(Received for publication, August 3, 1995; and in revised form, November 14, 1995)
From the
We studied the topogenic properties of five hydrophobic segments
(H5-H9) in the COOH-terminal third of Na,K-ATPase subunit
using in vitro insertion of fusion proteins into endoplasmic
reticulum membranes. These fusion proteins consisted of several
different lengths of truncated
subunit starting at Met
and a reporter protein, chloramphenicol acetyltransferase, that
was linked in frame after each hydrophobic segment. We found that
membrane insertion of the newly synthesized COOH-terminal third was
initiated by H5 and terminated by H9, indicating that here only H5 and
H9 have topogenic function. The other three, H6-H8, did not have
topogenic function in the native context and were translocated into the
endoplasmic reticulum lumen. These results were in striking contrast to
the previous models in which four or six hydrophobic segments were
proposed to cross the membrane. Furthermore, the findings suggest a
novel mechanism for achieving the final membrane topology of the
COOH-terminal third of the
subunit.
Na,K-ATPase consists of two non-covalently linked subunits: a
larger non-glycosylated subunit (about 100 kDa) and a
glycosylated
subunit (approximately 55 kDa) (Brotherus et
al., 1983), both of which are transmembrane proteins. The complete
amino acid sequence of the
subunit from different sources has
been cloned (see review by Mercer(1993)). Hydropathy analysis of the
sequence has shown nine hydrophobic segments (H1-H9). Four
segments (H1-H4) are in the NH
-terminal third and the
rest (H5-H9) are in the COOH-terminal third of the subunit.
However, H5 is often proposed to cross the membrane twice as H5 and H6,
so that the remaining segments are often named H7-H10.
The
four hydrophobic segments in the NH-terminal third have
been demonstrated to be transmembrane spans by immunochemical studies
(Kano et al., 1990; Arystarkhova et al., 1992; Ning et al., 1993; Mohraz et al., 1994; Yoon and Guidotti,
1994). The sidedness of the NH
terminus has been determined
to be cytoplasmic by immunochemical methods (Felsenfeld and Sweadner,
1988; Antolovic et al., 1991; Ning et al., 1993;
Canfield and Levenson, 1993; Yoon and Guidotti, 1994). The orientation
of these four hydrophobic segments in the lipid bilayer has been
determined by the in vitro ER (
)membrane insertion
experiments (Xie and Morimoto, 1995).
In contrast to the
NH-terminal third, the membrane topology of the
COOH-terminal third of the
subunit has been quite controversial.
Since this region is involved in several important functions of the
Na,K-ATPase such as cation binding and occlusion (Karlish et
al., 1990), ouabain binding, and conformational changes during the
E1 to E2 transitions that occur during the catalytic cycle (see review
by Vasilets and Schwartz(1993) and Lingrel and Kuntzweiler(1994)),
structural information on this particular region is crucial for
understanding the structure function relationship of the enzyme.
In
this paper, we describe studies on the transmembrane disposition of the
COOH-terminal third of the subunit using an experimental strategy
based on the topogenic property of hydrophobic segments. The
fundamental principle of this approach is that the transmembrane
disposition of a polytopic protein is achieved cotranslationally by the
action of a series of alternating insertion signals that initiate
translocation of downstream portions of the polypeptide, and of halt
transfer sequences that block the translocation of the downstream
sequences (Blobel, 1980; Sabatini et al., l982). Since the
hydrophobic segments in the COOH-terminal third occur after a long
cytoplasmic stretch, this principle implies that the first
transmembrane segment in this region must be an insertion signal that
has also anchoring function. Fusion proteins were made consisting of
several different lengths of truncated
subunit, linked in frame
at their COOH termini to a reporter protein, chloramphenicol
acetyltransferase (CAT), that has a consensus N-linked
glycosylation site (Gorman et al., 1982). Fusion proteins were
designed such that the topogenic properties of all the hydrophobic
segments could be examined individually and sequentially in their
native context. Occurrence of N-linked glycosylation and
sensitivity to protease digestion of the reporter protein were used as
markers for luminal and cytoplasmic disposition, respectively. The
results provide both structural (orientation with respect to the
membrane) and functional (an insertion signal, a halt transfer signal,
or an inactive element as signals) information on the hydrophobic
segment in the context tested. Furthermore, these findings shed light
on the membrane insertion mechanism of polytopic proteins.
The COOH-terminal side of the primer that contains BamHI site is given below.
These PCR products, which had been digested with PmlI and BamHI, were ligated to pSG5rGH, which had been digested with Eco47III and BamHI.
All plasmids encoding a fusion protein have been examined by DNA sequencing, the size of their primary translation products and immune reactivity to anti-CAT antibody in order to confirm the in frame ligation. These fusion proteins are shown schematically in Fig. 2.
Figure 2: Schematic drawing of the constructs used in the study. H, T7, SP6, and GH indicate hydrophobic segment, T7 RNA polymerase, SP6 RNA polymerase, and signal sequence of rat growth hormone, respectively.
For preparation of
the total membrane fraction, cells from four wells were used. After
transfection, followed by pulse-chasing as described above, cells were
washed three times with cold phosphate-buffered saline, and scraped in
0.3 ml/well of 10 mM Tris-HCl, pH 7.4, 10 mM KCl, 0.5
mM MgCl, 1 mM phenylmethylsulfonyl
fluoride, 1 mM dithiothreitol, and 2 µg/ml aprotinin and
immediately homogenized in a tight fitting Dounce homogenizer with 15
strokes. The homogenate was centrifuged at 40,000 rpm for 30 min at 4
°C in a Beckman Ti-50 rotor. The pellet was suspended in 200 µl
of 0.25 M sucrose, 10 mM Tris-HCl, pH 7.4, 3 mM Tetracaine, incubated at room temperature for 10 min, and then
subjected to various treatments.
In vitro transcription, in vitro translation, isolation of microsomes from the translation mixture, removal of globin from the translation reaction mixture, Endo H treatment, immunoprecipitation with anti-CAT antibody, protease digestion, and SDS-PAGE were carried out as described by Xie and Morimoto(1995).
Figure 1:
Amino acid sequence deduced from the
nucleotide sequence of rat brain Na,K-ATPase -1 cDNA (Hara et
al., 1987) and its hydropathy plot. Amino acids are numbered beginning at Gly
of the mature protein and preceded by
5 amino acid residues present in the primary translation product.
Hydrophobic segments H1-H9 are in boldface and are also
indicated by a dashed line. The site at which truncated
subunits are linked in frame with COOH-terminal reporter protein CAT at
cDNA level is indicated by an upward arrow. Hydropathy plot
was based on the Kyte and Doolittle algorithm(1982). Hydrophic regions
are above and hydrophilic regions are below the x axis. Underline and closed circle indicate respectively
core hydrophobic segments and the initiator methionine of the fusion
proteins C-5, C*-5, C*-6, C*-7, C*-8, and
C*-9.
To
examine whether the H5 in the truncated subunits initiates
translocation of the downstream peptides in living cells, we
constructed two eukaryotic expression vectors: (a) pMT2-N5,
which encoded a fusion protein(N-5) of NH-terminal 981
amino acid residues (containing five hydrophobic segments, H1, H2, H3,
H4, and H5 in the native context) and the CAT protein (219 amino acid
residues); and (b) pMT2-C5, which encoded a fusion protein
(C-5) consisting of a truncated sequence (89 amino acid residues
including H5) starting at Met
and CAT (see Fig. 2). These plasmids were expressed transiently in COS-1
cells.
As shown in Fig. 3, immunoprecipitation of the total
membrane fractions by anti-CAT antibody yielded a single band with a
molecular mass of about 110 kDa from pMT2-N5-transfected cells and two
bands with molecular masses of 40 and 37 kDa from pMT2-C5-transfected
cells. Upon treatment with Endo H, the 110-kDa protein band did not
shift, but the 40-kDa protein band distinctly shifted to 37 kDa. The
relative amount of the 40 kDa protein was about 50% of the total
immunoprecipitates. When the membrane fractions were digested with
proteases, followed by immunoprecipitation with anti-CAT antibody, two
bands at 33 and 30 kDa were obtained from both transfected cells. The
33-kDa band shifted to a band at 30 kDa by Endo H treatment, but no
shift was observed in the 30-kDa band by the same treatment. This
membrane-protected 30-kDa protein corresponded to the expected size of
the unglycosylated fusion protein C-5 (37 kDa) from which the
NH-terminal flanking region was cleaved. Since the
consensus N-linked glycosylation site was only one in these
fusion proteins and located within the CAT molecule downstream of H5,
the results clearly demonstrated that H5 initiated translocation of
both fusion proteins, and that the CAT protein was translocated across
the membrane into the lumen where it was glycosylated. Since the
membrane-protected 30-kDa protein was recovered by anti-CAT antibody as
a major product from both pMT2-N5- and pMT2-C5-transfected cells, the
37-kDa protein from pMT2-N5-transfected cells was apparently
translocated but failed N-linked glycosylation for unknown
reason. Therefore, the extent of glycosylation was low, but the
majority of the fusion proteins were considered to be translocated.
Figure 3:
Hydrophobic segment H5 initiates membrane
translocation of the newly synthesized COOH-terminal third of the
subunit. Top, total membrane fractions were prepared from
cells that had been transiently expressed with pMT2N5 or pMT2C5,
followed by metabolic labeling with
[
S]methionine as described under
``Experimental Procedures.'' Each membrane fraction was
divided into two aliquots. One aliquot was treated with proteases and
the other was used as a control. Both aliquots were subjected to
immunoprecipitation. Each immunoprecipitate was dissolved in 30 µl
of 1% SDS, 50 mM dithiothreitol, incubated in boiling water
for 3 min, and then mixed with an equal volume of 0.2 M citrate buffer, pH 5.5. Each aliquot was further divided into two.
One was treated with Endo H, and the other was the control. After
incubation at 37 °C overnight, samples were analyzed in SDS-PAGE as
described under ``Experimental Procedures.'' Bottom,
schematic drawing shows the orientation of the inserted chimera before (-P) and after (+P) protease digestion. Solid hearts, N, and C indicate glycosylated
forms, NH
terminus, and COOH terminus,
respectively.
Translation of the pC5-derived mRNA in the absence of microsomes and subsequent immunoprecipitation by anti-CAT antibody yielded: 1) a major protein with a molecular mass of about 36 kDa (Fig. 4, C-5, lane 1), which was the predicted size of the primary translation product (defined as the product that has not been modified co- and/or post-translationally, and generally referred to as the translation product obtained in the absence of microsomes) of the fusion protein C-5, and 2) a few minor proteins that could be polypeptides initiated by internal methionine residues, or degradation products. When the same mRNA was translated in the presence of microsomes, an additional protein with a molecular mass of about 39 kDa was obtained (Fig. 4, C-5, lane 2). This protein shifted its molecular weight down to about 36 kDa by Endo H treatment (lane 3). From the density of the band, the glycosylated form was estimated as about 20% of the total immunoprecipitate. This was much smaller than the glycosylated form obtained in the pN1-derived mRNA translation (about 80%) (Fig. 4, N-1, lanes 2 and 3).
Figure 4:
Mutation of 1 proline residue in the
hydrophobic segment H5 greatly increases its translocation ability in vitro. Left column, transcripts of pN-1, pC-5,
pC*-5, and pCB*5-6 were translated in the absence (lane
1) and presence (lanes 2-6) of microsomes. The
latter products were divided into two portions. One portion was used
for Endo H treatment (lanes 2 and 3), and the other
portion was further divided into three (lanes 4, 5,
and 6) and used for protease digestion. After treatment, the
samples were subjected to immunoprecipitation, followed by SDS-PAGE as
described under ``Experimental Procedures.'' Open circles indicate the position of glycosylated forms. Right
column, schematic drawing presents the orientation of the inserted
fusion proteins of lane 2. Solid hearts, N,
and C indicate glycosylated form, NH terminus, and
COOH terminus, respectively.
When
the translation product shown in lane 2 was digested with
proteases in the absence of Triton X-100, and membrane-protected
fragments were recovered by anti-CAT immunoprecipitation, three major
bands with molecular masses of 39, 36, and 30 kDa were observed. From
the size of the NH-terminal flanking region of H5 in the
fusion protein C-5 (53 amino acid residues) and the shift in molecular
mass by N-linked glycosylation (about 3 kDa), the 30-kDa
protein corresponded to the expected size of the unglycosylated form
from which the NH
-terminal flanking region was cleaved. The
36-kDa form was probably an undigested, glycosylated form which was
initiated by the internal Met
, although the size appeared
to be smaller than the size calculated from the position of the
internal methionine residue, or the undigested, nonglycosylated form.
The largest molecular mass, 39 kDa, corresponded to the expected size
of the undigested, glycosylated form. No fragments were recovered by
immunoprecipitation upon digestion in the presence of Triton X-100 (C-5, lane 6). However, the possibility that the
peptides in lane 5 were produced by incomplete digestion could
not be ruled out. That these proteins were inserted into microsomal
membranes was confirmed by their resistance to alkaline (pH 11.5)
extraction according to Fujiki et al.(1982), using membrane
insertion of the fusion protein N-1 and X-c as positive and negative
controls, respectively (data not shown here).
The
immunoprecipitates from the translation of the pC*5 transcript in the
presence of microsomes contained two major proteins with molecular
masses of 36 and 39 kDa at a mass ratio of about 1: 1 (Fig. 4, C*-5, lane 2). Endo H treatment resulted in the shift
of 39 kDa band to 36 kDa (lane 3), indicating that the
relative amount of the glycosylated form was about 50%. This value was
much higher than the value obtained in the pC5 translation.
Interestingly, the Endo H treatment also shifted the molecular mass of
the 36-kDa protein to 33 kDa. This shift was presumably due to
deglycosylation of a fusion protein that was initiated by an internal
Met. These results clearly demonstrated that the mutation of
Pro improved the translocation efficiency of H5. Thus,
the plasmids that encoded fusion proteins containing
``Pro
-mutated H5'' (referred to as H*5) were
used for testing the topogenic properties of the downstream hydrophobic
segments H6-H9.
Two proteins (39 and 42 kDa) were immunoprecipitated by anti-CAT antibody from translation of the pCB*5-6 transcript in the presence of microsomes (Fig. 4, B*5-6, lane 2). The 42-kDa protein was converted by Endo-H treatment to 39 kDa, which was the expected size of the primary translation product of the fusion protein (lane 3). From the comparison of the translocation efficiency between fusion proteins C*-5 and B*5-6, the H5-H6 loop was considered to be translocated into the ER lumen. Therefore, we concluded that there were no hydrophobic segments within the loop that functioned as a halt transfer signal. However, it cannot be ruled out completely the possibility that some regions in the loop may be inserted post-translationally to microsomal membranes together with H6, H7, and/or H8.
Figure 5:
Membrane insertion of newly synthesized
COOH-terminal third of the subunit that was initiated by
hydrophobic segment H5 was halted by hydrophobic segment H9. Left
column, transcripts of pC*-6, pC*7, pC*8, and pC*9 were translated
in the absence (lane 1) and presence (lanes
2-6) of microsomes. The latter products were divided into
two parts and analyzed as described in the legend to Fig. 4. Open triangles indicate the position of the glycosylated
forms. Right column, schematic drawing presents the
orientation of the inserted fusion proteins of lane 2. Solid hearts and diamond indicate glysocylated and
unglycosylated forms, respectively. N and C represent
NH
and COOH termini,
respectively.
As shown in Fig. 6, a single protein with the same molecular weight as the primary translation product (33 kDa) was obtained from the translation of pXc transcript in the presence of microsomes (X-c, lane 2). This protein was converted to a 30-kDa protein by Endo H treatment (X-c, lane 3). Protease treatment in the absence of Triton X-100 did not cause any change in the molecular mass of the protein, but the same treatment in the presence of detergent digested the protein (lanes 4 and 5). Since the signal sequence of rat growth hormone has 26 amino acid residues and the shift of molecular mass in SDS-PAGE due to one N-linked glycosylation was about 3 kDa, the translation product in the presence of microsomes would be almost the same size as the primary translation product if the translocation of a fusion protein initiated by the signal sequence was not halted by the downstream segment, and this product would be completely protected from digestion by exogenously added proteases. However, if the translocation was halted by the downstream segment, the translation product would be smaller by about 3 kDa than the primary translation product and digested by exogenously added proteases into fragments that could not immunoprecipitate with anti-CAT antibody. Therefore, the results showed that the translocation of the fusion protein X-c was initiated by the cleavable signal sequence and the following CAT protein was translocated into the ER lumen where it was glycosylated.
Figure 6:
Topogenic nature of the hydrophobic
segments observed in the native context are intrinsic. Top,
transcripts of the plasmids pX-c, pX-4, pX-5, pX-6, pX-7, pX-8, and
pX-9 were translated in the absence (lane 1) and presence (lanes 2-5) of microsomes. The products translated in
the presence of microsomes were divided into two portions. One portion
was used for Endo H treatment (lanes 2 and 3); the
other portion was used for protease digestion (lanes 4 and 5). After treatment, the samples were subjected to
immunoprecipitation, followed by SDS-PAGE as described under
``Experimental Procedures.'' Open circle (X-9)
indicates the position of the inserted fusion protein from which the
signal sequence was cleaved. Bottom, schematic drawing shows
the orientation of the inserted fusion proteins before (no mark) or
after (+P) protease digestion. Solid hearts and diamonds represent glycosylated and unglycosylated forms,
respectively. N, C, and GH represent
NH terminus, COOH termini, and signal sequence of rat
growth hormone, respectively.
The primary translation products of pX7, pX8, and pX9 transcripts showed a single protein with a molecular mass of 36 kDa (Fig. 6, lane 1). The immunoprecipitates from the pX5 and pX6 translation products showed two major proteins and one minor protein (lane 1). The largest ones corresponded to the expected size of the respective fusion proteins; the other two could have been the products that were initiated by internal methionines or degradation products. Translation of the pX5 and pX8 transcripts in the presence of microsomes yielded, respectively, the same size of the protein as obtained in the absence of microsomes (lane 2). Translation of the pX6 and pX7 transcripts yielded additional proteins (39 kDa in fusion proteins X-6 and X-7, 33 kDa in fusion protein X-7) and the translation of the pX9 transcript yielded a single protein with smaller molecular mass (lane 2). Endo H treatment resulted in the decrease by about 3 or 6 kDa of the molecular mass of each fusion protein, except the pX9 translation products, whose molecular mass was not changed (lane 3). Protease treatment did not cause any change in the molecular mass of any of the fusion protein except the smaller protein of the pX5 translation products (X-5, lanes 2 and 4) and the pX9 translation products (X-9, lane 4), both of which were digested into the fragments that did not immunoprecipitate by anti-CAT antibody. These data have confirmed the previous observation that among hydrophobic segments H6-H9, only H9 has a halt transfer signal function. It should be pointed out here that H5 functions as an insertion signal like H1, but, as opposed to H1, H5 has a weak halt transfer function as seen in Fig. 6(X-5, lanes 2 and 4).
Figure 7:
Hydrophobic segments H6, H7, and H8 that
were translocated in the ER lumen seem to remain there. COS-1 cells
were transfected with plasmids pX-c, pX-5, pX-6, pX-7, pX-8, and pX-9.
After metabolic labeling with [S]methionine,
followed by chasing, cell lysates were subjected to immunoprecipitation
with anti-CAT antibody and half of the immunoprecipitates was treated
with Endo H. At the same time the culture media were analyzed in the
same way to examine whether the translocated peptides may be secreted.
All the samples were analyzed in SDS-PAGE as described under
``Experimental Procedures.'' C and M represent cell lysate and medium,
respectively.
The immunoprecipitates from cells
expressing plasmids pX5, pX8, and pX9 showed a single protein with
molecular masses of 37, 36, and 36 kDa, respectively, and those from
cells expressed with pX6 and pX7 showed three proteins with molecular
masses of 33, 36, and 39 kDa (lane C). Endo
H treatment did cause a decrease by 3 or 6 kDa in the molecular mass of
each of the products except for the pX9 translation product, which
remained unchanged (lane C
). No proteins were
recovered from their culture media by the immunoprecipitation (lane
M
), even after a 5-h chase (data not shown).
These results have confirmed that all hydrophobic segments show the
same topogenic properties both in the native context and in the
different context. Interestingly, the fusion protein containing either
H5, H6, H7, or H8 was totally translocated into the ER lumen, but no
products were secreted into the culture medium, while the pXc products
were secreted like regular secretory proteins. The only difference
between the pXc translation product and pX5, -6, -7, and -8 translation
products was that the latter group of the fusion proteins contained a
hydrophobic segment (about 20 amino acid residues) at the ligation site
between the truncated growth hormone peptide and the CAT protein. Since
the glycosylated form was sensitive to Endo H treatment, the products
may remain in the ER lumen by unknown mechanisms.
Why H6, H7, and H8 did not function as a halt transfer signal can
not be explained by factors that are usually considered to be
determinants of topogenic properties of hydrophobic segments. For
example, the average hydrophobicity indices of the hydrophobic segments
calculated using the residue hydrophobicity index taken from the data
by Kyte and Doolittle(1982), were 2.19 (H1), 1.87 (H2), 2.42 (H3), 2.00
(H4), 2.07 (H5), 1.48 (H6), 1.69 (H7), 1.43 (H8), and 1.17 (H9). The
values for H6, H7, and H8, which had no topogenic function, were
significantly lower than those for H1, H2, H3, H4, and H5, which had
topogenic function. However, the values for H6, H7, and H8 were higher
than the value for H9, which had topogenic function. Thus, the
topogenic properties of hydrophobic segments could not be defined by
their average hydrophobic indices. Distribution of charged amino acids
in the NH- and COOH-terminal flanking regions of each
hydrophobic segment is also considered to be an important factor that
affects the topogenic properties of the segment (review by von
Heijne(1994)). However, as seen in Fig. 1, there were no
distinct differences in the distribution of charged amino acids of the
flanking regions among the segments that had topogenic function and
those who did not. These observations suggest that there must be other
factors that determine the topogenic properties of the hydrophobic
segments H6, H7 and H8. Similar examples have been reported with CHIP28
water channel protein (Skach et al., 1994), human
P-glycoprotein (Skach et al., 1993) and
H
,K
-ATPase (Bamberg and Sachs, 1994).
Another hydrophobic segment that displayed unexpected topogenic
properties was H5. This segment could initiate translocation of
COOH-terminal third of subunit, but the efficiency of
translocation was much lower than that of H1 (Fig. 4), and the
halt transfer function was much weaker than that of H1 ( Fig. 4and Fig. 6). When these two segments were compared,
their hydrophobicity indices were similar, but H5 contained 3 proline
residues while H1 did not contain any. Since proline is known as an
helix breaker, the unusual topogenic properties of H5 may be to
some extent due to the proline residues. This possibility was supported
by the present observation (Fig. 4) that mutation of Pro
to leucine resulted in a significant increase in the
translocation efficiency (Fig. 4). Homareda et
al.(1992, 1993) observed that increase in translocation efficiency
was proportional to the number of proline residues mutated.
When the
10 residues flanking the NH termini of H1 and H5 were
compared, H1 had 2 positively and no negatively charged amino acid
residues, but H5 had no positively and 1 negatively charged residue (Fig. 1). Since H5 initiates translocation of the COOH-terminal
third of the
subunit after a long cytoplasmic stretch, it is
reasonably considered to have the same topogenic function as H1 in
membrane insertion of its downstream peptide. Therefore, the positively
charged amino acid residues in the NH
-terminal flanking
region may play an important role in membrane insertion as indicated by
several investigators (review by von Heijne(1994)). However, the
NH
-terminal flanking region of H5 had only negatively
charged residue. Such a distinct difference from H1 might be at least
partially accounted for the unexpected topogenic properties of H5.
In
addition to the present data, five independent studies showed that the
loop between H6 and H7 was located in the extracellular side. These
studies are as follows. (a) Mohraz et al.(1994)
showed that the domain Trp-Phe
was
located on the extracellular side by immunogold method using site
specific antibody IIC9 and large membrane fragments rich in
Na,K-ATPase. (b) Ning et al.(1993) localized the
domain Asn
-Gln
on the extracellular
side by immunoelectron microscopy using an epitope-specific antibody. (c) Yoon and Guidotti(1994) demonstrated extracellular
location of Gln
. (d) Schultheis et
al.(1993) showed that Arg
was a portion of ouabain
binding site in the COOH-terminal third. (e) Lemas et
al.(1994) demonstrated that a stretch of 26 amino acid residues
(Asn
-Ala
), which included the
beginning of H7, were involved in the assembly with
subunit.
Contradictory results have been also reported on the sidedness of
the H7-H8 loop. Our data (Fig. 5) that showed the
extracellular localization of the loop agreed with the results by
Canfield and Levenson(1993). These authors found that using an epitope
insertion a peptide inserted at Gly was positive to the
peptide-specific antibody in the absence of detergent when the mutated
cDNA was expressed transiently in cells. However, Yoon and Guidotti
(1994) demonstrated that using the same ``epitope insertion''
the residues Val
and Phe
were located in
the cytoplasmic side. Furthermore, Fisone et al.(1994) found
that Ser
was phosphorylated by protein kinase A, which
indicates the cytoplasmic location of this amino acid residue.
Concerning the sidedness of the H8-H9 loop, our data indicated
that it was in the extracellular side, but Canfield and Levenson(1993)
showed by using ``epitope insertion'' method that Leu was located on the cytoplasmic side. The sidedness of the
COOH-terminal flanking region of H9 was shown to be in the cytosolic
side of the membrane by immunochemical method (Antolovic et
al., 1991), epitope insertion method (Canfield and Levenson,
1993), and the present topogenic analysis of the hydrophobic segments.
Most of contradictory results stem from studies on the localization of antigenic sites by immunofluorescence microscopy of intact and permeabilized cells, and studies on the protease digestion of Na,K-ATPase-rich vesicles in the presence or absence of detergent. Positive results obtained by these methods in the absence of detergent can be safely interpreted as extracellular location. However, those obtained only in the presence of detergent can not always be interpreted as intracellular location. For example, if an antigenic site on the extracellular side is folded in such a way that it is covered by other domains of the peptide, it will not react with antibodies in the absence of detergent. However, when cells are permeabilized, conformation of membrane spanning helices may induce unfolding of the peptide which covers the antigenic site. Even the antigenic sites that are partially embedded in the lipid bilayer from the surface but not exposed to the cytosolic side will become exposed.
The unique feature of this model is that the membrane
insertion of the subunit is achieved by combination of three
cycles of alternate initiation and termination of translocation to
establish transmembrane spans and co- or post-translocational
interaction of H6, H7, and H8 within the luminal surface of the ER to
establish partially embedded membrane spans (Fig. 8).
Figure 8:
A model for the membrane topology of the
Na,K-ATPase subunit. Hatched structures with numbers
1-9 represent hydrophobic segments H1-H9 (see Fig. 1). N and C indicate NH
and
COOH termini, respectively. Lengths of NH
- and
COOH-terminal flanking regions and those of the loops between two
adjacent hydrophobic segments are approximately proportional to the
number of the constituent amino acid
residues.
Recently Bamberg and Sachs(1994) reported a study of the topological
analysis of H,K
-ATPase
subunit
using a method similar to the one used in the present study to identify
transmembrane segments of the subunit. 10 hydrophobic segments
(M1-M10) were chosen from either biochemical analyses or
hydropathy plots, and their topogenic properties were examined. The
results showed that four segments M1-M4 in the
NH
-terminal portion were inserted into microsomal membranes
as in the case of four NH
-terminal hydrophobic segments
(H1-H4) of Na,K-ATPase
subunit. Unexpectedly, M5, M6, and
M7 did not have topogenic function like H6, H7, and H8 of Na,K-ATPase.
M8 and M10 had a halt transfer function but not an insertion signal
function, and M9 functioned as both an insertion and a halt transfer
signals. Based on these results, the authors have proposed a model that
H
,K
-ATPase is inserted
co-translationally (M1-M4 and M9-M10) by three cycles of
alternate initiation and termination of translocation and
post-translationally (M5-M8) by a different mechanism. This
indicates that both H
,K
-ATPase
subunit and Na,K-ATPase
subunit seem to be inserted into
microsomal membranes by similar mechanisms except that the insertion
signal of the third cycle of translocation is M9 for
H
K
-ATPase and H5 for Na,K-ATPase and
the sidedness of the post-translational insertion is cytosol for
H
,K
-ATPase and endoplasmic reticular
lumen for Na,K-ATPase. An interesting question is whether such
mechanisms of membrane insertion are common to P-type ATPase. To answer
to this question, topological studies of the other P-type ATPases using
the method similar to the one used by Bamberg and Sachs(1994) and us
will be required. In fact, there was a recent report on the membrane
topology of the Neurospora plasma membrane H
-ATPase by
Lin and Addison(1995). These authors described that the ATPase had 10
putative transmembrane segments M1-M10 and that fusion proteins
with various combinations of transmembrane segments were all integrated
co-translationally into microsomal membranes. However, as opposed to
their conclusion, the results were not supportive primarily because of
the lack of combination of transmembrane segments for constructing
fusion proteins. Interestingly, the results are rather suggestive of
the possibility that the enzyme may also be inserted into microsomal
membranes by the mechanisms similar to those of
H
,K
-ATPase and Na,K-ATPase.
What
membrane topology of the COOH-terminal third can be predicted from the
present studies and the primary structure of the subunit shown in Fig. 1? Hydrophobic segment H6 has 2 consecutive glycine
residues in the middle, and both flanking regions of the segment are
definitely located on the extracellular side (Fig. 4). Since
glycine is an
-helix breaker, the segment may be embedded in the
lipid bilayer from the lumen forming a hairpin structure in which the
two glycine residues make a turn. The hydrophobic segment H7 has 1 Asp
near the COOH terminus and H8 has 2 Glu residues in the middle of the
segment. The H7-H8 loop has 16 amino acid residues, of which 5
are positively charged and 2 are prolines, but no negatively charged
amino acid residues are present. The H8-H9 loop has 7 amino acid
residues, of which 2 are positively charged and 2 are proline residues,
but no negatively charged amino acid residues are present. Since H9 was
established as the last transmembrane segment whose COOH-terminal
flanking region was on the cytoplasmic side, the H8-H9 loop must
be on the luminal side. Therefore, if H7 and H8 are embedded in such a
way that the NH
terminus of H7 and COOH terminus of H8 face
the lumen, the H7-H8 loop, which is hydrophilic, would be folded
between H7 and H8, possibly by interaction between negatively charged
amino acids in the segments and positively charged amino acids in the
loop. The embedded H7 and H8 may form a complex with the other
hydrophobic segments in the COOH-terminal third as well as hydrophobic
segments in the NH
-terminal third.
Among hydrophobic
segments that have topogenic function, only H5 has 3 proline residues.
Because of this, the segment had weak halt signal function. As the
COOH-terminal third of subunit may be involved in occlusion and
cation transport (Karlish et al., 1990), and this portion pops
out when incubated at 50 °C (Arystarkhova et al., 1995),
it is reasonable to consider that the complex formed by H6, H7, and H8
is movable within the lipid bilayer and the nature of the hydrophobic
segment H5 may facilitate the movement of the complex. Thus, the unique
nature of H5 found in this study may contribute not only to the folding
process of the COOH-terminal third of the peptide during the enzyme
biogenesis but also to the movement of the folded peptide complex
within the membrane, coupling it to the gating activity of the enzyme.
Such complexity in the structure-function relationship of the
COOH-terminal third of the
subunit might have given rise, at
least partially, to the contradictory results on the sidedness of the
loops. Further studies on the role of the 3 proline residues in the
topogenic function of H5, the role of hydrophobic segments H5, H6, H7,
H8, and H9 in the folding process during enzyme biogenesis, and the
involvement of
subunit in these processes, which are currently in
progress, will provide crucial information on the conformational
changes of the folded peptide complex that accompanies the ion
transport function of the enzyme.