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INTRODUCTION |
In eukaryotic cells, membrane proteins are co-translationally
inserted into the endoplasmic reticulum membrane, and their final
membrane topologies are determined during membrane integration. They
are targeted to the endoplasmic reticulum membrane by the signal
recognition particle and interaction with the signal recognition particle receptor. A polar, transmembrane channel (the translocon) consisting of several subunits permits the passage of hydrophilic protein domains as well as the integration of hydrophobic membrane segments into the lipid bilayer (for recent reviews, see Refs. 1-3).
It has been proposed that membrane proteins integrate into the membrane
by a series of alternating signal anchor
(SA)1 or stop transfer (ST)
sequences. However, increasing evidence suggests that the acquisition
of a correct membrane topology of polytopic membrane proteins is often
more complicated and involves intermolecular interactions that require
specific sequence determinants within or adjacent to the hydrophobic
sequences. Furthermore, in oligomeric proteins, subunit assembly may
play a role in the correct packing of polytopic proteins.
We studied the in vivo membrane insertion of the polytopic
-subunit of H,K-ATPase which, in its mature form, is associated with
the
-subunit, a type II membrane protein. Little is known as to how
the assembly of the
-subunit may influence the membrane topology of
transmembrane segments of the
-subunit. However, it is clear that
association with the
-subunit is needed for the structural
maturation of the
-subunit of oligomeric P-type ATPases such as
H,K-ATPase (HKA
) and Na,K-ATPase (NKA
) because only
-subunit-assembled
-subunits become trypsin-resistant, are
protected from cellular degradation, and become functionally active (4,
5).
There is general agreement that the HKA
and NKA
have 10 transmembrane segments with the N and the C termini located at the cytoplasmic side in the mature
-associated enzyme (for reviews and
references, see Refs. 6-8). The hydropathy profile of the two enzymes
is quite similar but differs significantly in the level of
hydrophobicity in M7, M8, M9, and M10. Thus, M7, for example, has a
lower hydrophobicity value in HKA
than in NKA
, and M9 has a
higher hydrophobicity value in HKA
than in NKA
. An in
vitro transcription/translation assay showed that the putative transmembrane segments M1, M2, M3, M4, and M9 of the gastric HKA
can
act both as signal anchor (SA) and stop transfer (ST) sequences, M8 and
M10 can act only as ST sequences, and M5, M6, and M7 have no membrane
insertion properties (9). Thus, there is a gap of three known
membrane-inserted sequences in a sequential folding model determined
entirely by the hydrophobicity of the membrane segments. Similarly,
in vitro translation assays of M5 and M6 of the Na,K- (10)
or endoplasmic reticulum Ca-ATPase
-subunits (11) have been unable
to show efficient SA and ST properties of these segments. In
vivo translation/insertion experiments in Xenopus
oocytes showed that M5, M7, and M9 of NKA
exhibit partial SA
function. In the mature enzyme, the
-subunit is closely associated with the extracellular loop immediately preceding M8 in both NKA
and
HKA
, and the assembly of the
-subunit plays an important role in
the correct packing of the C-terminal membrane domain in NKA
(12).
The in vitro transcription/translation system may lack
factors present in the intact cell, and it is difficult to study the effect of
-subunit association in the in vitro system.
Therefore, we reassessed the SA or ST properties of the transmembrane
sequences of HKA
after expressing them in Xenopus oocytes
in the presence or absence of the
-subunit. As a test for membrane
insertion, we assessed the presence or absence of glycosylation of
chimera containing single or multiple transmembrane segments of
-subunits and the ectodomain of the
-subunit that contains
several glycosylation sites. We also assessed the stability of the
synthesized proteins by pulse-chase experiments. Whereas the three
N-terminal membrane segments are efficiently and stably integrated into
the membrane during synthesis, the C-terminal membrane segments do not
conform to the sequential signal anchor-hydrophilic segment-stop
transfer motif. Furthermore, association with the
-subunit plays a
role in the correct packing of the C-terminal membrane domain and the stabilization of HKA
, as has also been observed in NKA
(12). However, the specific requirements for the proper folding of the C-terminal membrane domain are distinct in the two P-type ATPases.
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MATERIALS AND METHODS |
Constructs--
The rabbit gastric HKA chimera containing
single or multiple putative transmembrane segments of the
-subunit
(see Table I) and the 177 C-terminal amino acids of the rabbit HKA
-subunit were prepared as described previously (9), but all
constructs were subcloned into the pSD5 vector containing a modified
Xenopus 5'-untranslated region, which improves the
translation of foreign cRNA in Xenopus oocytes as described
previously (13).
For the preparation of truncated
constructs lacking the HKA
ectodomain, we introduced the modified Xenopus
5'-untranslated region into the original pGEM7zf+ chimeric M1 construct
(e.g. containing the cDNA sequence for the first 139 amino acids of the
-subunit ending in the extracellular loop
following M1 and for the ectodomain of the HKA
-subunit; Ref. 9) by
using appropriate restriction sites. By polymerase chain reaction
mutagenesis, we then introduced a stop codon at the junction between
the
and the
sequence upstream of the artificial
BglII/HindIII linker. The variable domains
containing the putative transmembrane segments were excised from the
chimeric constructs by BglII and HindIII digestion and introduced into the BglII/HindIII
linker of this new vector. Finally, the constructs were subcloned from
the pGEM7zf+ vector to the pSD5 vector using EcoRI
restriction sites. All constructs generated by polymerase chain
reaction amplification were sequenced by dideoxy sequencing.
Expression of HKA Constructs in Xenopus Oocytes and
Immunoprecipitation of
- and
-Subunits--
Stage V-VI oocytes
were obtained from Xenopus laevis females (Noordhoek) as
described previously (5). In vitro synthesized RNA (cRNA)
was prepared as described by Melton et al. (14). Routinely,
8-10 ng of mutant HKA
cRNA and/or 2 ng of rabbit gastric HKA
cRNA were injected into oocytes. Oocytes were metabolically labeled
with 0.6 mCi/ml [35S]methionine (Hartmann Analytic) for
24 h and, in some instances, subjected to a 48-h chase period.
Microsomal fractions were prepared, and the pellets were taken up
in 0.5% digitonin and subjected to immunoprecipitation under
nondenaturating conditions (5) with polyclonal antibodies that
recognize the
- and
-subunits of HKA (15). Endoglycosidase
H (Endo H) (Calbiochem) treatment was performed as described previously
(16). SDS-polyacrylamide gel electrophoresis was performed on the
immunoprecipitated proteins without or with Endo H treatment, and the
protein pattern was quantified by densitometry with a LKB 2202 Ultrascan.
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RESULTS |
Comparison of the Topographic Properties of Transmembrane Segments
of the
-Subunit of Gastric H,K-ATPase Synthesized in Intact Cells
and in Vitro--
To follow the topogenic properties of membrane
segments of HKA
synthesized in vivo, we used the reporter
glycosylation scanning approach. This method has been used successfully
for topological mapping of several polytopic membrane proteins (6, 17,
18). The presence or absence of glycosylation of a C-terminal,
topographically neutral reporter sequence indicates whether a
translated sequence ends on the luminal or the cytoplasmic side of the
membrane, if one assumes that all membrane-inserted protein is
glycosylated. Glycosylation of putative membrane-spanning sequences
containing the last 177 amino acids of the HKA
-subunit at their C
terminus including five glycosylation sites was assessed after their
expression in Xenopus oocytes by cRNA injection, radioactive
labeling, and immunoprecipitation. Endo H that specifically cleaves
N-linked high mannose core sugars was used to distinguish
glycosylated protein species from nonglycosylated protein species to
confirm the origin of the shift in the protein migration on SDS
polyacrylamide gels.
Fig. 1 shows the Endo H sensitivity of
several membrane-spanning sequences of HKA
(summarized in Table
I) expressed in Xenopus oocytes after cRNA injection and labeled for 24 h with
[35S]methionine. M1 (lanes 1 and
2), M2 (lanes 3 and 4), M3
(lanes 7 and 8), M4 (lanes 9 and
10), and M9 (lanes 11 and 12), which were synthesized in vivo from cRNAs that are preceded by the
first 101 amino acids (cytoplasmic N terminus) of rabbit gastric
HKA
, were all efficient SA sequences that were Endo H-sensitive due to the glycosylation of the total protein population. In the oocyte, as
in vitro (9), glycosylation of M2 was prevented when it was
preceded by M1 (M1/2, lanes 5 and 6),
indicating that in this construct, M2 acts as a ST sequence, and the
first membrane loop is firmly anchored in the membrane.

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Fig. 1.
Membrane insertion of individual membrane
segments of H,K-ATPase -subunits expressed in Xenopus
oocytes. Oocytes injected with 8 ng of chimeric cRNA coding for
individual or combined transmembrane segments M1-M10 of the
-subunit of rabbit gastric H,K-ATPase ( HKA) and the
ectodomain of the HKA -subunit were labeled with
[35S]methionine for 24 h. Microsomes were prepared,
and immunoprecipitations were performed under nondenaturing conditions
with an antiserum that recognizes HKA and HKA (15).
Immunoprecipitates were subjected or not subjected to Endo H treatment,
which reduces the size of glycosylated signal anchor sequences but not
that of nonglycosylated stop transfer sequences. Autoradiograms of
immunoprecipitates subjected to SDS-polyacrylamide gel electrophoresis
are shown. Some artifactual bands (dots) are observed in
lanes 7-10, 13, and 14 that migrate in front of
the antibody heavy chains and probably represent actin. In lane
13, the coreglycosylated, Endo H-sensitive M10 is indicated by an
asterisk. Right, the position of marker proteins
is indicated. ng, nonglycosylated; cg, core
glycosylated. One of three similar experiments is shown.
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A set of larger constructs (Table I) was used to test whether and how
naturally preceding membrane-spanning segments influence the
orientation of C-terminal membrane sequences. The total population of a
M1-3 translation product was Endo H-sensitive (Fig.
2B, lanes 1 and 2),
showing that M3 also acts as an efficient SA sequence when preceded by
the membrane-integrated M1/2 pair. M4, which acts as an efficient SA
sequence when synthesized alone (Fig. 1, lanes 9 and
10), became partially but not entirely protected from
glycosylation when preceded by M1-3. Also, the M1-4 protein showed
aberrant migration on SDS-polyacrylamide gels with a lower molecular
mass than the M1-3 protein (Fig. 2A, lanes 3 and
4), as noted previously in vitro (9).

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Fig. 2.
Membrane insertion of combined membrane
segments of H,K-ATPase -subunits and topogenic effects of
-subunit assembly. Oocyte injection, metabolic labeling, sample
preparation, and immunoprecipitation were performed as described in the
Fig. 1 legend. A, membrane insertion of combined membrane
segments of chimeric HKA . Similar to results obtained in
vitro (9), M1-4 consistently showed aberrant migration on
SDS-polyacrylamide gel electrophoresis. B, effect of
H,K-ATPase -subunit ( HKA) co-expression on membrane
insertion of C-terminal membrane segments of HKA . The position of
the - and the -subunits is indicated. Oocytes were co-injected
with HKA cRNAs and 2 ng of HKA cRNA. cg, core
glycosylated HKA species; ng, nonglycosylated HKA
species. One of three similar experiments is shown.
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The observation that M5, M6, and M7 following the large cytoplasmic
loop had neither SA nor ST function in an in vitro
translation system led to the conclusion that M5-M7 must be inserted
into the membrane by a different mechanism than the other seven
transmembrane segments (9). One possibility that was considered was the
insertion of this region in an already folded state into a membrane
domain that included M1-M4 and M9-M10. However, when synthesized in
Xenopus oocytes, about 40-45% of the total HKA M1-5
population was glycosylated (Fig. 2A, lanes 5 and
6), suggesting that HKA M5 has some intrinsic SA function
in vivo. The addition of M6 (M1-6) completely
prevented glycosylation (lanes 7 and 8),
indicating that M6 can form a pair with M5 that may be membrane-bound
or retained in the cytoplasm.
In combined HKA
constructs, M9 of gastric HKA
displays SA
function and M7 does not display SA function when synthesized in
vitro and in vivo in intact cells. When synthesized in
the oocyte, HKA M1-7 and M1-8 proteins were Endo H-resistant and thus were not glycosylated (lanes 9 and 10), whereas
40-45% of the total M1-9 population was Endo H-sensitive and
glycosylated (lanes 13 and 14).
Individual M10, which was devoid of SA activity in vitro
(9), became partially glycosylated when expressed in the oocyte (Fig.
1, lanes 13 and 14). Thus, in vivo,
M10 can adopt a Nout-Ccyt as well as a
Ncyt-Cout orientation. Although M10 added to M1
could not act as a ST sequence in intact cells (Fig. 1, lanes
15 and 16), it completely prevented glycosylation when
added to M1-9 (Fig. 2A, lanes 15 and 16,
M1-10), indicating that M10 can form a pair with M9 that is
either retained in the cytoplasm or membrane-bound. The results shown
in Fig. 2 are also summarized in Table I.
The differences in the membrane insertion efficiency of certain
membrane segments of HKA
synthesized in vitro or in
intact cells cannot be easily explained. It is possible that due to the longer synthesis time allowed in intact cells compared with that used
in in vitro translations, a steady-state level of folding and membrane insertion of certain membrane segments can only be achieved in the former and not in the latter translation system. In
this context, however, it is interesting to note that P-glycoprotein synthesized in two different in vitro translation systems
adopts a different topography, possibly due to the presence or absence of certain soluble cytoplasmic factors (19). Whatever might be
responsible for the subtle differences between the in vivo and in vitro systems does not change the main conclusions
that can be drawn from the reporter glycosylation scanning assays. The
results obtained in both systems indicate that the formation of
N-terminal and C-terminal membrane pairs in HKA
is governed by
different mechanisms: the former is governed by sequential signal
anchor and stop transfer mechanisms, and the latter is governed by more
complicated interactions. We tested the role of
-subunit
interactions on the correct packing of the C-terminal membrane domain
of HKA
.
Effect of
-Subunit Assembly on the Topography of H,K-ATPase
-Subunit--
A domain that interacts with the
-subunit has been
identified in the extracellular loop just before M8 in the
-subunit
of both H,K-ATPase (20, 21) and Na,K-ATPase (22). The effect of
-subunit assembly on the membrane insertion of HKA
was determined by the co-expression of HKA
with HKA M1-7-M1-10
-proteins.
Endo H sensitivity and thus the apparent topography of HKA M1-7,
M1-8, or M1-10
-proteins were similar when they were expressed alone or together with
-subunits (compare Fig. 2B, lanes 1-4, 7, and 8 with Fig. 2A, lanes 9-12, 15, and
16) showing an absence of glycosylation. In contrast, the
M1-9 protein consistently showed a change in the ratio between
glycosylated, Endo H-sensitive and nonglycosylated, Endo H-resistant
species when it was expressed with
-subunits. The percentage of
glycosylated species increased from 40-45% in M1-9 expressed alone
to about 80-85% in M1-9 co-expressed with
-subunits (compare Fig.
2B, lanes 5 and 6 with Fig. 2A, lanes
13 and 14; Table I). This result suggests that assembly with
-subunits increases the efficiency of membrane integration of
M9 in the HKA M1-9
-protein. To further analyze the role of
-subunit assembly in the membrane integration of the HKA
, we determined the resistance against cellular degradation of various HKA
constructs synthesized in the absence or presence of
-subunits, an indicator of correct folding of polytopic integral
membrane proteins.
Effect of
-Subunit Assembly on the Stability of H,K-ATPase
-Subunits--
To test the stability of HKA
proteins synthesized
in the presence or absence of
-subunits, we prepared truncated
HKA
constructs lacking the glycosylation reporter sequence because
we have previously observed that the glycosylation reporter sequence
itself can destabilize chimeric NKA
constructs when it is exposed to
the cytoplasm (12), leading to cellular degradation. Fig.
3A shows pulse-chase
experiments performed in oocytes expressing truncated HKA M1-3-M1-10
-proteins in the absence of
-subunits. M1-3 proteins were
completely resistant to cellular degradation (Fig. 3A, lanes
1 and 2), consistent with the efficient membrane
insertion of this construct observed in the reporter glycosylation
scanning assay. M1-4 constructs produced two different protein
populations. One of the proteins had a lower molecular mass than the
M1-3 protein and likely corresponds to the aberrantly migrating M1-4
protein that shows an inefficient ST activity in the reporter
glycosylation scanning assay (Fig. 2A, lanes 3 and
4). This protein population was significantly degraded
during a 48-h chase period (Fig. 3A, lanes 3 and
4). The second protein population synthesized from the M1-4
construct exhibited the expected molecular mass, which was greater than that of the M1-3 protein, and it was relatively resistant to cellular degradation (lanes 3 and 4). This result
indicates that the particular M1-4 construct used in this study is
indeed able to form an
-protein population in vivo that
corresponds to a stably inserted M1-4 membrane domain. It is possible
that the removal of the hydrophilic glycosylation reporter sequence
favors the ST function of M4 in this particular construct.

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Fig. 3.
Cellular degradation of truncated H,K-ATPase
-subunits and effect of -subunit assembly. Oocytes were
injected with 10 ng of HKA cRNA coding for combined transmembrane
segments M1-M10 in the absence (A) or presence
(B) of 1 ng of HKA cRNA, labeled with
[35S]methionine for 24 h (odd-numbered
lanes), and subjected to a 48-h chase (even-numbered
lanes). Microsomes were prepared, and immunoprecipitations were
performed as described in the Fig. 1 legend. The position of the -
and the -subunits and the molecular mass of marker proteins are
indicated. HKA is present in its core-glycosylated (cg)
form during the pulse and the chase period and partially in its fully
glycosylated (fg) form during the chase period due to the
endoplasmic reticulum exit of unassembled HKA (26). The band
indicated by a dot and visible in lanes 1, 2, and
4 corresponds to endogenous oocyte NKA , which is
co-immunoprecipitated and assembled with HKA . One of three similar
experiments is shown.
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All other truncated HKA
proteins comprising C-terminal membrane
segments up to M10 (Fig. 3A, lanes 5-16) synthesized in the absence of
-subunits were almost completely degraded during a 48-h
chase, similar to the full-length
-subunit (lanes 17 and 18). These results are consistent with the inefficient
membrane insertion of M1-5-M1-10.
Co-expression with the HKA
had no effect on the stability of the HKA
M1-7
-proteins (Fig. 3B, lanes 1 and 2) but
partially protected the M1-8 (lanes 3 and 4) and
M1-9 proteins (lanes 5 and 6) from cellular
degradation. Furthermore, HKA M1-10 proteins (lanes 7 and
8) were completely stabilized after co-expression with
-subunits, similar to the full-length HKA
(lanes 9 and 10). These results confirm that association with the
-subunit is an important factor for correct membrane insertion and
stabilization of the C-terminal membrane domain of the HKA
.
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DISCUSSION |
Topological Properties of HKA
Membrane Segments--
The
present study shows a membrane insertion process of HKA
that is
characterized by major differences in the mechanisms that govern
membrane integration of the four N-terminal and the six C-terminal
membrane segments. The former segments act as alternate SA and ST
sequences and are sequentially inserted into the membrane to form
stable membrane pairs, whereas the latter segments need both
intramolecular interactions and assembly with
-subunits for correct
membrane packing and protection against cellular degradation.
Full SA activity of HKA
M1 and M3 and an efficient ST activity of M2
were observed in vivo (this study) and in vitro
(9), which provide the basis for stable membrane integration of a
domain comprising a M1/M2 pair and M3. This was also confirmed by the observation that M1-3
-proteins are completely resistant to
cellular degradation. On the other hand, the particular sequence used
in this study for HKA M4 produced aberrant migration on
SDS-polyacrylamide gels and degradation of part of the M1-4 protein
population, and it had inefficient ST activity in vivo. This
contrasts with the efficient ST activity observed in vitro
(9). This result may indicate that pair formation of M3 and M4 in
vivo is necessary but is not sufficient to determine the
four-membrane segment topology of the HKA
N terminus in contrast to
recent results obtained for NKA
synthesized in Xenopus
oocytes (12). Alternatively, it is more likely that M4 in the HKA M1-4
protein that ends at Leu-357 may be too short to assure its membrane
retention as an efficient ST sequence. According to a current model
(Ref. 8; see Table I), HKA M4 would end at Ala-359; consequently, the M1-4 protein lacks two positively charged residues that follow the
transmembrane domain and may be important for the ST properties of M4
in the HKA M1-4 protein synthesized in vivo. Positive
charges following a membrane-inserted domain have been shown to be
important for ST function (6). Nevertheless, part of the HKA M1-4
-protein population was able to adopt a relatively stable
membrane-inserted conformation with a correct molecular mass,
supporting the hypothesis that the N-terminal membrane domain of HKA
is formed by sequential insertion of the two membrane pairs M1/M2 and
M3/M4, as in NKA
(12).
M5, M7, and M9, which should act as SA sequences according to the
10-transmembrane segment model, produced a mixture of glycosylated and
nonglycosylated species in HKA M1-5 and M1-9
-proteins and only
nonglycosylated species in the M1-7
-protein synthesized in
vivo in the absence of
-subunits. Similar observations were made with NKA
, but the insertion efficiency of NKA M5, M7, and M9
differed from that of HKA membrane segments. The NKA M1-5
-proteins produced only about 5% glycosylated species, but M1-7 and M1-9 produced about 50% glycosylated species (12), perhaps due to the
greater hydrophobicity of M7 in NKA
.
Consistent with the poor membrane insertion properties, all
-proteins containing C-terminal membrane segments of both HKA
and
NKA
(23) were rapidly degraded during a chase period. Because we
have recently shown that major domains of NKA
are not exposed to the
lumen of the endoplasmic reticulum during synthesis (12), it is likely
that unassembled
-subunits of P-type ATPases are degraded from the
cytoplasmic side, as recently suggested for misfolded polytopic
membrane proteins (24). The degradation of the
-subunit, which is
likely to start in the C-terminal domain and/or the central cytoplasmic
loop, also destabilizes the intrinsically stable N-terminal domain and
renders it susceptible to degradation because a proteolytic fragment
corresponding to the N-terminal domain is never observed.
The poor efficiency of membrane insertion and the associated
instability of the C-terminal membrane domain likely reflect a
distinct, low level of hydrophobicity of HKA
and NKA
M5, M7, and
M9 that reduces interactions with nonpolar surfaces. In NKA
, the
presence of proline, polar, and/or charged residues in NKA M5, M7, and
M9 was shown to be responsible for the partial membrane insertion of
these membrane segments (12). As a consequence of their moderate
hydrophobicity, membrane insertion of M5, M7, and M9 of HKA
and
NKA
must be mediated by other factors, for instance, interaction
between membrane peptide segments in the translocon during translation.
Such peptide-peptide interaction is consistent with the recently
proposed two-dimensional crystal structure of the Ca-ATPase (25) that
would place two or three membrane segments in a structure where their
surfaces interact largely with other transmembrane segments, whereas
the other segments face the hydrophobic core of the membrane.
Furthermore, we show in this study and in previous studies of NKA
(12, 23) that in oligomeric P-type ATPases, association with the
-subunit plays an important role in the stabilization of the
moderately hydrophobic membrane pairs in the membrane.
Effect of
-Subunit Assembly on the Membrane Topography and
Stabilization of HKA
--
Co-expression of HKA
proteins with
-subunits had two major effects. On the one hand, it increased the
efficiency of membrane insertion of M9 in a M1-9
-protein, and on
the other hand, it partially protected M1-8 and M1-9
-proteins and
completely protected M1-10 proteins against cellular degradation.
Together with the observation that the interaction of the
-subunit
with the region of the extracytoplasmic loop between M7 and M8
participates in the stable membrane insertion of the M7/M8 pair in the
NKA
(12), this result indicates that in hetero-oligomeric
P-type ATPases, not only intramolecular but also intermolecular
interactions between subunits participate in the correct packing of the
C-terminal membrane domain of the
-subunits.
A Model for Stable Membrane Integration of the C-terminal Membrane
Domains of HKA and NKA
-Subunits--
Our data indicate that the
precise role of
-subunit assembly and intramolecular interactions in
the stable membrane insertion of HKA
and NKA
may vary, possibly
due to the differences in the intrinsic SA and ST function of M7 and
M10, respectively, in the two proteins. According to the reporter
glycosylation scanning assay, HKA M7 in a HKA M1-7
-protein has no
visible SA function (Ref. 9 and this study), but NKA M7 in a NKA M1-7
-protein has partial SA function and produces 50% glycosylated
forms (12). In both
-subunits, the addition of M8 produces only
nonglycosylated forms. These observations suggest that in NKA M1-8
-proteins, an equilibrium exists between membrane-inserted and
cytoplasmic forms of the NKA M7/M8 pair, whereas in HKA M1-8 proteins,
the equilibrium may be significantly shifted in favor of the
cytoplasmic forms of the HKA M7/M8 pair, as illustrated in the model in
Fig. 4. In consequence, the probability
of
-subunit assembly with the extracellular loop between M7 and M8
in M1-8 proteins is smaller in HKA
than in NKA
. This may be
reflected by the partial stabilization as compared with the complete
stabilization of the HKA M1-8 and NKA M1-10
-proteins,
respectively, when they are co-expressed with
-subunits. The
difference in the stabilizing effect of the
-subunit on HKA and NKA
M1-8
-proteins also indicates that M9/M10 pair formation has a
distinct role in the correct folding of the HKA
and NKA
. In
HKA
, co-expression with
-subunits favors membrane insertion of
M9, but only M1-10 proteins are stabilized (Fig. 4). This infers that
M9/M10 pair formation and probably its interaction with the M7/M8 pair
are necessary for an efficient membrane insertion of HKA M7/M8 and
consequently for an efficient
-assembly and stabilization of HKA
.
On the other hand, in NKA
,
-assembly with the M7/M8 extracellular
loop is sufficient to stabilize the entire
-subunit, although it
restrains M9 membrane insertion (Fig. 4). In NKA
, the formation of
the M9/M10 pair may only be possible due to the better ST function of
NKA M10 as compared with that of HKA M10.

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Fig. 4.
Comparison of membrane insertion properties
and stability of HKA and NKA and effect of -subunit
assembly. The membrane retention and cellular degradation of HKA
(this study) and NKA (12, 25) M1-8, M1-9, and M1-10 -proteins
synthesized in the absence or presence of -subunits are shown. In
HKA and NKA , the four N-terminal membrane segments are stably
inserted into the membrane due to sequential SA and ST mechanisms
independent of the -subunit. On the other hand, insertion of the six
C-terminal membrane segments is mediated by interactions between
membrane segments and by -subunit assembly with the extracytoplasmic
loop (el) between M7 and M8. Our results are compatible with
a model in which, in the absence of the -subunit, the C-terminal
membrane pairs of M1-8, M1-9, and M1-10 exist as membrane-inserted
and cytoplasmic forms in a dynamic equilibrium (shown in the diagrams
and indicated by an arrow). The membrane insertion of the
NKA and HKA M7/M8 pair in the M1-8 protein cannot be directly
quantitated, but due to the poor SA function of HKA M7, it can be
predicted that the equilibrium of the HKA M7/M8 pairs is
significantly shifted toward the cytoplasmic forms. On the other hand,
the NKA M7/M8 pairs are evenly distributed between the membrane and
the cytoplasm. From these data, it may be inferred that -subunit
interaction with the extracellular loop between M7 and M8
(el) is more likely to occur in NKA M1-8 -proteins than
in HKA M1-8 -proteins. This is supported by the observation that
HKA M1-8 is only partially protected, but NKA M1-8 is completely
protected against cellular degradation after co-expression with
-subunits. According to the reporter glycosylation scanning assay,
HKA and NKA M9 in M1-9 -proteins have a partial SA function.
-Subunit assembly favors HKA M9 membrane retention and impedes that
of NKA M9. Nevertheless, -subunit assembly only partially protects
HKA M1-9 -proteins but fully protects NKA M1-9 proteins from
degradation similar to HKA M1-8 and NKA M1-8, respectively. Finally,
membrane retention of the M9/M10 pairs in HKA and NKA M1-10
-proteins in the absence of -subunit assembly cannot be
predicted, but it is fully achieved in the presence of -subunit
assembly, because NKA M1-10 as well as HKA M1-10 become completely
protected from cellular degradation. Together with the observation that
HKA M1-5 and M1-6 -proteins are completely degraded independent of
the presence of -subunits, these results also imply that after
assembly with -subunits, the HKA M5/M6 pair is correctly
membrane-inserted in the HKA M1-10 -protein. For further
discussion, see text.
|
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In conclusion, in both HKA
and NKA
, the correct membrane
integration of the N-terminal domain appears to be determined by the
properties of the individual segments, whereas the insertion and
stabilization of the C-terminal membrane domain depend on interactions
with other membrane segments as well as on intermolecular association
with the
-subunit. However, subtle differences exist between
the two P-type ATPases in the fine control of the insertion process.