From the Department of Biochemistry and Molecular
Biology, Oregon Health Sciences University, Portland Oregon 97201-3098 and the § Center for Ulcer Research, West Los Angeles
Veterans Affairs Medical Center, Los Angeles, California 90073
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ABSTRACT |
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The integral membrane protein, the gastric
H,K-ATPase, is an Recent functional studies of P-type ATPases have focused on two
major activities, the transmembrane transport of cations and the
hydrolysis of ATP via a phosphoenzyme intermediate, which appear to be
carried out by the actions of two separate protein domains, the
intramembrane domain that binds cations and the major cytoplasmic loop
responsible for ATP binding and hydrolysis (1, 2).
P-type ATPases can be classified into two subgroups based upon their
function and structure (3). A mechanistic aspect of most
P2-type ATPases is occlusion of the transported cations
during the reaction cycle (4). Such occlusion is pictured as an
intermediate state where cations are bound within the transmembrane
segments of the protein without free access to either aqueous
compartment. Occlusion was also recently shown to be retained in a
post-tryptic preparation of the Na,K-ATPase composed of the
transmembrane segments and lacking most of the cytosolically exposed
protein (5).
Chemical modification and site-directed mutagenesis studies of
expressed P2-ATPases have indicated that the M5M6
transmembrane region may have a special importance in cation occlusion
and transport (6-10). In studies on tryptic membrane preparations of
the Na,K-ATPase obtained in the presence of K+, mere removal of
K+ ions resulted in the selective release of the M5M6 hairpin
from the membrane to the aqueous phase (11). This membrane
stabilization of the M5M6 region by the counter-transported cation
suggested that the organization and folding of the M5M6 hairpin and the interactions between M5M6 and the rest of the protein may be associated with the transport function (11). The uniqueness of this region of the
membrane is also shown by the failure of the M5M6 hairpin to behave as
an independent unit in the process of synthesis and insertion in
in vitro translation studies of the H,K-ATPase (12). Similar
conclusions were derived from Cos-1 cell translation of segments of the
Na,K-ATPase and from in vitro translation of the endoplasmic
reticulum Ca-ATPase (13, 14), suggesting a general property of this
membrane domain of the P2-type ATPases.
The present studies were undertaken to determine whether or not
cation-dependent stabilization of the M5M6 hairpin in the membrane was also a feature of the H,K-ATPase, another
P2-ATPase. Because this protein can be obtained in a sealed
vesicular inside out orientation, the directionality of the release of
the M5M6 hairpin from the membrane (toward the cytoplasmic or
extracellular space) following K+ removal could also be
determined, unlike right side out oriented vesicles achieved in
microsomal preparations of Na,K-ATPase (15). A preliminary report of
this work has been presented (16).
Materials--
TPCK-treated1
trypsin, NaCl, KCl, Na2ATP, bovine serum albumin, sucrose,
ultra pure urea, Trizma (Tris) base, and Tricine were purchased from
Sigma. Enzyme Preparation and Activity Assay--
The H,K-ATPase was
isolated from hog gastric mucosa (17). Protein was determined by the
method of Lowry et al. (18). Vesicles were aliquoted and
stored (3-5 mg/ml protein) in a 34% sucrose solution buffered with 50 mM PIPES (pH 6.8) and frozen at Trypsin Digestion--
H,K-ATPase (1 mg/ml) was suspended in
medium containing 1 mM EDTA, 200 mM KCl, 20 mM Tris/HCl (pH 6.8) and incubated at 4 °C for 16-24 h.
Then TPCK-treated trypsin (1:10 w/w with respect to H,K-ATPase) was
added, and incubated at 37 °C for 1.5 h. The reaction was
terminated with the addition of the serine protease inhibitor AEBSF (2 mM); AEBSF was present at 1 mM in all
subsequent steps. Soluble peptide fragments were separated from
membrane-bound fragments by centrifugation (436,000 × g, 30 min, 4 °C). The membrane fraction was resuspended
with buffer and pelleted once more. The pellet (~500 µg) was
resuspended in buffer containing 1 mM EDTA, 20 mM Tris (pH 6.8), and 250 mM sucrose. Samples
were split in half and diluted to 1 mg/ml with buffer containing either
200 mM KCl or 5 mM MgPi; the
[sucrose] was adjusted accordingly to maintain isosmotic conditions.
Samples were incubated (37 °C, 15 min) and centrifuged (436,000 × g, 30 min, 4 °C). Supernatants containing peptides
from the extravesicular space were removed and saved. Pellets were
resuspended (1 mg/ml) in the same buffer (including KCl and
MgPi, respectively) with the addition of 0.05% SDS (w/w)
and incubated (37 °C, 15 min) and then centrifuged (436,000 × g, 30 min, 4 °C). Supernatants containing peptides from
the intravesicular space were removed and saved. The pellet from the sample-containing KCl tube was resuspended in buffer (1 mg/ml), but
MgPi was added instead of KCl. The suspension was incubated (37 °C, 15 min) and then centrifuged (436,000 × g,
30 min, 4 °C). The supernatant was removed and saved. All
supernatants were treated with 1 mM CPM for better
visualization of low molecular weight peptides (15). Peptides were
precipitated with acetone (9:1, v/v) at The two specific questions addressed by our studies were (i) in
the H,K-ATPase post-tryptic preparation does the removal of K+ ions lead to the destabilization and selective release
of the M5M6 hairpin from the membrane and (ii) if such release occurs, is the hairpin released to the cytoplasmic or extracellular
compartment?
-
heterodimer, with 10 putative transmembrane
segments in the
-subunit and one such segment in the
-subunit.
All transmembrane segments remain within the membrane domain following
trypsinization of the intact gastric H,K-ATPase in the presence of
K+ ions, identified as M1M2, M3M4, M5M6, and M7, M8, M9,
and M10. Removal of K+ ions from this digested preparation
results in the selective loss of the M5M6 hairpin from the membrane.
The release of the M5M6 fragment is directed to the extracellular phase
as evidenced by the accumulation of the released M5M6 hairpin inside
the sealed inside out vesicles. The stabilization of the M5M6 hairpin
in the membrane phase by the transported cation as well as loss to the
aqueous phase in the absence of the transported cation has been
previously observed for another P2-type ATPase, the
Na,K-ATPase (Lutsenko, S., Anderko, R., and Kaplan, J. H. (1995)
Proc. Natl. Acad. Sci. U. S. A. 92, 7936-7940). Thus,
the effects of the counter-transported cation on retention of the M5M6
segment in the membrane as compared with the other membrane pairs may
be a general feature of P2-ATPase ion pumps, reflecting a
flexibility of this region that relates to the mechanism of transport.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-Mercaptoethanol, SDS, ammonium persulfate, and Coomassie
Brilliant Blue R-250 were from Bio-Rad.
4-(2-Aminoethyl)-benzenesulfonyl fluoride hydrochloride (AEBSF) was
from ICN. Acrylamide and bisacrylamide were from Roche Molecular
Biochemicals. Rainbow gel electrophoresis standards were from Amersham
Pharmacia Biotech. 7-Diethylamino-3-(4'-maleimdylphenyl)-4-methyl coumarin (CPM) was from Molecular Probes. Polyvinylidene difluoride (PVDF) electroblotting membrane was from Millipore.
80 °C until used.
H,K-ATPase activity was measured in a standard assay medium containing
1 mM EGTA, 20 mM KCl, 3 mM
MgCl2, 3 mM Na2ATP, 40 mM Tris/HCl (pH 6.9), and 250 mM sucrose in the
presence or absence of 1 µg/ml nigericin. The suspension was
incubated for 15 min at 37 °C (10 mg protein/ml), and the liberation
of Pi measured as described by Brotherus et al.
(19). The difference in activity seen upon the addition of nigericin is
a measurement of vesicle integrity. Vesicles used were >70% sealed
and oriented cytosolic side out. Maximal enzyme activity was also
determined by using 100 mM NH4Cl instead of
KCl, with NH4+ acting as a
K+ congener. Vesicles do not have to be permeablized for
NH4+ to reach the inside because
NH3 can freely diffuse across the bilayer. In experiments
to determine the appropriate [SDS] required to permeabilize the
vesicles, enzyme activity was measured at various ratios of SDS to
protein (see Fig. 2) and compared with maximal activity with nigericin
or NH4Cl.
20 °C for 16 h and
then were run on a 16.5% Tricine gel (20). After electrophoresis,
protein fragments were transferred onto PVDF membranes by
electroblotting in 10 mM CAPS, 10% MeOH (pH 11.0) (21).
Fluorescent CPM-labeled bands were cut from the PVDF and subjected to
N-terminal amino acid sequencing.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Scheme showing the methods used to determine
the directionality of the M5M6 hairpin loss from a post-tryptic
membrane preparation of porcine gastric H,K-ATPase. 1,
intact cytoplasmic side out vesicle preparation containing
intact gastric H,K-ATPase and
subunits. 2,
post-tryptic membrane preparation of H,K-ATPase showing the remaining
segments: M1M2, M3M4, M5M6, and M7-M10, as well as an essentially
intact
-subunit. 3, the two potential outcomes depending
on the directionality of release. a, expected result if M5M6 release is
to the cytoplasmic side; b, expected result if M5M6 is released to the
extracellular side. 4, if the M5M6 segment is released to
the extracellular side (intravesicular side) then permeablization of
the vesicles is required to release the "trapped" peptide.
SDS Treatment of the H,K-ATPase Vesicle Preparation--
As shown
in Fig. 1, SDS treatment of the vesicle preparation is used to gain
access to the intravesicular (extracellular) space. If release of M5M6
occurs to the extracellular space the vesicles will need to be
disrupted in order for the M5M6 hairpin to be seen in the supernatant;
if release is to the cytoplasmic space, disruption of the vesicles by
detergent will not be required, and centrifugation alone will separate
the released hairpin from the vesicles. It was necessary first to
determine an appropriate SDS concentration that would permeabilize the
vesicles but not denature or disrupt the H,K-ATPase. To do this, we
made use of the fact that for maximal H,K-ATPase activity
K+ ions must gain access to the intravesicular space. Fig.
2 demonstrates that SDS ratios 0.1%
(w/w) were able to disrupt vesicle integrity without denaturing the
H,K-ATPase.
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Directionality of Release of the M5M6 Hairpin--
Just as was
originally reported for the Na,K-ATPase (5), extensive tryptic
digestion of the H,K-ATPase in the presence of K+ ions
produces a membrane residue that contains the -subunit (largely
undigested) and four sets of transmembrane segments, M1M2, M3M4, M5M6,
and M7-M10 (22). The post-tryptic preparations of both the Na,K- and
H,K-ATPases retained the ability to occlude K+ ions (5, 23). In
the Na,K-ATPase, removal of K+ ions from this preparation
resulted in a loss of the ability to occlude K+ associated
with the release of the M5M6 hairpin from the membrane to the aqueous
phase (11). Fig. 3 shows a single fluorescently labeled peptide (~10
kDa) that was released from the post-tryptic H,K-ATPase preparation
upon the removal of K+ ions. N-terminal amino acid analysis
gave a single sequence (NAADMIL ... ) that corresponds to the
residues in the M5M6 hairpin (Table I).
It was shown earlier that both the N and C termini of this fragment are
cytoplasmic (24), whereas either (or both) cysteine 813 and 822 within
this stretch are located at the extracytoplasmic side (25). These
observations demonstrated that the M5M6 segment indeed spans the
membrane twice. Furthermore, this peptide was not released to the
supernatant until after SDS treatment of the proteolyzed vesicles, thus
it is released toward the intravesicular (extracellular) space (Fig.
3A). In some experiments, a faint band was apparent (~10
kDa) in the supernatant prior to SDS treatment (data not shown). This
peptide was most likely the M5M6 hairpin, because it was only observed
when K+ ions were not present in the trypsinized
preparation (i.e. treatments outlined in 3a of
Fig. 1). Additionally, because the intensity of this band appeared to
vary inversely with the fraction of tight vesicles (as determined by
K+ ionophore stimulated ATPase activity), we conclude that
the M5M6 hairpin is released to the intravesicular space and then exits from leaky vesicles. Consistent with this conclusion is that SDS permeablization of these preparations subsequently revealed a vivid
band at 10 kDa that subsequent amino acid sequencing confirmed as M5M6
(Table I).
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These findings cannot directly rule out an SDS effect on the post-tryptic H,K-ATPase preparation. That is, does SDS itself promote the release of the M5M6 fragment? To answer this question, we also treated post-tryptic H,K-ATPase vesicles with SDS in the presence of 200 mM K+. Under these conditions, we observed no peptide release (Fig. 3B, lane 1). However, when K+ was subsequently removed from these permeablized vesicles the M5M6 hairpin appeared in the supernatant (Fig. 3B, lane 2).
In the experiments discussed above, the post-tryptic membrane
preparation was obtained in the presence of 200 mM KCl. In
two initial experiments, when trypsin digestion was performed in the presence of 20 mM KCl, we obtained two separate peptide
sequences from the released fragment (~10 kDa; Table I). The first
sequence corresponded to M5M6, whereas the second sequence corresponded to the M7M8 transmembrane pair (Table I). It has been previously shown
that in the absence of K+ ions an additional trypsin cleavage
takes place in the H,K-ATPase between M8 and M9 (22). It seems that
this cleavage also takes place at low K+ (i.e.
20 mM), thus separating the M7M8 hairpin from the ~21-kDa C terminus.
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DISCUSSION |
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The membrane domain of the P2-type ATPases contains
the ion transport pathway, and the results of a large number of studies have been interpreted as showing involvement of M4, M5, M6, and perhaps
M8 in ion translocation and ion occlusion (2, 7, 26). Various methods
including sequencing of membrane-embedded tryptic fragments (5, 22),
labeling with sided thiophilic reagents (23), or extracytoplasmic
photoactivatable reagents (27) and in vitro translation
provided strong evidence for 10 membrane segments in the -subunit of
the P2-type ATPases.
Hydropathy algorithms of P2-type ATPases all predict four membrane sequences in the N-terminal sector but predict only a single sequence in the M5M6 region and vary in terms of prediction of the last four segments (3). In accord with the ambiguity of these predictions, in vitro translation of individual segments or truncated constructs of the H,K-ATPase were not able to demonstrate transmembrane insertion of M5, M6, and M7 (13). Expression of segments or truncated constructs of the Na,K-ATPase also showed that M5 and M6 were not membrane inserted (14, 28). These results suggested that peptide-peptide interactions were more significant for these segments as compared with the N-terminal segments. Indeed, it was suggested earlier that the M5M6 hairpin of the Na,K-ATPase was involved in protein-protein interactions with other transmembrane segments within the lipid portion (11). Furthermore, our results (Ref. 11 and this work) suggest that cations are also required for stabilization in the membrane of the M5M6 hairpin of P2-type ATPases.
In the case of the P2-ATPases, results from chemical modification (8) and mutagenesis (6-10) have suggested that amino acid residues in M5M6 are intimately associated with cation occlusion and transport, and this hairpin is now believed to play a central role in cation binding and complexation. The loss of the M5M6 hairpin from the membrane and its prevention by K+ (Ref. 11 and this work) suggest that the occluded K+ ions play a role in causing a rearrangement of these membrane segments. One plausible mechanism for the action may be the neutralization of repulsive negative charges in Asp residues of M6 and the Glu in M5. Cation complexation by the electron-donating residues of M5M6 substitute for hydration and stabilize the presence of the charged cation in the membrane. At the same time the positive cation apparently serves to stabilize the anionic and hydrophilic transmembrane segments of M5 and M6 within the membrane.
The finding that the M5M6 hairpin of about 10-11 kDa (or about 90 amino acid residues) is lost to the extracellular space shows that this redistribution involves the mobilization across the membrane of a considerable number of charged and hydrophilic amino acid residues. We believe that M5M6 is probably surrounded by other protein helices rather than the membrane lipid (3, 11). In this way protein-protein interactions would be greatly modified as cations (e.g. K+) are bound to and released by the pump. Another line of evidence suggested mobility in this region of the enzyme. Pantoprazole (5-difluoromethosy-2-[3,4-methosy-2-pyridyl)methylsulfinyl]-1H-benzimidazole) con-verts to a cationic thiophilic sulfenamide and binds to Cys813 and Cys822 in the loop joining M5 and M6 forming disulfide bonds. Prior to this covalent reaction tryptic cleavage occurs largely at Arg775. After derivatization, cleavage is found only at Lys791 (29), showing that formation of disulfides within the connecting loop of M5 and M6 results in a change in accessibility of the N-terminal region of M5 to trypsin. Furthermore, the exposure of Cys983 in M10 of the Na,K-ATPase to sulfhydryl-reactive probes (but not of Cys residues in M1, M2, or M4) following the loss of the M5M6 hairpin also provides evidence for interactions between C-terminal segments and M5M6 (15). Similar conclusions have been reached from in vitro translation studies of both the Na,K-ATPase and the Ca-ATPase (13, 14).
In addition to the release of the M5M6 hairpin, the M7M8 transmembrane
pair was also released from the membrane following digestion by trypsin
in low K+ conditions (Table I) where the 21-kDa fragment is
cleaved between M7M8 and M9M10, also suggesting a weak interaction
between the bilayer and this pair of transmembrane segments. Prior to
cleavage between M8 and M9, the M5M6 tryptic fragment remains
associated with the 21-kDa C terminus; conversely, in the absence of
K+, when cleavage occurs between M8 and M9 the association
vanishes, suggesting that M5 and M6 are in the vicinity of M7-M10
(22). Further, although M8 acts as a stop transfer sequence in in
vitro translation, M7 is unable to membrane insert either as a
stop transfer or a signal anchor sequence (its putative role). However, in in vivo expression in frog oocytes, M7 is able to act as
a membrane inserted sequence provided the -subunit is co-translated, consistent with the strong interaction observed between the region preceding M8 and the
-subunit of the H,K-ATPase (12, 30). The
hydropathy profile (31, 32) of the M7 segment in the H,K-ATPase is
significantly lower than that of corresponding segment of the Na,K-ATPase consistent with its easier release from the membrane after
cleavage of the connecting cytoplasmic linkage.
The striking mobility of the M5M6 hairpin with respect to the membrane
that is modified by the presence of the occluded and transported cation
probably plays a significant role in the transport cycle. Indeed, the
direct link between the M5M6 hairpin and the large intracellular loop,
shown to contain the ATP binding site (33-35), is consistent with a
role in coupling ATP hydrolysis with cation transport. We have
speculated earlier (11) that movements of M5M6 in the Na,K-ATPase
protein in a direction that is perpendicular to the plane of the
membrane may play a role in active transport; on the basis of
the present results such a mechanism may apply to other
P2-ATPases.
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FOOTNOTES |
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* This work was supported by Grants HL30315 and GM39500 (to J. H. K.), Grants K 46917, 41301, and 53462 and a U. S. Department of Veterans Affairs Senior Medical Investigator grant (to G. S.), and National Research Service Award HL09972 (to C. G.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, L224, Oregon Health Sciences University, 3181 S.W. Sam Jackson Park Rd., Portland OR 97201-3098. Tel.: 503-494-1001; Fax: 503-494-1002; E-mail: kaplanj{at}ohsu.edu.
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ABBREVIATIONS |
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The abbreviations used are: TPCK, L-1-tosylamido-2-phenylethyl chloromethyl ketone or tosylphenylalanyl chloromethyl ketone; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; AEBSF, 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride; CPM, 7-diethylamino-3-(4'-maleimdylphenyl)-4-methyl coumarin; PVDF, polyvinylidene difluoride; PIPES, 1,4-piperazinediethanesulfonic acid; CAPS, 3-(cyclohexylamino)propanesulfonic acid.
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REFERENCES |
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