrane domains of
the
subunit, was detected with a peptide-directed antiserum
(757) raised against the first 12 amino acids of the
subunit. This fragment also appeared after 1 min of trypsinolysis, and
as a doublet still containing the N-terminal epitope at longer times.
This is seen in Fig. 2, which shows digestion in the absence of
added ligands. In both figures, there was reduction of total staining
by the UBI
1 antibody of the 60-kDa
subunit and its 50-kDa
fragment, which could be explained by digestion of epitopes very close
to the C terminus.
(
)
Thus, while exposure of the
subunit could not be detected, a site in the middle of the
subunit was easily accessible at the extracellular surface, as well as
a site near the C terminus.
Figure 2:
Digestion of the extracellular domain of
the subunit in right-side-out vesicles. Vesicles (33 µg) were
digested with trypsin without external ligands present. Lanes 1 and 5 contained no trypsin. Aliquots were taken at 1 min
( lanes 2 and 6), 10 min ( lanes 3 and
7), and 30 min ( lanes 4 and 8) of
proteolysis. Samples containing 2.5%
-mercaptoethanol
( 1-4) or without reducing agent (5-8) were loaded
on 5-15% gradient gels. Blots were stained with UBI
1
antibody to detect the C-terminal half ( A) and reprobed with
antibody 757 to detect fragments containing the N terminus
( B). The
subunit and its 50-kDa and 16-kDa fragments are
denoted by arrows. The asterisk indicates the
position of trypsin. C shows the sequence of the
subunit
within the first disulfide bridge and in the close vicinity.
Vertical arrows indicate proposed sites of initial tryptic
cleavage, whereas arrowheads denote additional positions for
tryptic attack at 30 min of proteolysis.
Similar tryptic digestion patterns were
observed when other physiological ligand combinations were used. The
tested conditions are listed in . The pretreatments did not
cause the opening of more than a small fraction of the sealed vesicles,
as determined by measuring the latency of ATP hydrolysis. Regardless of
conditions, the subunit was resistant to digestion. Cleavage of
the
subunit was restricted to formation of the N-terminal 16-kDa
and C-terminal 50-kDa fragments at early times, with some additional
cleavage at the C-terminal end of each fragment.
subunit fragments detected when gels were
run under reducing ( lanes 1-4) and nonreducing
( lanes 5-8) conditions. In A, the blot was
probed with the antibody against amino acids 150-302, while in
B it was reprobed with antibody against amino acids
1-12. It appears that the initial digestion occurred within a
stretch of protein stabilized by a disulfide bond, which prevented the
separa-tion of the fragments when
-mercaptoethanol was omitted.
These results are in agreement with previous reports
(19, 34, 35) . By 30 min, however, another
cleavage occurred outside the region connected by the disulfide,
permitting the sepa-ration of the fragments. The delayed appearance of
frag-ments on nonreducing gels is probably because cleavage at the
initial points (Arg-135, Arg-142, or Lys-146, i.e. within the
disulfide bond between Cys-125 and Cys-148) causes the protein to lose
its tight globular structure, allowing trypsin access to Arg-149 or
Arg-151 (Fig. 2 C).
subunit to limited tryptic digestion, as well as the
inaccessibility of the extracellular surface of the
subunit, and
establish that there is little effect of ligands which alter
Na,K-ATPase conformation.
Digestion of Vesicles with Various Proteases
A
variety of other specific and nonspecific proteases was tried for
digestion of either subunit at the extracellular surface;
summarizes the results. The subunit was not cleaved
with any of them. This indicates how inaccessible its surface is and
also serves as an internal control establishing that proteolysis did
not occur during gel electrophoresis. Digestion patterns of the
subunit were different with different proteases. No digestion was
detected with papain, which was surprising because it had been reported
previously to cleave the
subunit at the extracellular surface
(15, 36) . Like trypsin, chymotrypsin and elastase
digested some, but not all, of the
. Subtilisin, ficin, proteinase
K, and bromelain caused extensive digestion, leaving only 15-20%
of
intact. A 16-kDa N-terminal fragment like that generated by
trypsin was detected after digestion by chymotrypsin, elastase,
subtilisin, and ficin, indicating that this marks a generally
accessible site on the surface of the protein. A new N-terminal
fragment with an apparent molecular mass of 40 kDa was detected after
digestion by chymotrypsin, ficin, and proteinase K, marking a second
exposed region. After digestion by bromelain, no major fragments were
seen, indicating extensive digestion at secondary sites.
Proteolysis of Vesicles after Heat and
Reduction
The most complete trypsinolysis of the subunit
described in the literature was achieved after first treating it with
300 m
M
-mercaptoethanol to disrupt tertiary structure by
reduction of its three disulfide bonds
(37) . We hypothesized
that such a reorganization of the extracellular domain of
could
result in exposure of hidden stretches of the
subunit.
Preincubation of vesicles with 100 m
M DTT at 37 °C did not
inhibit the activity of the Na,K-ATPase and did not make the vesicles
leaky, as measured by the latency of ATP hydrolysis. Trypsinolysis
after this treatment resulted in typical cleavage of the
subunit
into 16-kDa and 50-kDa fragments and no digestion of the
subunit.
Increasing the concentration of DTT to 200 m
M and varying the
duration of treatment and the temperature over the range from
4-37 °C did not promote digestion of
. Although at least
partial reduction of
's disulfide bonds should occur under
some of these conditions, as shown for purified enzyme
(38) ,
the digestion pattern in vesicles was unchanged. Heating of the
vesicles to 50 °C during incubation with 200 m
M DTT or 250
m
M
-mercaptoethanol, however, led to significant changes
in the subsequent tryptic digestion of both subunits, accompanied by
loss of most of the ATPase activity. Table III demonstrates the
correlation between the disappearance of the 16-kDa fragment of the
subunit and the exposure of the tryptic site on the
subunit.
subunit was seen in lanes
6-8 but not in lanes 2-4, thus demonstrating
access to a new cleavage site. As calculated from several experiments,
the fragment of the
subunit was 8-9 kDa smaller than the
intact protein. Similar fragments were generated by digestion with
chymotrypsin. Site-specific antibodies were used to determine whether
the cleavage point was near the N or C terminus (Fig. 4). The doublet
was stained by the antibody against the N terminus ( A,
lanes 8 and 9), while staining of the
subunit
by the antibody against the C terminus was reduced ( B). When
DTT was omitted and pretreatment consisted only of heating at 50
°C, no digestion of
was seen ( lanes 5 and
6). Proteolysis 8-9 kDa from the C terminus would place
the cleavage site approximately between Lys-907 and Asn-937.
Figure 3:
Dual effect of DTT and high temperature on
digestion of the subunit. Right-side-out vesicles were
preincubated with 200 m
M DTT for 15 min at 37 °C
( lanes 1-4) or at 50 °C ( lanes 5-8).
Trypsinolysis was then performed at 37 °C for different periods of
time: 1 min ( lanes 2 and 6), 10 min ( lanes 3 and 7), and 30 min ( lanes 4 and 8).
Lanes 1 and 5 were without trypsin. Samples were
electrophoresed on 5-15% gradient gels. Blots were stained with
the K1 antiserum. Arrows denote the positions of the intact
subunit and the fragment resulting from digestion. The
unidentified 70-kDa band again appeared as in Fig. 1, but it was not
digested.
Recently, Goldshleger and Karlish
(22, 23) reported
that cleavage of the subunit occurred if pig kidney vesicles were
heated at 55 °C for 30 min prior to trypsinolysis. Proteolysis
resulted in a 91-kDa fragment, and digestion was near the C terminus,
as found here. When we used those conditions, as shown in
Fig. 4
( lanes 11-12), a larger fraction of the
subunit in rat kidney vesicles was digested than after heating to
50 °C in DTT, but the resulting fragment was similar in size.
According to densitometric analysis of antibody-stained blots, as
little as 15-20% of intact protein was left undigested, whereas
60-70% of the
subunit remained uncleaved in DTT/50
°C-treated samples.
Figure 4:
Location of cleavage sites in and
. Vesicles (9 µg) were digested with trypsin or chymotrypsin
(1 h, 37 °C) with or without prior treatment as indicated at the
bottom of the figure. Samples were analyzed on a 7.5%
polyacrylamide gel ( A and B) or on a 16%
polyacrylamide Tricine gel ( C). Blots were stained with McK1
antibody, which detects the N terminus of rat
1, amino acids
14-22 (16) ( A); peptide-directed antiserum 845, which
detects the C terminus of
, amino acids 1003-1013
( B); and peptide-directed antibody 757, which detects the N
terminus of
( C). The digestion fragments are marked with
arrows. The asterisk denotes the position of
chymotrypsin, which was evidently autodigested in the presence of DTT
( lane 9). As with trypsin, the basis for ECL detection of
chymotrypsin is not understood. Autodigestion of trypsin prevented its
visualization in this experiment (see Fig.
1).
To detect fragments of the subunit,
16.5% polyacrylamide Tricine gels were used. Fig. 4 C illustrates staining with the antibody against amino acids
1-12. Digestion patterns in control vesicles and those only
heated at 50 °C were identical: undigested
and the 16-kDa
fragment could be detected, and with chymotrypsin, the 40-kDa
N-terminal fragment was also seen. In contrast, proteolysis of the
glycoprotein was altered in conditions where digestion of the
subunit was observed. If DTT treatment at 50 °C preceded digestion,
about twice as much
remained intact with both trypsin and
chymotrypsin. This may be due to enhanced autodigestion of the
proteases in the presence of the reducing agent. For the fraction of
that was digested, however, the yield of the 16-kDa N-terminal
fragment was greatly reduced, and instead there was an N-terminal
fragment of approximately 6 kDa. After heating the vesicles to 55
°C, the
subunit became entirely sensitive to digestion by
both proteases (Fig. 4 C). There was a major N-terminal
fragment of 6-7 kDa in trypsin, suggesting exposure of a cleavage
site just above the membrane; Goldshleger et al. (22) reported detection of a 10-kDa fragment of pig
subunit in similar conditions. We detected three similar fragments with
chymotrypsin, suggesting the exposure of multiple sites. Two identical
blots of the fragments generated by trypsin treatment were stained with
an antibody (FP
1) against amino acids 150-302 ( lanes
1-6) and an antibody
(757) against amino acids
1-12 ( lanes 7-12). It can be seen that the
staining of the 50-kDa fragment was greatly reduced and no major
subfragments were seen, indicating that there was further digestion
that removed the epitopes for this antibody (Fig. 5).
Figure 5:
subunit in DTT-treated vesicles is more resistant to proteologysis. Vesicles (9 µg were heated as indicated in the presence or absence of 200 mM DTT prior to proteoloysis. After 1 h of digestion (37 °C), samples were separated on a Tricine gel, and blots were stained with the polyclonal antibody FP
1, which detects the C-terminal half of
(lanes 1-6), or with peptide-directed antibody 757, which detects its N terminus (lanes 7-12). The
subunit and its fragments are denoted with arrows.
correlated
with the most thorough digestion of
.
Protection of the C-terminal Domain of
K+(and its congener
Rb+) is known to stabilize the Na,K-ATPase against
denaturation by heat
(39, 40) , to stabilize the
disulfide bonds in the by
Rb+
subunit against reduction
(41) ,
and to stabilize a 19-kDa segment at the C terminus of
to
digestion by trypsin
(42) . Goldshleger et al. (22) have reported that Rb+also protects
against proteolysis of sites on pig
at the extracellular surface,
which is logical since the cleavage point in
that is exposed by
50 °C/DTT or 55 °C lies within the 19-kDa fragment (8-9
kDa from the C terminus). Fig. 6 shows that 10 m
M Rb+fully protected the C-terminal domain of the
rat
subunit from trypsinolysis in vesicles that had been heated
at 55 °C for 30 min. Moreover, digestion of the
subunit under
the same conditions followed the pattern of the control sample, in
which a single cleavage generating an N-terminal 16-kDa fragment was
the final product of proteolysis. Choline chloride at 10 m
M did not provide protection for either
or
subunits
(data not shown). Table IV confirms that the presence of Rb+during heating of pig kidney vesicles to 55 °C also protected
[
H]ouabain binding in parallel with ATPase
activity.
Figure 6:
Rb+ ions protect a native configuration of the C-terminal domain of and the N-terminal domain of
. Vesicles were trypsinized (1 h, 37 °C) in the presence or absence of 10MM RbCl with or without prior hearting as indicated. Samples were separated on a 7.5% polyacrylamide gel (A) or a Tricine gel (B). Blots were stained with the monoclonal antibody of McK1, which detects the
N terminus of
(A), or site-specific antibody 757, which detects the N terminus of
(B). The positions of
,
,
and the fragments of digetsion are denoted with arrows. Digestion products after
heating in Rb+ were indistinguishable from those in the control.
Exposure of Kinase Phosphorylation Sites
Does the
extracellular cleavage site on become accessible because of
loosening of compact structure, or is there a more fundamental
reorganization of the C-terminal domain caused by the harsh treatment?
The recent identification of Ser-938 (rat numbering) as the site of
phosphorylation by protein kinase A
(43, 44) provides a
way to address this, since the cleavage site is predicted to be near
this residue, and exposure of one site would be predicted to be
accompanied by exposure of the other. Evidence that Ser-938 is normally
exposed on the intracellular surface is that expression of mutagenized
protein in transfected cells blunted the modest inhibition of
Na,K-ATPase activity elicited by stimulation of protein kinase A
(43) . Phosphorylation of Na,K-ATPase
subunit by protein
kinase A has a peculiarity, however. Once the cell is disrupted and
membranes are isolated, phosphorylation requires the presence of a
detergent like Triton X-100
(45) , even when there are no intact
vesicles. The requirement for detergent is not understood, but suggests
that Ser-938 is close to the membrane or is affected by conformation
changes brought about by interaction of transmembrane segments with
their lipid or detergent environment. The detergent alone can inhibit
Na,K-ATPase activity
(46) .
(
)
The presence of Rb+during both
pretreatment and phosphorylation reduced phosphorylation in all cases,
including in the control with Triton, where a 25% reduction was
observed. The effect of Rb+was most pronounced,
however, in the absence of Triton. Fig. 7 B shows
antibody staining of the same blot to demonstrate that variation in
subunit recovery did not account for the differences seen. Under
the same conditions, treatment with Triton X-100 caused essentially
complete loss of ATPase activity, while the DTT/50 °C and 55 °C
pretreatments caused 80-90% loss (data not shown). Preincubation
with 1 m
M ouabain before heating, like Rb+,
prevented the exposure of the phosphorylation site in medium without
Triton (also not shown).
Figure 7:
subunit of Na,K-ATPase treated with
heat and reduction can be phosphorylated by protein kinase A catalytic
subunit without detergent. Rat kidney microsomes (1 µg) were
phosphorylated by protein kinase A in the presence or absence of Triton
X-100 or 10 m
M RbCl, with or without prior treatment, as
indicated. A shows an autoradiogram of 32P-labeled
subunit after SDS-gel electrophoresis and electrophoretic
transfer of the samples. The same blot was restained with the antibody
McK1 ( B). Although the original blot was still radioactive,
the time required to expose x-ray film for visualization of ECL signal
is too short (5-20 min) for autoradiographic detection of the
32P (which required 16 h). The harsh treatments were able to
substitute for Triton X-100.
The critical question is whether the
phosphorylation site remains hidden at the intracellular surface in
right-side-out vesicles. Only a lack of phosphorylation would be
consistent with the retention of both normal topology and intact
vesicles. Since trypsin did not obtain access to the many tryptic
cleavage sites on on the inside of the vesicles after heating and
reduction, and since protein kinase A is much larger than trypsin, the
harsh treatments were unlikely to have made the vesicles leaky to the
kinase. Fig. 8 A shows, nonetheless, that phosphorylation of
rat kidney vesicles by kinase in the extravesicular space occurred
after heating and reduction even in the absence of detergent. In
control vesicles, phosphorylation was seen only after addition of
Triton X-100. By densitometry, 49% of the Na,K-ATPase in vesicles
treated at 50 °C with DTT became phosphorylated, and 59% of that
treated at 55 °C. These proportions are too large to have been
derived from the 15% open vesicles in the preparation. Fig. 8 B shows the constant amount of
subunit present per lane. The
incomplete level of phosphorylation after heating and reduction
corresponds with the incomplete cleavage of
to the 90-kDa
fragment seen in other experiments. The data suggest that in a
significant proportion of the Na,K-ATPase units, a radical conformation
change has resulted in the movement of the kinase and tryptic sites to
the outside.
Figure 8:
Protein kinase A phosphorylation of subunit in right-side-out vesicles reveals a structural reorganization of the C-terminal domain. Phosphyorylation of rat kidney vesicles (10 mg) by protein kinase A catalytic subunit was performed int he presence or absence of Triton X-100 with or without prior treatment as indicated. A blot
is an autoradiogram of 32P-labeled
subunit; straining of the same blot with McK1 antibody is presented in B. After harsh treatments, the phosphorylation site became accessible from the outside.
subunit
C terminus. In Fig. 9 A, staining by an antibody (McK1)
against the N terminus of
illustrates the production of the
90-kDa fragment. In Fig. 9 B, it is seen that
in
the heated and undigested sample was phosphorylated by protein kinase A
catalytic subunit. In the digested sample, the cleaved 90-kDa fragment
was not phosphorylated, as predicted if the cleavage site is on the
N-terminal side of Ser-938. Uncleaved
was also not
phosphorylated, as predicted if only those enzyme units that have
undergone a radical conformation change are susceptible to both trypsin
and the kinase. The data suggest that the pretreatment used was not
sufficient to achieve 100% conversion of
subunits to the altered
conformation. In Fig. 9 C, staining with antibody against the C
terminus of
illustrated that only very small amounts of small
cleavage products could be detected, at 22 kDa and 15.2 kDa. Both of
these may have been derived from enzyme in open vesicles, and since
they were not phosphorylated by protein kinase A in the absence of
Triton X-100, they were more likely to be derived from undenatured
enzyme than the heat-modified enzyme. Their predicted N termini would
be Asn-833
(42) and Trp-889
(23) (rat numbering).
Autoradiography of the same gel to detect 32P
(Fig. 9 D) showed that there was a small amount of a
phosphorylated fragment of approximately 17.5 kDa which did not
coincide with either of the fragments stained by antibody against the C
terminus; we cannot be certain whether this fragment was derived from
Na,K-ATPase or from a phosphorylated contaminant of 45 kDa also seen in
Fig. 9D. There was a smudge of phosphorylated fragments
of molecular mass around 8.5 kDa, also not stained by the antibody, and
also in low yield (Fig. 9, C and D). The
results indicate that the phosphorylated portion removed from
to
generate the major trypsin-stable 90-kDa fragment was further digested,
and that neither phosphate- nor ETYY-containing peptides were recovered
in useful amounts.
Figure 9:
The fragment containing the
phosphorylation site and the C terminus can be further digested. Heated
or untreated rat kidney vesicles were phosphorylated by protein kinase
A catalytic subunit without the addition of Triton X-100. After
phosphorylation, portions were digested with trypsin, and all samples
were electrophoresed on a 7.5% polyacrylamide gel ( A and
B) or a 16% polyacrylamide Tricine gel ( C and
D). Blots were first exposed to x-ray film for autoradiography
of the 32P ( B and D). The positions of the
intact subunit and its 90-kDa fragment are indicated with
arrows ( B): only the intact, undigested
contained 32P. The asterisk in D shows the
position of the smallest fragment containing 32P. The yield
of 32P-labeled fragments was very low, suggesting losses due
to further digestion. The blots were then stained with monoclonal
antibody McK1 ( A) to detect the intact
subunit and its
90-kDa fragment ( A) or with anti-ETYY to detect intact
and any C-terminal fragment ( C). The yield of C-terminal
fragment was negligible and did not coincide with any 32P,
indicating that the epitope was cleaved off.
An alternative hypothesis, that the affected
Na,K-ATPase units could be denatured and entirely exposed at the
extracellular surface, is not viable because of the resistance of the
90-kDa major fragment to extravesicular trypsin. Further evidence that
normal topology was maintained for the rest of the subunit was
obtained by using another enzyme, protein kinase C, as a probe for the
exposure of an intracellular site closer to the N terminus
(47, 57) . Fig. 10 shows that protein kinase C does
phosphorylate a fraction of the Na,K-ATPase in right-side-out vesicles
( lane 1), presumably because of unsealed vesicles in the
preparation. Treatment with 0.016% SDS to open the vesicles
significantly increased the level of subsequent phosphorylation
( lane 4). Pretreatment with heating and reduction decreased
the overall level of phosphorylation by 30% in SDS-opened vesicles
( lanes 5 and 6), indicating either some denaturation
of the kinase target site or a DTT effect on the kinase. It can be seen
clearly, however, that the heating did not increase phosphorylation,
compared to the samples treated with DTT but not heated, in vesicles
that had not been opened with SDS (compare lane 2 with
lane 3). Similar results were obtained when vesicles were
heated at 55 °C (data not shown). The failure to detect, in
lanes 2 and 3, phosphorylation comparable to that in
lanes 5 and 6, allowed us to conclude that the
integrity of the lipid bilayer was retained, and that the protein
kinase C phosphorylation site, unlike the protein kinase A site, was
not exposed at the extravesicular surface.
Figure 10:
Exposure of the protein kinase C phosphorylation site is not increased in sealed vesicles. Phosphorylation
of vesicles by protein kinase C without or without permeabilization with SDS
was performed without or without prior incubation with heat and 200 mM DTT, as indicated.
A shows an autoradiogram of 32P-labeled protein.
Arrows indicate the position of the phosphorylated
subunit and autophosphorylated protein kinase C. Staining of the same blot
with McK1 antibody is presented in B. A basal level of phosphorylation was seen due to open vesicles, but no
increase was caused by the harsh treatment.
Exposure of the
As shown
above (Fig. 9 C), the absence of anti-ETYY staining of
fragments suggested that the C terminus had been digested from the
extracellular surface after heating and reduction. The extravesicular
exposure of this site was tested by a competition enzyme-linked
immunosorbent assay, in which antibody that binds to membrane-bound
antigen in solution is removed by centrifugation, and residual antibody
is tested for binding to antigen that has been denatured by adsorption
to a polystyrene plate. The anti-ETYY antibody is known to bind only to
denatured enzyme
(28) . We hypothesized that heating and
reduction would denature the C terminus enough to permit it to bind,
and we verified this using rat kidney microsomes (data not shown). The
experiment was then performed with sealed right-side-out vesicles, with
and without permeabilization (Fig. 11). Without prior heating,
anti-ETYY bound very little to the vesicles (high concentrations of
vesicles were required to reduce binding), and permeabilization had
little effect. The concentration of SDS used here was sufficient to
open vesicles and unmask the epitope for antibody McK1 near the N
terminus
(10, 16) , but it did not affect the epitope
for anti-ETYY. After heat treatment of the vesicles, however, antibody
bound well, even to vesicles that had not been opened by detergent
treatment. The conclusion is that the C terminus, like the protein
kinase A phosphorylation site, became exposed at the extracellular
surface.
Subunit C Terminus
Figure 11:
Heating exposes the C terminus of
at the extravesicular surface. Anti-ETYY antibody (1:1,000 dilution)
was preincubated in solution with various amounts of sealed rat kidney
vesicles ( open circles and squares) or SDS-treated
vesicles ( closed circles and squares, with
( squares) or without ( circles) prior heating at 55
°C. After removing the antigen-antibody complex by centrifugation,
the residual soluble antibody was assayed by conventional enzyme-linked
immunosorbent assay using rat kidney microsomes as the antigen bound to
the plate. Vesicles competed effectively for antibody only after they
had been heated, and sealed right-side-out vesicles were almost as
effective as those opened with SDS.
Implications for Na,K-ATPase
Previous attempts to demonstrate proteolysis of the
Na,K-ATPase Subunit
Structure
subunit at the extracellular surface have not been
successful, but cleavage at sites that are predicted to be
extracellular has been observed when digestion is performed with enzyme
that is accessible from both sides (Arg-880 between H7 and H8
(9, 37) ; Arg-974 between H9 and H10
(19, 37) ). This led us to seek new conditions that
would permit digestion from the outside. Although the original
rationale for heating and reduction was to disrupt the
subunit to
expose the natural surface of the
subunit, we observed a discrete
step of
subunit denaturation. What makes the result significant
is that the denaturation event affected the exposure of only a portion
of the
subunit near its C terminus.
-helices have been complicated by low hydropathy
values and by an unconvincing alignment of predicted segments from
different members of the ATPase gene family
(48) . Attempts to
determine topology by the detection of epitopes, or the insertion of
exogenous epitopes, have resulted in models
(10, 29, 49, 50, 51) that
conflict in some instances with models resulting from proteolytic
digestion studies
(9, 34, 37, 52) . Not
all of the predicted membrane spans in this region can act as signal
anchor sequences or stop-transfer signals during biosynthesis in
vitro (53, 54, 55) . There is also
controversy about whether H9 and H10 span the bilayer
(19, 52, 56) . In sum, the physical evidence for
6
-helical membrane spans in this region is not compelling.
subunit
after treatment is consistent with its substantial unfolding. To what
extent the
subunit was denatured is not known, but ATP hydrolytic
activity and ouabain binding were destroyed, whereas the membrane
barrier was not. A small portion of the
subunit, normally hidden,
was exposed at the extracellular face. A minimum of three sites within
this region became accessible: the cleavage site generating the 90-kDa
N-terminal fragment, the protein kinase A phosphorylation site, and a
cleavage site near the C terminus. Strictly speaking, we do not know
whether these sites are normally hidden by compact folding at the
extracellular surface, by burying them within the transmembrane
portion, or by exposing them on the intracellular surface. For this
reason, the structure is represented as a black box in Fig.
12 B.
Location of the First Cleavage Site
The exact site
of cleavage is not yet known, since we were unable to recover a
C-terminal fragment to determine its sequence. From the difference in
electrophoretic mobility between intact and the N-terminal
fragment, we calculated that the cleavage site was 8-9 kDa from
the C terminus. The closest predicted sites are Lys-933, Arg-935, or
Arg-936. The fact that the phosphorylation site (Ser-938) was removed
by proteolysis rules out any cleavage sites closer to the C terminus.
The next nearest sites are at Arg-906 and Arg-907, but cleavage here
would be predicted to reduce the size of
by 12,000 kDa.
Phosphorylated fragments of approximately the expected size, 8.5 kDa,
were seen on Tricine gels, but they were present in very low yield.
Since they were not stained by an antibody against the C-terminal amino
acids ETYY, we cannot be certain that they were not derived from an
unrelated protein.
Figure 12:
Folding models for the Na,K-ATPase. A shows a conventional 10-helix model for the subunit, and a
1-helix model for the
subunit. The N and C termini are indicated,
and the P marks the location of phosphorylation by protein
kinase A. The oval marks the segment beginning at Trp-887,
which has been identified as the location of the epitope for antibody
IIC
and also as a site important for the assembly of
with
. Two hot spots for proteolysis of the
subunit are
marked with arrows; the
subunit is resistant to
digestion. B shows an alternative model for
. The last 3
predicted spans are replaced by an unspecified structure, buried within
the protein core of the
subunit. The site of IIC
binding and
-
assembly ( oval) is still
oriented to the extracellular surface, and the phosphorylation site to
the cytoplasm, but the C terminus of
is suggested to be buried.
C shows the structure obtained after heating and reduction.
The phosphorylation site is exposed on the extracellular side of the
membrane, as well as the C terminus. New proteolytic digestion sites
( arrows) on
result in the cleavage of both the
phosphorylation site and the C terminus from the extracellular surface.
Additional cleavage sites on the
subunit are also exposed. The
association between
and
is assumed to be disrupted. The
harsh conditions may have also altered structure at the intracellular
surface, but this is not shown for
simplicity.
Goldshleger and Karlish
(23) digested preparations of pig
kidney vesicles after heating to 55 °C; they reported detection of
4 fragments (7.4-14.5 kDa) containing ETYY. The yield of the
peptides was not reported. The peptides were sequenced, and the largest
was the result of cleavage at Trp-889 (rat numbering). Although
initially interpreted in terms of an 8-span model
(22) ,
cleavages were later shown to occur between H7 and H8, H8 and H9, and
H9 and H10, implying that H8 and H9 popped out of the membrane to the
extracellular surface.(
)
Our failure to detect
ETYY-containing phosphorylated peptides could be explained if the rat
Na,K-ATPase is more labile than pig Na,K-ATPase and undergoes more
complete digestion in similar conditions.
Location of the Protein Kinase A Phosphorylation
Site
The protein kinase A phosphorylation site at Ser-938 became
accessible as a result of the treatment, and it is adjacent to the
predicted cleavage site
(43, 44, 57) . Two
different groups have inserted epitopes at this location to determine
its topology, but reached opposite conclusions
(49, 51) . Some consideration should be given to the
possibility that Ser-938 isn't a physiological kinase regulatory
site, since phosphorylation cannot be observed in vitro without the addition of Triton X-100, and Triton X-100 has
inhibitory effects on ATPase activity. If the site were actually on the
extracellular side, but buried, it would be plausible for both heating
and Triton X-100 to cause similar changes in its exposure. In this
event, the harsh treatments used here would have performed as
originally predicted: exposing sites that were already extracellular,
but masked. The weight of opinion, however, is that the phosphorylation
site normally lies on the inside, leading to the conclusion that in
these conditions it popped out.
Location of the C Terminus
If only a hairpin pair
of transmembrane helices (H8 and H9) containing the protein
kinase A phosphorylation site and the new tryptic cleavage site was
extruded during heating and reduction, the C terminus of the
subunit would be predicted to remain protected on the intracellular
surface. The paucity of any digestion fragments staining for the
epitope at the very C terminus (ETYY), however, implies that the C
terminus became accessible to trypsin in the extravesicular space. The
accessibility of the C terminus was confirmed directly by showing that
heated vesicles acquired the ability to bind anti-ETYY antibodies in
solution, and that opening the vesicles caused very little additional
exposure of sites. Thus, all of the data support the heat-treated
structure modeled in Fig. 12 C.
subunit near the C terminus as shown here is
not known. Covalent labeling studies with pyridoxal phosphate
(21) or lactoperoxidase followed by carboxypeptidase digestion
(59, 60) are consistent with an intracellular location,
but are not quantitative enough to prove that all of the C termini are
normally exposed, rather than buried. Other evidence that the C
terminus must be on (or close to) the cytoplasmic surface is that a
cDNA fusion product between the
and
subunits could be
expressed as active enzyme
(61) . The flexibility of the linker
may have allowed repositioning of the Na,K-ATPase C terminus, but it
seems unlikely that it could have allowed it to move from the outside
to the inside without disruption of enzyme activity.
chains that were neither cleaved to 90 kDa nor phosphorylated
suggests, however, that the exposure of the primary cleavage site, the
phosphorylation site, and the C terminus was a single concerted event.
Implications for Na,K-ATPase
The identification of fragments of the untreated Subunit
Structure
subunit revealed that it has two proteolytic hot spots, one yielding an
N-terminal fragment of 16 kDa described before
(34, 35) , and the other yielding an N-terminal fragment
of about 40 kDa. The location of the second site can be predicted to be
in the vicinity of residue 238, if each amino acid has an average
molecular mass of 110 and each of the three carbohydrate groups changes
the electrophoretic mobility by the equivalent of 9 kDa. The 40-kDa
fragment would thus contain 75-80% of the protein and two
carbohydrate groups. In both cases, the cleavage sites would lie within
disulfide bridges. Consequently, the cleavages would not be expected to
have a major impact on Na,K-ATPase tertiary structure.
subunit still
stained the ~6-kDa trypsin digestion product. Trypsin cleaves off 4
amino acids from the N terminus only when it has access to the
intracellular surface
(18, 34) . In the presence of
Mg2+/P
, or in the presence of
Ca2+, both of which promote more extensive digestion of
and
, the first 26 amino acids are removed
(9, 19) , which would remove the binding site for this
antibody. The site's continued presence here implies that the N
terminus of
is still inaccessible. Goldshleger et al. (22) reported that their 10-kDa N-terminal fragment of pig
subunit began at the N-terminal alanine, also demonstrating that
trypsin had not entered the vesicles. The 6-kDa fragment seen after
tryptic digestion here should be long enough to span the membrane if
cleavage was at Arg-70. The difference in apparent molecular mass (6
kDa versus 7.7 kDa predicted) could be due to anomalous
mobility of hydrophobic fragments on the gel.
subunit is stabilized by three disulfide bonds, one would expect its
cleavage to be more complete after reduction than after heating alone.
In the absence of reduction, digestion after treatment at 50 °C for
15 min resulted in cleavage of
at one site like the control,
while after 55 °C for 30 min, it was digested extensively. Addition
of reducing agent in the milder conditions promoted more extensive
digestion of at least some of the
subunit. The most salient
observation was that pretreatment at 55 °C did a good job of making
sensitive to proteolysis without DTT.
Implications for
There is
strong evidence that the -
Interaction
subunit associates specifically with
portions of the C-terminal third of the
subunit
(62, 63, 64) . Chimeras of Na,K-ATPase with
Ca2+-ATPase have narrowed down an essential region to
26 extracellular amino acids between the 7th and 8th spans of the
10-span model, from Asn-891 to Ala-916
(63) . We have observed
that an antibody (IIC
) that maps to the same region binds
to native enzyme only under certain circumstances, suggesting that the
region is subject to conformational changes
(10) . An antibody
to the H,K-ATPase, mAb 146-14, has been shown to interact with
both the
subunit and the
subunit at the extracellular
surface near H7, in the region implicated in
-
interaction in
the Na,K-ATPase
(50) . The region of the
subunit most
important for assembly appears to be within the N-terminal half
(4, 5) and has been narrowed down to 96 amino acids
just on the C-terminal side of the membrane span by construction of
chimeras
(65) . Other sites are also implicated
(66, 67) , but may have their effect through influence
on conformation. Within the required region, only amino acids
68-77 have a high degree of conservation between species and
isoforms, a property expected of the assembly site because of the
interchangeability of
subunits.
-
interaction sufficiently to expose new tryptic cleavage
sites at or near the interaction site. The ~6-kDa N-terminal
tryptic fragment of the
subunit should result from cleavage at
RVAP, just above the membrane span and within the most likely site of
assembly (amino acids 68-77). The
subunit cleavage site
that leaves a 90-kDa N-terminal fragment should be just on the
C-terminal side of the
assembly site, in a region that appears to
be stabilized by the complex with
, but extruded when
-
interactions are disrupted. The observation that conditions that expose
one subunit to digestion also expose the other subunit supports the
hypothesis that
and
interact closely at these sites.
, and next best in the
presence of Na+, whereas phosphorylation is lower in
the presence of K+and is markedly inhibited by ouabain
(44) . The conditions that are best for phosphorylation thus are
those that are worst for Rb+occlusion.
and
subunits go through condensed and expanded conformations in a concerted
way. Within this framework it is logical to propose that, after
heating, the transmembrane spans of the
subunit that do not
directly participate in interaction with the
subunit remain
stably associated with the membrane, while those that do interact with
unfold into the extracellular space.
Table I
Tryptic cleavage of and resistance of
is unaffected by Na,K-ATPase ligands
Table II
subunit
subunit.
Table III
and
subunits of Na,K-ATPase in
right-side-out vesicles
subunit) or peptide-directed antibody 757 (
subunit).
Table IV
mg of protein-1 min-1) was heated at 55 °C in the presence
or absence of 10 m
M RbCl. [
H]Ouabain
binding was measured at equilibrium (37 °C, 1 h, in the presence of
5 m
M Mg2+and 5 m
M P
),
and was determined to be 6.5 nmol/mg of protein in the control sample.
The results represent the average of two independent experiments.
1 antibody (the equivalent of the UBI
1 antibody used here) are localized within the C-terminal quarter
(amino acids 230-302) of the
subunit. More direct
confirmation was obtained in experiments using the peptide-directed
antibody 940 against amino acids 294-302 (gift of W. J. Ball,
Jr.), which indicated that the extreme C terminus of the
subunit
can be easily clipped off when rat kidney right-side-out vesicles are
proteolyzed even under mild conditions. In similar experiments
performed with pig kidney vesicles, we did not see any loss of staining
of the
subunit or its 50-kDa fragment with monoclonal antibody
M17-P5-F11 (gift of W. J. Ball, Jr.), which recognizes amino acids
234-236 in
. Data not shown.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.