(Received for publication, July 18, 1995; and in revised form, August 9, 1995)
From the
The membrane topology of the rat endoplasmic reticulum (ER) and
sarcoplasmic reticulum (SR) Ca ATPases were
investigated using in vitro transcription/translation of
fusion vectors containing DNA sequences encoding putative
membrane-spanning domains. The sequences of these Ca
ATPases are identical except for the COOH-terminal end, which
contains an additional predicted transmembrane segment in the ER
ATPase. The MO and M1 fusion vectors (Bamberg, K., and Sachs, G.(1994) J. Biol. Chem. 269, 16909-16919) encode the
NH
-terminal 101 (MO vector) or 139 (M1 vector) amino acids
of the H,K-ATPase
subunit followed by a linker region for
insertion of putative transmembrane sequences and, finally, the
COOH-terminal 177 amino acids of the H,K-ATPase
subunit
containing five N-linked glycosylation consensus sequences.
The linker region was replaced by the putative transmembrane domains of
the Ca
ATPases, either individually or in pairs.
Transcription and translation were performed using
[
S]methionine in a reticulocyte lysate system in
the absence or presence of canine pancreatic microsomes. The translated
fusion protein was identified by autoradiography following separation
using SDS-polyacrylamide gel electrophoresis. When testing single
transmembrane segments, this method detects signal anchor activity with
M0 or stop transfer activity with M1. The first four predicted SERCA
transmembrane domains acted as both signal anchor and stop transfer
sequences. A construct containing the fifth predicted transmembrane
segment was able to act only as a stop transfer sequence. The sixth
transmembrane segment did not insert cotranslationally into the
membrane. The seventh was able to act as both a signal anchor and stop
transfer sequence, and the eighth showed stop transfer ability in the
M1 vector. The ninth transmembrane segment had both signal anchor and
stop transfer capacity, whereas the tenth transmembrane segment showed
only stop transfer sequence properties. The eleventh transmembrane
sequence, unique to the ER Ca
ATPase, had both signal
anchor and stop transfer properties. These translation data provide
direct experimental evidence for 8 or 9 of the 10 or 11 predicted
transmembrane sequences in the current topological models for the SR or
ER Ca
ATPases, respectively.
The eukaryotic P type ATPases consist of a single catalytic
subunit and, on occasion, a second,
, subunit(1) .
All of these P type ATPase
subunits have a hydropathy profile
indicative of a polytopic integral membrane protein (Fig. 1).
The mammalian Na,K-, H,K-, and Ca
-ATPases seem to
share a characteristic hydropathy profile despite widely differing
amino acid composition(1) . This profile is also similar to
that of the fungal H
-ATPases, with the exception that
the H5 and H6 regions are more hydrophobic and also distinct with an
intervening hydrophilic sequence(2) . Based on this hydropathy
profile, the catalytic subunit of these enzymes has a large cytoplasmic
domain, a membrane domain composed of several membrane-spanning
segments, and a small extracytoplasmic
domain(3, 4, 5) . Hydropathy predictions are
unequivocal in defining the first four putative transmembrane segments
but are less certain in the COOH-terminal one-third of these pumps.
Whereas the SR (
)Ca
-ATPase was thought to
have 10 transmembrane segments(3) , both the Na,K- and
H,K-ATPases were predicted to have eight transmembrane segments when
they were sequenced(4, 5) . The Neurospora H
-ATPase has been proposed to have 8, 10, or 12
such segments(2, 6, 7) . No standard
algorithm predicts 10 segments. In particular, only a single
transmembrane segment is predicted for the H5/6 region of the mammalian
pumps, in contrast to the two segments predicted for the Neurospora H
-ATPase(2) .
Figure 1:
The hydropathy profile
and the primary sequence of the ER Ca-ATPase deduced
from its cDNA sequence. At the top of this figure, the Kyte
and Doolittle hydropathy profile is shown using a moving average of 11
amino acids. The 11 possible transmembrane domains are shaded.
The sequences used for insertion are underlined, with dotted lines for the H5 long sequence and solid lines for all the other fragments.
Numerous experimental methods have been used to determine the composition of the membrane domain of these pumps, such as protease cleavage, epitope mapping, mutagenesis, chemical labeling and in vitro translation scanning(8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18) . It appears that no single method provides unequivocal data.
Mapping
of the Na,K-ATPase with epitope insertion has been interpreted as
indicating eight transmembrane segments(19) . A mobile
COOH-terminal pair of transmembrane segments has been proposed that
become exposed on the outside surface upon heating, based on tryptic
access and phosphorylation at Ser-938 by protein A kinase(20) .
A similar heat-dependent translocation of the last extracytoplasmic
loop was detected during trypsinization of the
Na,K-ATPase(21) . Insertion of epitopes into the Na,K-ATPase
provided evidence for the first eight transmembrane segments but was
not used to determine the location of the last two hydrophobic
segments(22) . Epitope mapping of the SR
Ca-ATPase showed that the postulated extracytoplasmic
loop between H7 and H8 was indeed present in the interior of the SR
vesicles(13, 14) . Tryptic digestion of this ATPase
provided experimental evidence for the first eight transmembrane
segments that had been predicted, but an unexpected cleavage between H9
and H10 was observed(12) . Mutagenesis to generate
Ca
transport mutants provided evidence that H4, H5,
H6, and H8 were in the membrane
domain(15, 16, 17, 18) . The crystal
structure of the SR Ca
-ATPase at 14-Å
resolution shows that the membrane domain can accommodate 10
membrane-spanning segments(23) . Tryptic cleavage showed the
presence of eight transmembrane segments in the gastric
H,K-ATPase(24) . Extracytoplasmic labeling with covalent
reagents provided evidence for the same
segments(24, 25) . However, in vitro translation showed the presence of an additional pair of
transmembrane segments, to give an experimental consensus for 10
membrane segments in this enzyme (9) . In vitro translation of regions of the Neurospora enzyme showed
that the COOH-terminal domain contained six membrane insertion
sequences (8) although it was concluded that pairwise insertion
had to occur(26) . A similar protein, the
Mg
-ATPase of Salmonella typhimurium, was
analyzed by generating different fusion proteins containing cytoplasmic
(
-galactosidase) or extracytoplasmic (
-lactamase) markers at
the beginning or end of putative transmembrane segments. It was
concluded that this protein had 10 transmembrane segments(27) .
The particular Ca-ATPase cDNA used in this study
is from the ER(28) . This SERCA2 pump differs from the SERCA1
pump from the SR only by having 42 more amino acids on the COOH
terminus as a result of alternative RNA
splicing(3, 28) . This extended COOH-terminal region
contains an additional hydrophobic sequence, which is not present in
the SR Ca
-ATPase and which contains an N-linked glycosylation consensus sequence, NFS at position
1036(28) . If this last sequence is membrane-spanning, the
orientation of the carboxyl terminus of the ER pump with respect to the
membrane would be extracytoplasmic, in contrast to all other known
mammalian P type ATPases. Epitope mapping has suggested that this is
indeed the orientation of the COOH-terminal domain(29) . The
rest of the membrane domain of the ER Ca
-ATPase is
expected to be identical to that of the SR Ca
-ATPase
with the exception of the COOH-terminal region.
Assembly of this type of polytopic integral membrane protein containing an even number of transmembrane segments is thought to require the presence and sequential insertion of topogenic signals into the membrane of the endoplasmic reticulum, odd numbered segments acting as signal anchor sequences, even numbered segments acting as stop transfer sequences(30) . In vitro translation using fusion vectors containing these sequences can therefore be used to determine the presence or absence of membrane insertion sequences, and this method has been applied to the SERCA2 ATPase in this paper.
Figure 2: In vitro translation of the vectors MO and M1 with insertion of the cyclo-oxygenase hydrophobic sequence and a M1 control. At the left of the figure are the autoradiograms resulting from translation of the MO and M1 constructs containing a fragment from the cyclo-oxygenase (amino acids 290-317) in the absence(-) and presence (+) of microsomes. At the right of the figure are the two lanes (- and + microsomes) showing a typical translation of the M1 construct.
We investigated a hydrophobic sequence of the soluble part of the sheep cyclo-oxygenase(35) . This sequence (positions 290-317) has been shown by crystal analysis (36) to be buried inside the structure of the enzyme and is not membrane-spanning even though it has a hydrophobicity level similar to Ca H7. This sequence had no effect in the M0 vector but almost completely prevented the glycosylation of M1 (Fig. 2). Because in the native protein there is no sequence preceding this stretch of amino acid that can act as a signal anchor sequence, the stop transfer activity of this region of the protein as demonstrated in our constructs is not diagnostic of a membrane-spanning segment in the cyclo-oxygenase.
Figure 3: In vitro translation of the vectors MO and M1 with insertion of the Ca H1, Ca H2, and the putative membrane pair Ca H1-2. The figure shows the autoradiograms of the proteins resulting from translation of the different MO and M1 constructs (Ca H1, C H2, and Ca H1-2) in the absence(-) and presence (+) of microsomes followed by SDS-PAGE.
The H2 transmembrane domain of the ATPase
(positions 82-109), inserted into M1, prevents the glycosylation
of the region, acting as a strong stop transfer sequence. When
inserted into MO, it promotes the glycosylation of the
region.
This sequence appears less effective in promoting glycosylation than
the H1 sequence, which is the natural signal anchor sequence. Hence, it
is a better stop transfer than signal anchor sequence.
Translation
of the H1/H2 construct resulted in the absence of glycosylation,
predicted from a sequence containing a signal anchor and a stop
transfer segment pair. The in vitro translation properties of
these segments correspond to what is expected both from the hydropathy
plot and from biochemical analysis of the SR
Ca-ATPase.
Figure 4: In vitro translation of the vectors MO and M1 with insertion of the Ca H3, Ca H4, and the membrane pair Ca H3-4. The figure shows the autoradiograms of the translated proteins resulting from translation of the different MO and M1 constructs (Ca H3, Ca H4, and Ca H3-4) in the absence(-) and presence (+) of microsomes followed by SDS-PAGE.
Figure 5: In vitro translation of the vectors MO and M1 with insertion of the Ca H5, Ca H6, the membrane pair Ca H5-6, and the Ca H5 longer construct. The figure shows the autoradiograms of the proteins resulting from translation of the different MO and M1 constructs (Ca H5, Ca H6, Ca H5-6, and the Ca H5 extended sequence) in the absence(-) and presence (+) of microsomes followed by SDS-PAGE.
The isolation of a fragment containing H5-H6, beginning at residue
Leu-776 and probably ending at residue Lys-835, following trypsinolysis
of intact, cytoplasmic side out vesicles (12) showed that these
sequences contain a pair of membrane embedded segments in the intact SR
Ca-ATPase.
Figure 6: In vitro translation of the vectors MO and M1 with insertion of the Ca H7, Ca H8, and the membrane pair Ca H7-8. The figure shows the autoradiograms of the lanes resulting from translation of the different MO and M1 constructs (Ca H7, Ca H8, and Ca H7-8) in the absence(-) and presence (+) of microsomes followed by SDS-PAGE.
As also shown in Fig. 6, the putative transmembrane domain, H8 (positions 881-932 or 889-921) did not show signal anchor properties but inhibited most of the glycosylation of the M1 vector, showing that it has activity as a stop transfer sequence. These data suggest the presence of the H7 and H8 pair of segments. The translation of the H7/H8 construct in M0 yielded predominantly a product similar to that found with H7 alone, suggesting that H8 interacted weakly with its H7 partner in this in vitro translation method.
Figure 7: In vitro translation of the vectors MO and M1 with insertion of the Ca H9, Ca H10, the Ca H11, and the membrane pair Ca H9-10. The figure shows the autoradiograms of the lanes resulting from translation of the different MO and M1 constructs (Ca H9, Ca H10, Ca H11, and the Ca H9-10 pair) in the absence(-) and presence (+) of microsomes followed by SDS-PAGE.
The summary of all the above results is presented in Table 3.
The different approaches that have been taken to define the
membrane domain of the Ca-ATPases have not provided
full information on the number of transmembrane sequences. Most of the
SERCA pump putative transmembrane domains appear to be amenable to in vitro translation analysis, as were those of the H,K-ATPase
and the H. pylori P type ATPase(9, 31) .
The translation results for the first four transmembrane segments show that they are able to act as both signal anchor and stop transfer sequences. H2 and H4 act as signal anchor sequences as demonstrated by glycosylation in the MO fusion protein despite the fact that their membrane orientation in the fusion protein is opposite to their orientation in the assembled enzyme. The same effect can be seen with H1 and H3 inserted into the M1 vector. Thus, the orientation of the segment appears not to be important in determining membrane insertion in these particular constructs. The same result has been found with the H,K-ATPase(9) . These sequences were able to insert individually in contrast to the pairing apparently required by the membrane integration of H1 and H2 of Neurospora ATPase(26) .
No region in the predicted H5, H6, or H5
with H6 acted as a signal anchor sequence. However, H5 with part of H6
was able to act as a stop transfer sequence. In order to interpret the
appearance of only a stop transfer sequence, where a signal anchor and
stop transfer pair was expected, we tested a sequence from
cyclo-oxygenase containing a hydrophobic stretch of amino acids that is
known not to be membrane-inserted(35, 36) . It acted
as a stop transfer sequence in M1 but not as a signal anchor sequence
in M0. Therefore, the finding of a stop transfer sequence in the H5
region of the Ca-ATPase, in the absence of a
preceding signal anchor sequence, should not be interpreted as evidence
for co-translational membrane insertion of this sequence in the
assembly of the protein. The H5 and H6 region of the H,K-ATPase also
did not show insertion properties in this translation
system(9) . This region of the Na,K-ATPase also did not
membrane-insert stably during in vitro translation until
leucine was substituted for all of the prolines(37) . However,
this region of the Neurospora H
-ATPase did
provide two membrane insertion sequences (8) as predicted by
hydropathy(2) .
In the case of the H,K-ATPase, the
subunit has been shown to interact strongly with the H7/H8
region(38) . Co-translation of the
subunit might
influence assembly of the
subunit by forcing translocation of
H7/H8 and H5/H6 as a bundle following interaction of the two nascent
subunits. Since the SERCA pumps have no
subunit, this
assembly hypothesis is made less likely by the data presented above
where H5/6 do not display their expected membrane insertion properties.
The H5 and H6 segments of the mammalian P ATPases contain many
hydrophilic amino acids and are shorter than other predicted
transmembrane segments. Perhaps these segments have stronger
interaction with other regions of the protein than with the translocon
and are post-translationally inserted. Interaction of H5/H6 with
subsequent membrane-inserting sequences would then precede their
membrane insertion.
The H7 of the Ca-ATPase acted
both as a signal anchor sequence and also as a stop transfer sequence.
The H7 of the H,K-ATPase had no membrane insertion properties in these
vectors and is more hydrophilic than this region of the
Ca
-ATPases(9) . The H8 region of the gastric
H,K-ATPase acted only as a stop transfer sequence(9) . In the
case of the H8 of the Ca
-ATPase, it appears that it
is able to act as a stop transfer sequence when assembled in the M1
vector. Given both of these properties, this H7-H8 region of the
Ca
-ATPase fulfills the expectations for a pair of
membrane-inserted segments.
The ninth transmembrane segment, which
is as hydrophobic as the NH-terminal segments, acted as
both signal anchor and stop transfer sequence. H10, which follows this
sequence, acted only as a stop transfer sequence with its natural
partner and with gastric H1. This pair of transmembrane sequences
membrane-inserts as expected in the assembled ATPases deduced from the
different algorithms (Table 2). The finding of these two membrane
segments supports the 10-transmembrane segment model for the SR
Ca
-ATPase when combined with epitope localization,
mutagenesis, and trypsinolysis
data(3, 10, 11, 12, 13, 14, 15, 16, 17, 18) .
The hydrophobic sequence representing the 11th putative transmembrane domain of the SERCA2 ATPase acted as both a signal anchor sequence and a stop transfer sequence. The former property would place this as a transmembrane segment. Consistent with this, antibody epitope localization (28) has demonstrated that the COOH terminus is inside the ER. This unusual orientation for the COOH-terminal end of this ATPase suggests that the N-linked glycosylation sequence is utilized and is perhaps a retention signal for this pump in the ER, whereas the SR ATPase, without this COOH-terminal glycosylation, is able to exit from the ER(43) .
All signal anchor sequences were found to have stop transfer properties. However, some sequences of the enzyme acted only as stop transfer signals. It seems that sequences only able to stop transfer are less hydrophobic or shorter than signal anchor sequences. This in vitro translation method assumes sequential insertion of the transmembrane segments since the glycosylation signal is COOH-terminal. It does not assay the possibility that an even number of transmembrane segments of lesser hydrophobicity might insert only as a bundle rather than individually. However, absence of membrane insertion of known transmembrane segments during translation may indicate a different means of assembly of these segments, for example post-translational rather than co-translational.
From the above, 8 of the 10 putative helical transmembrane segments
of the SR Ca-ATPase and 9 of the 11 putative helical
transmembrane segments of the ER Ca
-ATPase displayed
properties indicating co-translational insertion. The exception of the
fifth and sixth transmembrane segments might be interpreted as
indicative of post-translational insertion, determined by the
succeeding sequence of the protein. In combination with trypsinolysis
as well as with mutagenesis and epitope mapping, the above data provide
experimental justification for the transmembrane region of the
Ca
-ATPase as originally proposed(3) .