From the Department of Biological Chemistry, Weizmann Institute of Science, Rehovot, 76100 Israel
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ABSTRACT |
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This study characterizes disulfide cross-links
between fragments of a well defined tryptic preparation of Na,K-ATPase,
19-kDa membranes solubilized with C12E10
in conditions preserving an intact complex of fragments and Rb
occlusion (Or, E., Goldshleger, R., Tal, D. M., and Karlish,
S. J. D. (1996) Biochemistry 35, 6853-6864).
Upon solubilization, cross-links form spontaneously between the Renal Na,K-ATPase consists of a catalytic The spatial organization of trans-membrane segments of the catalytic
subunits is unknown although such information is essential for defining
the structure of the cation transport path. Recently published
structures of Ca-ATPase and H-ATPase (12, 13), at 8-Å resolution, show
that the 10 trans-membrane segments are This paper describes experiments utilizing
CuP1-catalyzed S-S bridge
formation. Older work (17-20) showed that treatment of detergent-solubilized Na,K-ATPase leads primarily to a 1:1 A potential problem in cross-linking membrane proteins is the
possibility of intermolecular cross-linking via random collisions in
the membrane. Detergent solubilization can overcome this problem because the soluble protein can be diluted. Indeed Sarvazyan et al. (23, 24) have reported that treatment of digitonin-solubilized 19-kDa membranes with CuP yielded two cross-linked products, dimers of
fragments containing M7/M10 and M1/M2 (22 kDa:11 kDa) and trimers containing these two fragments with the 19-kDa membranes lack ATP-dependent functions, but retain
cation occlusion and ouabain binding (8, 22, 25). Recently we described
a procedure for solubilizing 19-kDa membranes with the non-ionic
detergent C12E10 which preserves intact the
complex of fragments with occluded Rb ions and bound ouabain (1). The intact soluble complex contains one copy of each fragment. After solubilizing 19-kDa membranes in the absence of Rb ions and ouabain, neither Rb occlusion nor the complex of fragments are intact (1). Because cross-linking in the latter condition, as in Ref. 23, might
reveal non-native interactions between fragments, one of our objectives
has been to characterize cross-linking of the
C12E10-solubilized intact complex of fragments
containing occluded Rb ions. A second objective has been to identify
cross-linked cysteines.
Na,K-ATPase was prepared from pig kidney red outer medulla (26)
and stored at Cross-linking Catalyzed by CuP--
The solubilized preparation
(0.4 mg/ml) was treated at 20-22 °C with CuP (final concentration:
0.5/2.5 mM), added in 5 aliquots every 12 min. After 1 h solid urea was added to 2 M and the pH was readjusted to
8.0 with solid Tris base. Free cysteines were blocked by adding
iodoacetamide to 40 mM and free Cu2+ was
chelated with 10 mM EDTA(Tris). After 30 min at
20-22 °C the mixture was acidified to pH Gel Electrophoresis--
Precipitated protein was collected by
centrifugation at 9700 × g for 1 h at 4 °C,
dried under a stream of nitrogen, and dissolved in loading buffer.
Samples were resolved by Tricine-SDS-PAGE as in Refs. 22 and 27. Either
14-cm short or 23-cm long gels were used. In nonreducing conditions
glutathione and mercaptoethanol were omitted from sample and running buffers.
Purification of CuP-catalyzed Cross-linked
Products--
Precipitated protein (15 mg) was dissolved in 2 ml of
nonreducing sample buffer and resolved on two 1.5-mm thick long 10% Tricine gels. Gels were briefly fixed, stained, and destained and bands
of interest were cut out and equilibrated with 660 mM Tris-Cl, pH 8.9, for 1 h. Protein was eluted into 50 mM NH4HCO3, 0.1% SDS using a
Bio-Rad Model 422 Electro-Eluter, run at 8 mA/tube overnight. Eluted
protein was precipitated with 4 volumes of methanol at Proteolytic Digestions--
Protein (0.8 mg/ml) in 35 mM Tris-Cl, pH 8.0, 0.07% SDS was digested with trypsin
(10% w/w) at 37 °C in the presence of 2 M urea and 10 mM CaCl2. Digestions with chymotrypsin (10%
w/w) were done at 37 °C in 25 mM Tris-Cl, 1 mM EDTA, pH 8.0, 0.05% SDS. Proteolytic digestions were
stopped with 5 mM phenylmethylsulfonyl fluoride.
Phosphorylation by PKA--
Purified protein (0.1-0.25 mg/ml)
was incubated at 30 °C for 30 min in 20 mM Tris-Cl, pH
7.5, 0.02% SDS, 0.1% octyl glucoside, 10 mM
MgCl2, 50 µM [ Digestion with N-Glycosidase F--
Free or cross-linked Immunoblotting--
Protein samples were separated on short
Tricine gels, which were electroblotted onto PVDF (Semi-Phor TE70,
Hoefer Scientific Instruments) (28). PVDF sheets were incubated with
antisera (22). Immunoblots were stained by diaminobenzidine with metal ion enhancement (29).
Sequencing--
Bands of interest were cut out of the gel,
equilibrated in 660 mM Tris-Cl, pH 8.9, for 1 h and
transferred to PVDF by either of two procedures. (a) Bands
were separated on a second 1.5-mm thick short 10% Tricine gel and
electroblotted onto PVDF (22). (b) Bands were cut into small
pieces, and protein was eluted into 1.7 ml of 1 M Tris-Cl,
0.1% SDS, pH 8.45, by mixing on a rotating wheel for 20 h.
Supernatants were transferred into 3-ml syringes connected to 13-mm
Swinnex filter holders (Millipore) and eluted proteins were blotted
onto PVDF (Immobillon PSQ Millipore) by centrifugation at
290 × g for 25 min. Sequencing of peptides blotted
onto strips of PVDF was done on an Applied Biosystems Model 475A
protein sequencer with an on-line Model 120A phenylthiohydantoin
analyzer. Each reported sequence was done at least twice.
Materials--
1,10-Phenanthroline-HCl was from BDH Chemicals,
L-1-tosylamido-2-phenylethyl chloromethyl ketone-trypsin
(bovine pancreas) was from Worthington, CuP-catalyzed Cross-linked Peptides--
Fig.
1 presents a representative experiment
showing components of C12E10-solubilized 19-kDa
membranes in reducing or nonreducing conditions and also cross-linked
products after treatment with CuP. In reducing conditions (Red),
untreated C12E10-solubilized 19-kDa membranes
reveal the standard 19-kDa and smaller (8-12 kDa) fragments of the
Fig. 2 presents an immunoblot using
anti-KETYY to detect cross-links in intact 19-kDa membranes. After CuP
treatment in the presence of Rb ions (+Rb) the antibody recognized the
19-kDa fragment but no higher molecular mass species, confirming the
absence of cross-links in intact 19-kDa membranes seen in Fig. 1. By
contrast, after CuP treatment in the absence of Rb ions (
The components of the 70-80-kDa cross-linked product have been
identified by sequencing (Table I) and immunoblots (e.g Fig. 3). Immunoblots showed that both
spontaneously formed and CuP-induced cross-linked product contain the
Identification of Cross-linked Residues and Segments--
The
following experiments demonstrate cross-linking of Cys44 of
the
One approach involved tryptic digestion of the 70-80-kDa product and
sequencing of cross-linked fragments. Cross-linked fragments were
identified as bands present in non-reducing but absent in reducing
conditions (Fig. 4). Several such bands
were observed (marked **, *, a and b). The bands
marked with an asterisk (*) were not sequenced for they were observed
also in digests of the
The evidence for the internal cross-link
Cys964-Cys983 in Table II excludes either
residue as a partner of Cys44 of the
The hypothesis just proposed was tested as follows (Figs.
5, 6, and Table
III). The 70-80-kDa cross-linked
product, or the 19-kDa fragment, were first incubated with protein
kinase A and [
In principle, the 6.5-kDa 32Pi-labeled
cross-linked fragment might extend forward from Ser936 and
include M9 (containing Cys964) or backward from
Ser936 and include M8 (containing Cys911 and
Cys930). In order to distinguish between these
possibilities we have sequenced the fragment, repeating the experiment
of Fig. 5 with 50-fold more protein (Fig.
6). The CuP cross-linked product (0.3 mg)
was labeled with 32Pi using PKA, digested with
chymotrypsin, and protein was separated on a 16.5/6% gel. Most of the
digest was separated under reducing conditions (Red), but for
comparison a portion was separated in nonreducing conditions (Non-red).
Because a large amount of protein was loaded onto each lane, the
labeled fragment could not be expected to run as a sharp band even in
the reducing conditions, as in Fig. 5, and indeed the autoradiograph in
Fig. 6 (Red) showed a relatively broad band of labeled material in the
region of 6-7 kDa. The broad labeled band was transferred to PVDF, and
sequenced (Table III). A mixture of two sequences was found, N terminus
Glu902 corresponding to a fragment containing M8 and N
terminus Arg972 corresponding to a fragment containing M10,
respectively. Only the former fragment contains Ser936, and
hence the radioactive label. Thus, Figs. 5, 6, and Table III
demonstrate that Cys44 of the Cross-linked Fragments of 19-kDa Membranes
CuP treatment of 19-kDa membranes solubilized with
C12E10 in the presence of Rb ions and ouabain
led to appearance of one major cross-linked band, which consists of a
mixture of CuP treatment of digitonin-solubilized proteolyzed dog kidney
Na,K-ATPase produces cross-linked fragments corresponding to a trimer
of CuP treatment of unsolubilized 19-kDa membranes revealed a
heterogeneous mixture of cross-linked products containing the 19-kDa fragment, in the absence of Rb ions, and suppression of the cross-links in the presence of Rb ions (Fig. 2). These cross-links are probably the
products of non-native interactions since the same fragments were
observed after thermal inactivation which disorganizes the fragments
(30, 31). K+, Na+, or ouabain protect against
CuP-catalyzed cross-linking of fragments of unsolubilized 19-kDa
membranes (24). These ligands also protect 19-kDa membranes against
thermal inactivation of Rb occlusion (30, 31). Thus protection against
CuP-catalyzed cross-linking presumably reflects cation- or
ouabain-induced stabilizing interactions between fragments which
prevent their disorganization.
Identity of Cross-linked Regions
The conclusion that Cys911 or Cys930 in
M8, is cross-linked to Cys44 of the Proximity of M8 and M subunit, 19- and 11.7-kDa fragments of the
subunit, containing
trans-membrane segments M7-M10 and M1/M2, respectively. Treatment with
Cu2+-phenanthroline (CuP) improves efficiency of
cross-linking. Sequencing and immunoblot analysis have shown that the
cross-linked products consist of a mixture of
-19 kDa dimers
(
65%) and
-19 kDa-11.7 kDa trimers (
35%). The
-
cross-link has been located within the 19-kDa fragment to a 6.5-kDa
chymotryptic fragment containing M8, indicating that
Cys44 is cross-linked to either Cys911 or
Cys930. In addition, an internal cross-link between M9 and
M10, Cys964-Cys983, has been found by
sequencing tryptic fragments of the cross-linked product. The
M1/M2-M7/M10 cross-link has not been identified directly. However, we
propose that Cys983 in M10 is cross-linked either to
Cys104 in M1 or internally to Cys964 in M9.
Based on this study, cross-linking induced by
o-phthalaldehyde (Or, E., Goldshleger, R., and Karlish,
S. J. D. (1998) Biochemistry 37, 8197-8207), and
information from the literature, we propose an approximate spatial
organization of trans-membrane segments of the
and
subunits.
INTRODUCTION
Top
Abstract
Introduction
References
subunit (112 kDa), a
glycosylated
subunit (33 kDa), and a
subunit (
6.5 kDa) (3).
The
subunit contains the ATP hydrolytic site and the Na+ and K+ transport sites. The
subunit is
required for correct post-translational processing and stabilizes the
subunit (4). The
subunit may be a tissue-specific regulator
(5). Recent studies on P-type pumps have focused on residues involved
in cation or ATP binding, or conformational transitions (3, 6). Cation
occlusion sites are located within trans-membrane segments (8), and
site-directed mutagenesis suggests that side chains within M4, M5, M6,
and M8 ligate cations (6, 9-11). In addition, a variety of studies have defined the topological organization of the catalytic subunits, comprising 10 trans-membrane segments (see Refs. 3 and 7 and references therein).
-helices and most helices
are tilted so that their spatial organization changes at different
levels in the membrane. A tentative model of helix packing of Ca-ATPase
has been proposed based on constraints of trans-membrane topology,
site-directed mutagenesis, and disulfide cross-linking (12, 14, 15).
Modeling of Na,K-ATPase has also been attempted based on hydrophobic
labeling and prediction of helix orientation with respect to lipid
(16). Despite these attempts, direct determination of helix proximity
is largely lacking. Recently, covalent cross-linking experiments have
begun to provide such specific information (2, 15).
cross-linked product. Identification of the cross-linked residues constitutes a formidable task for, whereas the
subunit has only one
free cysteine, Cys44, located within its single
trans-membrane helix (21), the
subunit contains 23 cysteines. The
problem could be simplified by using 19-kDa membranes which are
obtained by extensive tryptic digestion of Na,K-ATPase. This
preparation consists of fragments of the
subunit comprising mainly
trans-membrane segments (M1/M2, M3/M4, M5/M6, and M7/M10) connected by
the external loops, and a partially cleaved
subunit (8, 22). The
fragments contain only 10 cysteines located within or next to
trans-membrane segments.
subunit (
:22 kDa:11 kDa)
in 1:1:1 stoichiometries. These studies established that a cysteine
residue within Asn831-Tyr1016 of the
subunit (Cys911, Cys930, Cys964, or
Cys983) is cross-linked to the
subunit
(Cys44), but did not identify the cysteine.
EXPERIMENTAL PROCEDURES
80 °C. Protein and ATPase activity were determined as described (26). Specific activities were 13-18 units/mg of protein.
Before use enzyme was thawed and dialyzed overnight at 4 °C against
1000 volumes of 25 mM histidine, 1 mM
EDTA(Tris), pH 7.0. 19-kDa membranes were prepared by tryptic digestion
of Na,K-ATPase with trypsin (22), and resuspended at 3 mg/ml in 2 mM RbCl, 25 mM imidazole, 1 mM
EDTA, pH 7.5. The specific Rb occlusion capacity was 6 to 6.5 nmol/mg
of protein. 19-kDa membranes were solubilized with
C12E10 at a ratio of 2.2 (w/w) as described by
Or et al. (1). Before cross-linking with CuP the pH was adjusted to 8.0 with RbOH (final concentration, 0.8 mM).
6.0 with acetic
acid and protein was precipitated with 4 volumes of methanol/ether
(2:1) and stored at
20 °C overnight. When analytical amounts were
cross-linked, urea, Tris base, and acetic acid were omitted.
20 °C after
adjusting the pH to 7.0 with acetic acid. Pellets were collected by
centrifugation and dissolved in 50 mM Tris-Cl, pH 8.0, 0.1% SDS. Samples were analyzed on a minigel to check the amounts and
purities of the eluted proteins.
-32P]ATP, and
50 ng of catalytic subunit of PKA. Autoradiography was carried out
using a Fuji BAS 1000 PhosphorImager after transfer of proteins from
the gel to PVDF paper.
subunit (0.1 mg/ml) in 50 mM phosphate buffer, pH 7.5, containing also 0.01% SDS and 0.7% octyl glucoside, was treated with
2500 units of N-glycosidase F at 37 °C for 20 h. The
reaction was stopped by adding loading buffer.
-chymotrypsin was from
Merck, N-glycosidase F was from New England Biolabs, and
octyl glucoside from Calbiochem. Trypsin inhibitor (type 1-S from
soybean), phenylmethylsulfonyl fluoride, iodoacetamide, ouabain,
thioglycolate, Tricine, C12E10, and molecular
weight markers (SDS17 and VII-L) were from Sigma. Electrophoresis grade
reagents for SDS-PAGE and PVDF paper were from Bio-Rad and Millipore,
respectively. [
-32P]ATP was from Amersham. Antisera
recognizing Asn889-Gln903 and
Lys1012-Tyr1016 (anti-KETYY) and the catalytic
subunit of PKA were gifts from colleagues (see "Acknowledgments").
Antisera were raised against an 11.7-kDa peptide containing M1/M2
(N-terminal Asp68) and a 16-kDa fragment of the
subunit
(N-terminal Ala5) as described previously (1).
RESULTS
subunit (the latter are not well resolved on this 10% gel), an intact
subunit or its
50-kDa glycosylated and 16-kDa fragments (22). In
nonreducing conditions (Non-red) one major new band appeared, 70-80
kDa, and the intensity of the 19-kDa fragment was less than in reducing
conditions. The
50- and 16-kDa fragments were not seen because they
are internally cross-linked between Cys125 and
Cys148. After treatment of
C12E10-solubilized 19-kDa membranes with CuP
(Sol), the intensity of the 70-80-kDa band rose and that of the 19-kDa
peptide and
subunit decreased further (compare Sol with non-Red),
suggesting that this band contains both the 19-kDa peptide and
subunit. About 63% of the
subunit underwent cross-linking based on
the weight ratio of the cross-linked product and remaining
subunit
extracted from gels (
3.1, w/w), and on the composition of the
cross-linked product (see Table I).
Raising CuP concentration or incubation time did not improve the yield
(not shown). CuP treatment of unsolubilized 19-kDa membranes,
containing occluded Rb ions, did not produce the 70-80-kDa
cross-linked band (Mem). After denaturation of 19-kDa membranes with
SDS, CuP treatment did not produce the 70-80-kDa band (SDS), excluding
the possibility that the cross-link is the product of a nonspecific
association of its components after termination of the reaction. A
small amount of a higher molecular weight band (Fig. 1,
asterisk) was observed after C12E10
solubilization but the amount was not increased by CuP treatment. This
band was also observed with intact 19-kDa membranes. It may represent
the product of inter-molecular cross-linking between adjacent
complexes, and therefore it was not investigated further. In conditions
producing the 70-80-kDa band the remaining 19-kDa fragment ran a
little faster than in the other conditions, implying that the peptide
is more compact (compare Non-red and Sol with Mem, and SDS). This hints
at the possibility of an internal cross-link (see also Table
II).
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Fig. 1.
Spontaneous and CuP-catalyzed cross-links
detected in C12E10-solubilized 19-kDa
membranes. Samples (140 µg) were resolved on a long 10% gel.
Red and Non red-solubilized 19-kDa membranes were
electrophoresed under reducing or nonreducing conditions, respectively,
without prior CuP treatment. Sol, solubilized 19-kDa
membranes treated with CuP; Mem, unsolubilized 19-kDa
membranes (0.4 mg/ml) in 3.3 mM imidazole, 130 µM EDTA, pH 7.5, plus 5 mM RbCl and 45 mM choline-Cl were treated with CuP. SDS, 19-kDa
membranes in 3.3 mM imidazole, 130 µM EDTA,
pH 7.5, plus 5 mM RbCl, 10 mM ouabain were
denatured with 2% SDS prior to addition of
C12E10 and CuP treatment.
Sequences found in the major CuP cross-linked product
Sequence analysis of cross-linked tryptic fragments
Rb) the
amount of 19-kDa peptide was reduced, and a number of bands appeared (30.8-66 kDa), as well as a smear of material in the upper half of the
gel. Using antibodies raised against fragments containing M1/M2, M3/M4,
and M5/M6 all these bands were recognized by anti-M1/M2 and some of
them also by anti-M3/M4 or anti-M5/M6 (not shown). This heterogeneity
suggests that the cross-links might be nonspecific. In fact the same
pattern of anti-KETYY recognition was found for 19-kDa membranes in
which Rb occlusion had been thermally inactivated (30, 31) (Fig. 2,
Th.in). As discussed below these cross-links cannot be
assumed to represent proximities between fragments in a native
state.
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Fig. 2.
CuP-catalyzed cross-links detected in intact
19-kDa membranes. 19-kDa membranes (0.5 mg/ml) were suspended in
25 mM imidazole, 1 mM EDTA, pH 7.5, without
( Rb, Th.in) or with 30 mM RbCl
(+Rb). Membranes were either kept on ice (
Rb)
or were incubated at 37 °C for 25 min (+Rb, Th.in). All
samples were then treated with CuP as described under "Experimental
Procedures," and then dissolved in 2% SDS and treated with 40 mM iodoacetamide and 10 mM EDTA for 30 min at
room temperature. Protein was precipitated with 4 volumes of methanol.
The samples were resolved on a 10% gel in nonreducing conditions,
transferred to PVDF paper, and then probed with the anti-KETYY
antibody.
subunit 19-kDa peptide (M7-M10) and an 11.7-kDa peptide (M1/M2)
(not shown). N-terminal sequencing clearly identified four fragments
(Table I). Two sequences are derived from the
subunit (N terminus
Ala5 in 19-kDa membranes, Ref. 22), which is partially
cleaved to fragments of 16 kDa (N terminus Ala5) and
50
kDa (N terminus, Gly143). The sequence with N terminus
Ala5 represents both the 16-kDa fragment and intact
subunit, and about 65% is cleaved judging by the yields of 26 and 17 pmol/cycle, respectively. Two sequences are derived from the
subunit: 19 kDa (N terminus Asn831, 17 pmol/cycle) and 11.7 kDa (N terminus Asp68, 9 pmol/cycle). Note that the yields
of the components are not stoichiometric. The excess
subunit (26 pmol/cycle) over the 19-kDa fragment (17 pmol/cycle) implies that the
cross-linked product was not completely resolved from free
subunit.
The different yields of the 19- and 11.7-kDa fragments could imply that
the cross-linked product consists of either a mixture of
:19-kDa dimers (
65%) and
:19 kDa:11.7 kDa trimers (
35%) or a mixture of dimers,
:19 kDa and
:11.7 kDa, which are not well resolved on
the gel. Deglycosylation of the
subunit has allowed us to distinguish between these alternatives. The deglycosylated cross-linked product was clearly resolved into two bands of 62- and 54 kDa (Fig.
3A, Coom). Both bands were recognized by anti-
antibodies and anti-KETYY but only the upper 62-kDa band was recognized also by
anti-M1/M2. Thus the cross-linked product consists of a mixture of
:19-kDa dimers and
:19 kDa:11.7-kDa trimers.
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Fig. 3.
Identification of cross-linked peptides
following deglycosylation of the subunit. Purified CuP cross-linked product was treated or
not with N-glycosidase F (+ or
PNGase,
respectively), resolved on a short 10% gel and electroblotted onto
PVDF. Coom' lanes were stained with Coomassie
(A). Samples treated with PNGase were also probed with
antibodies recognizing the 19-kDa fragment (anti-KETYY), the 16-kDa
N-terminal fragment of the
subunit (anti-16 kDa), or the 11.7-kDa
peptide containing M1/M2 (anti-M1/M2) (B).
subunit to a cysteine in M8 (Cys911 or
Cys930), and an internal cross-link between
Cys964 (M9) and Cys983 (M10). We have not been
able to identify the M7/M10-M1/M2 cross-link, probably because the
:19 kDa:11.7-kDa trimer was formed in too low a yield (see
"Discussion").
subunit and represent fragments cross-linked
by internal S-S bridges (not shown). The two fragments a, 9.3 kDa, and
b, 7.7 kDa, were not observed in digests of the
subunit and were
sequenced (Table II). Fragment a contained three peptides derived only
from the
subunit, with N termini Met973 (M10),
Asn944, and Ile946 (M9), respectively. The
combined average yield of fragments containing M9 is 12.6 pmol/cycle
while that of the fragment containing M10 is 11.7 pmol/cycle. These
yields are measured against a background of about 1 pmol/cycle, i.e.
with a possible error of about 10%. Thus, the experiment demonstrates
that M9 and M10 are present in equimolar proportions, suggesting that
Cys964 within M9 and Cys983 within M10 are
cross-linked to each other. Sequencing of fragment b led to essentially
the same result suggesting that it is a truncated version of fragment a
(probably cleaved at Arg998 or Arg1003).
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Fig. 4.
Digestion of the CuP-catalyzed cross-linked
product with trypsin. The CuP cross-linked product (95 µg) was
digested with trypsin for 12 h (see "Experimental
Procedures"). Samples (~48 µg/lane) were resolved on a short 1-mm
16.5% T, 6% C gel in either reducing (Red) or non-reducing
conditions (Non-red).
subunit, unless
one makes an unlikely assumption that Cys44 can form S-S
bridges with different cysteines in the 19-kDa peptide. Thus, one could
predict that Cys44 is cross-linked to either
Cys911 or Cys930 in M8. However, direct
evidence was not obtained because we detected no cross-linked band
containing sequences from both
and
subunits. A hypothesis to
explain the paradox could be that the broad band (**) in Fig. 4
contains cross-linked fragments of both
and
subunits, but the
latter are also cross-linked by the internal S-S bridges to other
glycosylated fragments of the
subunit. Sequencing of this band (**)
showed indeed that it consists of a heterogenous mixture of peptides
which precludes simple interpretation (not shown).
-32P]ATP, in order to phosphorylate
Ser936 in the PKA site RRNS (32, 33), and the labeled
proteins were then digested with
chymotrypsin.2 The digestion
products were resolved on a 16.5% gel, blotted onto PVDF paper, which
was first autoradiographed (Fig. 5, 32Pi) and
then also immunostained with an antibody recognizing residues Asn889-Gln903 in the loop between M7 and M8
(Fig. 5, L7-8). For greater ease of understanding, the
scheme in Fig. 5 marks the positions of the
Asn889-Gln903 epitope and the phosphorylation
site of PKA in relation to the trans-membrane segments, M7-M10. In
nonreducing conditions only broad, poorly defined bands of
32Pi-labeled material (*) were observed
(Non-red). However, in reducing conditions, a discreet
32Pi-labeled band of 6.5 kDa appeared (Red).
The same 6.5-kDa band appeared also in a chymotryptic digest of
32Pi-labeled 19-kDa fragment resolved under
reducing conditions (19 kDa). Thus the 6.5-kDa band is a fragment of
the
subunit containing Ser936 which is cross-linked to
the broad band of material in nonreducing conditions and is released by
reduction of an S-S bridge. By contrast to the result with PKA
phosphorylation of Ser936, the
anti-Asn889-Gln903 antibody recognized an
8.0-kDa chymotryptic fragment, the size of which was not affected by
reduction (Fig. 5, L7-8). Thus, this fragment is not a
cross-linked species and does not contain the PKA site at
Ser936 (compare L7-8 with
32Pi).
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Fig. 5.
Phosphorylation and chymotryptic digestion of
the CuP-catalyzed cross-linked product. The CuP cross-linked
product (10 µg, 0.25 mg/ml) was labeled with 15 µCi of
[ -32P]ATP using PKA (see "Experimental
Procedures"). The protein was precipitated with methanol and then
digested with chymotrypsin at 0.2 mg/ml for 1.5 h. The digest was
divided and either reduced (Red) or not
(Non-red). The 19-kDa fragment (2 µg, 0.1 mg/ml) was also
labeled with [
-32P]ATP, digested with chymotrypsin,
and then reduced. Samples were resolved on a short 16.5% T, 6% C gel,
and blotted onto PVDF. The sheet was autoradiographed using a Fuji BAS
1000 PhosphorImager (32Pi) and then
immunostained with an antibody recognizing residues
Asn889-Gln903 (L7-L8).
Sequence of 32P label chymotryptic fragment derived from
the major cross-linked product
subunit is cross-linked
to either Cys911 or Cys930 in M8 (see
"Discussion").
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Fig. 6.
Preparation of the
32Pi-labeled chymotryptic fragment of the
cross-linked product used for N-terminal sequencing. The CuP
cross-linked product (300 µg, 0.2 mg/ml) was labeled with 51 µCi of
[ -32P]ATP by PKA, precipitated with methanol, and then
digested at 1 mg/ml with chymotrypsin for 70 min. Samples (50 µg)
were resolved on a long 16.5% T, 6% C gel under reducing (Red) or
nonreducing conditions (Non-red). The gel was
autoradiographed using a Fuji BAS 1000 PhosphorImager. The 6-7-kDa
32Pi-labeled band was transferred to PVDF and
sequenced.
DISCUSSION
:19-kDa dimers (
65%) and
:19 kDa:11.7-kDa trimers
(
35%) (Figs. 1 and 3, Table I). These cross-links reflect
proximities of fragments within the intact detergent-solubilized
complex. The fact that cross-links were formed in detergent-solubilized
but not in native 19-kDa membranes could imply either that the
detergent induced a degree of rearrangement of trans-membrane segments
which permit cross-links between the relevant cysteines or that
cysteines embedded in lipid, such as Cys44 of the
subunit, are not reactive in native membranes due to insufficient
exposure to oxygen and CuP, and only become exposed to oxygen or CuP
after solubilization. However, the crucial point is that Rb occlusion
is fully preserved in these C12E10-solubilized 19-kDa membranes (1). Therefore the organization of trans-membrane segments in the soluble complex of fragments, and proximities revealed
by cross-linking (M8/M
, M9/M10), must be essentially similar to
those in native 19-kDa membranes and Na,K-ATPase.
:22 kDa:11 kDa (1:1:1) and, in lower amounts, a dimer of 22 kDa:11 kDa3 (23). Our study
confirms the finding of a
:19 kDa:11.7-kDa trimer, although in lower
yield than a
:19-kDa dimer, but a 19 kDa:11.7-kDa dimer was not
observed (see Fig. 1). Presumably, the similarities and differences
between the two sets of observations reflect the state of the soluble
complex. Solubilization of pig kidney 19-kDa membranes with
C12E10, in the absence of Rb ions and ouabain,
leaves the
-M7/M10 pair tightly associated but the M5/M6 and M3/M4
fragments dissociate, while the M1/M2 fragment shows an intermediate
degree of interaction with the
:19-kDa pair (1). Thus,
solubilization of 19-kDa membranes by digitonin in the absence of Rb
ions and ouabain (23) should not have preserved an intact complex.
Tighter association of the 11-kDa fragment with the
:22-kDa pair
could explain why a
:22 kDa:11-kDa trimer was observed even in the
absence of Rb ions and ouabain (23).
subunit rests on a
combination of direct evidence and exclusion of alternatives. The
inference of the internal cross-link (Fig. 4, Table II, Fig. 1) suggest
that M9 and M10 form a hairpin with Cys964 and
Cys983 juxtaposed, and excludes both residues as partners
for the
subunit. Direct evidence has been obtained by utilizing PKA
to label Ser936 with 32Pi (32, 33).
After chymotryptic digestion of the
32Pi-labeled cross-linked product, reducing
conditions released a 6.5-kDa 32Pi-labeled
fragment, with N terminus Glu902 (Figs. 5 and 6,
Red; Table III). This 6.5-kDa fragment includes Cys911 and Cys930 in M8 and ends before
Cys964 in M9. In nonreducing conditions the fragment is
cross-linked to a mixture of partially digested glycosylated fragments
of the
subunit, held together by internal S-S bridges (Figs.
4-6).
is compatible with prior evidence that
and
subunit interact strongly at the extracellular surface primarily
within the short sequence SYGQ outside M8 (34, 35). It is also
compatible with our finding that o-phthalaldehyde
cross-links
and
subunits near M8 (2). Although we cannot say
which cysteine in M8 is cross-linked to the
subunit,
Cys911 is a more likely candidate. A helical wheel
representation of M
(Fig. 7) reveals a
sector of about 100° with short or non-hydrophobic side chains
(Gly43, Cys44, Gly47,
Gly51, Gln54, shaded boxes) near the
extracellular surface. The nonhydrophobic sector could participate in a
protein-protein interface with the remaining hydrophobic surface facing
the lipid. Thus M
may contact M8, including Cys911, near
the extracellular surface.
View larger version (42K):
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Fig. 7.
Helical wheel representation of the membrane
spanning helix of the subunit.
The cross-link between M7/M10 and M1/M2 fragments has not been
identified. Based on topological considerations, Sarvazyan et
al. (23) proposed that Cys983-Cys104
(M1-M10) or Cys138-Cys930 (M2-M8) are likely
pairs. At first sight a Cys983-Cys104
cross-link appears incompatible with the
Cys983-Cys964 internal cross-link (Table II).
However, the observation of a mixture of -M7/M10 dimers and
-M7/M10-M1/M2 trimers (Figs. 1 and 3 and Table I) leads to the
following hypothesis. Assume, that M1, M9, and M10 are in proximity, so
that S-S bridges can form either between Cys983 in M10 and
Cys964 in M9 or between Cys104 in M1 and either
Cys983 or Cys964. Then 19-kDa fragments
containing the Cys983-Cys964 internal
cross-link could not be cross-linked to the M1/M2 fragment via
Cys983-Cys104 or
Cys964-Cys104. Thus, the mutually exclusive
formation of S-S bridges provides an economical explanation of the
mixture of cross-linked product. By contrast, the assumption of a
Cys138-Cys930 cross-link does not readily
explain this observation. Sarvazyan et al. (23, 24) observed
the
:22 kDa:11 kDa trimer with 1:1:1 stoichiometry and the 22 kDa:11-kDa dimer, but no
:22-kDa dimer. This could imply that the
internal Cys964-Cys983 cross-link was not
formed in their conditions.
Spatial Organization of Membrane Spanning Helices of and
Subunits
Fig. 8 presents a tentative proposal for spatial organization of trans-membrane segments of Na,K-ATPase. The arrangement is based on direct and indirect evidence on helix proximity, indications that trans-membrane helices of class II P-type pumps are separated into domains, and the necessity for M1/M2, M3/M4, M5/M6, and M9/M10 to be paired due to their short extracellular loops. Obviously, Fig. 8 depicts only an approximate arrangement, due to lack of detailed information on helix proximity and tilt. The major objective is to illustrate the structural constraints discussed below.
|
The thick dashed line in Fig. 8 demarcates separate domains
comprising M1 to M6 and M7 to M10 with M, respectively, evidence for
which is provided by the following observations. (a)
Compared with class II eukaryotic P-type pumps with 10 trans-membrane
helices, prokaryotic class I P-type heavy metal pumps contain the
equivalent of only the first six helices and conserved cytoplasmic
regions (with two extra helices at the N-terminal side). It has been
proposed that sequences corresponding to M1 to M6 of class II pumps
represent a core structural and functional unit for cation transport
and energy transduction, while the extra four C-terminal segments of
class II pumps evolved to serve additional functions (3, 36).
(b) We have shown recently that Fe2+ (37) and
Cu2+ (38) ions catalyze selective oxidative cleavages of
Na,K-ATPase, providing information on spatial organization around the
bound metal ions. Iron-dependent cleavages of the
subunit occur at the cytoplasmic surface in conserved sequences located
between M2 and M3 and between M4 and M5 (37).
Copper-dependent cleavages occur at the extracellular
surface between M7 and M8, to a small extent between M5 and M6, and
between M9 and M10, and in the
subunit
(38). A comparison of iron- and
copper-catalyzed cleavages is highly suggestive of division of the
subunit into two domains comprising M1-M6 and M7-M10/M
, respectively
(38). (c) High resistance to digestion by selective and
unselective proteases of the C-terminal fragment (M7-M10) of
Na,K-ATPase (8, 22) and H,K-ATPase (39) implies that it is folded into
a compact domain.
Division into domains of M1-M6 and M7-M10 provides a strong constraint
on the way helices can or cannot be packed. For example, M7 and M8 are
unlikely to be widely separated in the molecule although they are
connected by a relatively long extracellular loop (40 residues)
which, in principle, could permit such a separation. The positions
allocated to M1-M10 of the
subunit and M
are based on
the following considerations.
M4, M5, and M6-- Site-directed mutagenesis indicates that these helices form the main cation occlusion sites in Ca-ATPase and Na,K-ATPase, supplying ligating groups to occluded cations (6, 11, 40, 41). Recent cross-linking studies on Ca-ATPase have shown directly that M4 and M6 interact intimately (15). Thus M4, M5, and M6 are drawn in triangular contact (as in Refs. 14 and 15).
M7, M8, and M--
Proximity of M7 to M5 is strongly implied by
our recent finding of a cross-link between cytoplasmic segments just
preceding M7 and M5 (2). M6 and M7 are connected by a fairly short
cytoplasmic loop, and charged residues within the loop may form the
entrance to occlusion sites in M4, M5, and M6 (42). Therefore M7 is
also placed near M6. As argued above, M7 and M8 are unlikely to be widely separated and proximity of M8 to M4, M5 and M6, is suggested by
mutations of Glu908 in Ca-ATPase (9, 43). Thus M7 and M8
are placed together. M
interacts with M8 as required by the CuP
cross-linking (
Cys44-Cys911 or perhaps
Cys44-Cys930), and the evidence for
interaction between
and
subunits near the entrance to M8 (35,
2). As seen in Fig. 7 a likely surface of interaction of M
occupies a sector of only 100° with the remaining hydrophobic sector
facing the lipid. This is suggestive of the triangular contact in Fig.
8 with M
placed between M7 and M8.
M9 and M10-- The M9/M10 pair are internally cross-linked through Cys964 and Cys983 (Table II). Helices M8 and M9 are connected by a fairly short cytoplasmic loop. Thus, M9 is placed near M8. M9 is also placed near cation sites, i.e. M4, M5, and M6, in order to explain cation-protected modification of Glu953 by dicyclohexylcarbodiimide (44), conformation-dependent changes in fluorescence of N-(p-(2-benzimidazolyl)phenyl)-maleimide, covalently attached to Cys964 (45), and chemical modification of Cys964 (46). A direct interaction of M10 with M5/M6 is suggested by chemical modification of Cys983 following dissociation of the M5/M6 hairpin from dog kidney 19-kDa membranes (46, 47) and there is evidence implying association of the M5/M6 and M9/M10 hairpins of H,K-ATPase (39).
M1, M2, and M3-- The major constraints on the locations of these helices are the indications for the two domains and the necessity for M3 to be close to M4 and M2 to M1. We have proposed above that M1 is cross-linked either to M10 (Cys104-Cys983) or M9 (Cys104-Cys964), the M1-M10 interaction (Cys104-Cys983) depicted in Fig. 8 is a more likely possibility due to other constraints on the interactions of M9 referred to in the previous paragraph.
Comparison of Fig. 8 with the helix packing model of Ca-ATPase (12)
shows a general similarity in positions of M1-M7 as well as a major
difference in that M8, M9, and M10 are placed on opposites sides of the
molecules. Conceivably the position of M7 relative to M8-M10 is
affected by the subunit which interacts strongly with the L7/8.
However, it is equally likely that the uncertainty is due to lack of
detailed information. Two structural constraints used in Ref. 12,
namely proximity of M4-M6 based on S-S bridges (15) and orientation of
nonconserved residues of trans-membrane helices toward the lipid, fit
either arrangement. A third line of evidence involved placing helices
identified as the cytoplasmic stalk, S2-S5, above M2-M5 in the middle
of the molecule (12). Without comparable structural data for
Na,K-ATPase it is not known whether this constraint applies. Specific
predictions in Fig. 8, are that M4 and M6 are largely surrounded by
neighboring helices rather than lipid (see Ref. 47), M1 is close to
M10, and there is no proximity between M1/M2 and M7/M8. Clearly, it now
becomes necessary to devise a means to test specific predictions and
assumptions in order to refine the models.
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ACKNOWLEDGEMENTS |
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We thank Prof. Jack Kyte (University of California at San Diego, La Jolla, CA) for providing anti-KETYY, Prof. Jesper V. Møller (University of Aarhus, Aarhus, Denmark) for providing anti-Asn889-Gln903, and Prof. Shmuel Shaltiel (Department of Biological Regulation, Weizmann Institute of Science, Rehovot, Israel) for a gift of the catalytic subunit of PKA.
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FOOTNOTES |
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* This work was supported by Grant 96-00022 from the US-Israel Binational Science Foundation.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.
Present address: Dept. of Biochemistry, Biozentrum, University of
Basel, Klingelbergstr. 70, Basel, Switzerland.
§ To whom correspondence should be addressed. Tel.: 972-8-9342278; Fax: 972-8-9344118; E-mail: bckarlis{at}weizmann.weizmann.ac.il.
The abbreviations used are: CuP, Cu2+-phenanthroline; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; PAGE, polyacrylamide gel electrophoresis; PVDF, polyvinylidene difluoride; C12E10, polyoxyethylene 10-laurylether; PKA, protein kinase A.
2 Chymotrypsin was used rather than trypsin in order to avoid cleavage before the PKA phosphorylation site.
3 The 22- and 11-kDa fragments of proteolyzed dog kidney enzyme are equivalent to the 19- and 11.7-kDa fragments of proteolyzed pig kidney enzyme.
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REFERENCES |
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