From the
Transmembrane disposition of the NH
Na,K-ATPase of animal cells is an integral plasma membrane
protein which plays an important role in maintaining the characteristic
ionic composition of cytoplasm and in generating electro-chemical
gradients necessary for normal cell function (see reviews by Jorgensen
and Andersen(1988) and Skou and Esmann(1992)). The enzyme consists of
two noncovalently linked subunits in an equimolar ratio (Brotherus
et al., 1983): a larger nonglycosylated
The complete amino acid sequence of the
Na,K-ATPase
In the present paper, we describe
the studies on the transmembrane disposition of the
NH
All cDNA constructs (pN-1, pN-2, pN-3, and pN-4) encoding
the fusion proteins of the truncated Na,K-ATPase
That all constructs encoding chimeras were
ligated in frame has been confirmed by DNA sequencing, size of the
primary translation products, and immunoreactivity of the primary
translation products to anti-CAT antibody.
To confirm if the hydrophobic segment H3
functions also as an anchor signal, the translation mixture performed
in the presence of microsomes was digested with trypsin and
chymotrypsin, and the digested sample was subjected to
immunoprecipitation with anti-CAT antibody. As predicted, only one
fragment (about 29 kDa) was recovered by the immunoprecipitation
(Fig. 3, lane 8), and no fragments were recovered upon
proteolysis in the presence of Triton X-100 (Fig. 3, lane
9). This verified that the 29-kDa band was the membrane-protected
fragment, and the results confirmed that the hydrophobic segment H3
functioned as a signal/anchor type II.
To ascertain that the CAT protein remained
untranslocated, the translation mixture of the pN-4 transcript, carried
out in the presence of microsomes, was digested with trypsin and
chymotrypsin, and the digested sample was subjected to
immunoprecipitation with anti-CAT antibody. No fragments were recovered
(Fig. 3, lane 11). Thus, protease digestion experiments
demonstrated that the CAT protein remained in the cytoplasmic side of
membranes and was digested by exogenously added proteases.
The primary translation product of the
control chimera X-c gave a single band with a molecular mass of about
33 kDa (X-c, lane 1). The product obtained in the presence of
microsomes also gave a single band with a molecular mass of about 33
kDa (X-c, lane 2); however, the molecular mass of this protein
was shifted to about 30 kDa by Endo H digestion (X-c, lane 3).
After digestion of the translation product with trypsin and
chymotrypsin, a 33-kDa protein was recovered by immunoprecipitation
(X-c, lane 4), but the translocated protein was digested into
undetectable fragments by immunoprecipitation when digested in the
presence of Triton X-100 (X-c, lane 5). These results show
that, as expected, the cleavable signal sequence of rat growth hormone
did very efficiently initiate translocation of the control chimera, and
almost all of the translocated proteins were glycosylated and fully
protected by membranes from proteolysis.
The primary translation
products of the plasmids pX-1, pX-2, pX-3, and pX-4 were about 37, 44,
37, and 47 kDa, respectively (Fig. 4A), and the
translation products carried out in the presence of microsomes were
about 34, 41, 34, and 44 kDa, respectively. These sizes were not
changed by Endo H treatment (Fig. 4A), suggesting that
all four chimeric proteins were inserted into membranes where their
signal sequences were cleaved and further translocation of their
downstream peptides was halted by the hydrophobic segments. Therefore
the CAT protein remained in the cytoplasmic side of the membranes. When
these translation products were digested with trypsin and chymotrypsin,
no fragments were recovered by immunoprecipitation with anti-CAT
antibody, which proved that the CAT protein remained in the cytosol and
was digested by exogenously added proteases (Fig. 4). Thus, the
data have clearly demonstrated that all four hydrophobic segments in
the NH
Homareda et al.(1989) used in vitro ER
membrane insertion of truncated
The number of transmembrane
domains in the NH
Although the first group of
chimeras showed the orientation of H2 and H4 ( Fig. 2and
Fig. 3
), the results were not conclusive, because the chimeras
lacked a marker for the luminal disposition of NH
The present
study has demonstrated that the experimental approaches employed here
are very effective in determining the topogenic nature of hydrophobic
segments in the native context of a polytopic membrane protein. Using
the same approaches, studies on the membrane topology of the
COOH-terminal third of Na,K-ATPase
We thank Dr. David D. Sabatini for encouragement and
support throughout this work. We also thank Dr. Y. Hara for the rat
brain Na,K-ATPase
-terminal third
of the Na,K-ATPase
subunit was studied using an experimental
approach that involved in vitro endoplasmic reticulum membrane
insertion of chimeras. These chimeras consisted of four truncated
amino-terminal segments of the
subunit linked at amino acid
residues 126, 179, 313, and 439 to chloramphenicol acetyltransferase
(CAT), a reporter protein, that contains a consensus sequence for
N-linked glycosylation. The fusion sites were located after
one of the four hydrophobic segments (H1-H4). The results showed
that the chimeras in which the
subunit was truncated at positions
126 and 313 were glycosylated, and the glycosylated peptides were
protected by membranes from proteolysis. However, the other two
chimeras were not glycosylated and the inserted peptides were digested
by protease into fragments which did not immunoprecipitate with
anti-CAT. These results clearly demonstrate that hydrophobic segments
H1 and H3 function as signal/anchor type II, and H2 and H4 function as
halt transfer signals. Furthermore, membrane insertion of the
NH
-terminal third of Na,K-ATPase
subunit is achieved
by a series of alternate signal/anchor type II and halt transfer
sequences.
subunit (about
100 kDa), and a glycosylated
subunit (about 55 kDa) (Glynn, 1985;
Jorgensen, 1982), both of which are transmembrane proteins (see review
by Ovchinnikov(1987)).
subunit has been deduced from the nucleotide sequence
of cDNA cloned from various sources (Shull et al., 1986;
Kawakami et al., 1985 and 1986; Noguchi et al., 1986;
Hara et al., 1987). The hydropathy analysis has shown that the
subunit has eight to ten hydrophobic segments. Four segments
(H1-H4) are present in the NH
-terminal third, and the
rest of four to six segments are present in the COOH-terminal third.
Orientation of the first four hydrophobic segments in the lipid bilayer
has been investigated by a variety of approaches, including
immunochemical methods (Farley et al., 1986; Ball and Loftice,
1987; Felsenfeld and Sweadner, 1988; Kano et al., 1990;
Arystarkhova et al., 1992; Mohraz et al., 1994),
proteolytic digestion (Ovchinnikov et al., 1987),
site-specific labeling (Jorgensen et al., 1982; Nicholas,
1984; Capasso et al., 1992; see also review by Mercer(1993)).
These results demonstrated that the NH
terminus is exposed
on the cytoplasmic side of the membrane and that the predicted four
hydrophobic segments appear to be transmembrane domain. Recently
Homareda et al.(1989) constructed truncated
subunits
containing two adjacent hydrophobic segments (H1 and H2, H3 and H4, or
H5 and H6) and examined if these truncated peptides were inserted into
dog pancreas microsomal membranes in
N-ethylmaleimide-sensitive fashion using a rabbit reticulocyte
lysate translation system. Their results have shown that at least two
insertion signals are present within the four hydrophobic segments in
the NH
-terminal third.
-terminal third of
subunit using an experimental
strategy based on the topogenic properties of the four hydrophobic
segments H1-H4. In this approach, a reporter protein
chloramphenicol acetyltransferase (CAT)
(
)
that
has a consensus N-linked glycosylation site is linked
sequentially in frame after each hydrophobic segment in four truncated
subunits of different lengths. CAT is chosen since it is a
cytosolic protein and does not contain either insertion or halt
transfer sequences. It can therefore be used as a faithful
COOH-terminal reporter protein (Lipp and Dobberstein, 1986, 1988). The
chimeras are designed such that topogenic properties of hydrophobic
segments can be examined sequentially in the native context.
Signal/anchor and halt transfer sequences are identified by
characterization of their COOH-terminal flanking sequence with respect
to the membrane. Two properties, occurrence of N-linked
glycosylation and membrane protection of the reporter protein from
proteolysis, are used as the marker for the luminal disposition. The
results, therefore, will provide information on both the orientation
(with respect to the membrane) and the function (an insertion signal, a
halt transfer signal or topogenically inactive element) of each
hydrophobic segment in the native context.
Materials
Restriction enzymes, SP6 RNA
polymerase, DNA polymerase I (Klenow enzyme), T4 DNA ligase, T4
polynucleotide kinase, RNasin, Taq DNA polymerase,
m7G(5`)ppp(5`)G, and calf intestinal alkaline phosphatase were obtained
from either Boehringer Mannheim or Life Technologies, Inc. Tetracaine,
phenylmethylsulfonyl fluoride, trypsin, chymotrypsin, and Trasylol
(aprotinin) were obtained from Sigma. The Geneclean kit was purchased
from BIO 101. Reticulocyte lysate and dog pancreas microsomal membranes
were obtained from Promega Corp. (Madison, WI).
[S]Methionine was obtained from DuPont NEN.
Rabbit antibody against CAT was obtained from 5 Prime
3 Prime
Inc. (Boulder, CO).
Plasmids and Construction of Mutants
Plasmids
pGEM-3Z and pCAT basic were obtained from Promega Corp., and pT7/T3 19R
was obtained from Life Technologies, Inc. Okayama-Berg vector
containing an entire coding region of rat brain Na,K-ATPase -1
subunit was supplied by Dr. Y. Hara (Tokyo Dental Medical School,
Tokyo).
subunit and CAT
as a reporter protein were derived from two plasmids: pF1 in which the
entire coding region of
-1 subunit was engineered into a plasmid
pT7/T3 19R, and pCAT, in which the entire coding region of CAT was
engineered into a plasmid pGEM-3Z. cDNA constructs pX-1, pX-2, pX-3,
and pX-4 were constructed from an in vivo and in vitro expression vector containing rat growth hormone cDNA (pSG5rGH) and
one of the pN-1, pN-2, pN-3, and pN-4.
pF1
The -1 subunit cDNA, engineered from
Okayama-Berg vector into pT7/T3 19R vector, contained about 200
nucleotides in 5` end-noncoding region. When the mRNA from the cDNA was
translated in a rabbit reticulocyte lysate system the translation
efficiency was very poor probably due to the long stretch of GC pairs
preceding the initiation codon. To improve the in vitro translation efficiency, a new 45-mer oligonucleotide,
AGCTTGATATCGATAACTGAATAGCTGCAGGACGATCGCCATGGG, was designed according
to Kozak's rule (Kozak, 1987) and engineered just before the
initiator codon of the
subunit by ligating the annealed
oligomers, the NcoI/SacI fragment from plasmid
Okayama-Berg
1 and the plasmid pT7/T3 19R cut with
HindIII and NcoI.
pCAT
The entire coding region of the CAT gene
obtained by cutting a plasmid pCAT basic with HindIII and
NlaIV was ligated into the plasmid pGEM-3Z cut with
HindIII and HincII.
pN-1
The 404-base pair fragment obtained by
cutting a plasmid pF I with PstI and RsaI was ligated
into the plasmid pCAT that had been cut with PstI and
HincII.
pN-2
A plasmid pF 1 was linearized by
NspV digestion, and its protruding ends were filled in with
Klenow enzyme. The linearized, blunt-ended pF 1 was then ligated to
8-mer SalI linkers, and the ligated sample was digested with
PstI and SalI. A 535-base pair
PstI/SalI fragment thus obtained was inserted into
the plasmid pCAT that had been cut with PstI and
SalI.
pN-3
A plasmid pF 1 was digested with
XhoI, and the protruding ends were filled in with Klenow
enzyme. After digestion with PstI, an approximately
0.97-kilobase pair fragment was isolated and inserted into the plasmid
pCAT that had been cut with PstI and HincII.
pN-4
A plasmid pF 1 was linearized with
AflII, and its protruding ends were filled in with Klenow
enzyme. After digestion with PstI, a fragment of about 1.35
kilobase pair was isolated and inserted into the plasmid pCAT that had
been cut with PstI and HincII.
pX-1, pX-2, pX-3, and pX-4
A fragment containing
one of four hydrophobic segments (H1, H2, H3, or H4) and CAT was made
by polymerase chain reaction using their corresponding linearized
plasmid pN-1, pN-2, pN-3, or pN-4 as the template and two
oligonucleotides described below. The polymerase chain reaction product
was digested with NaeI (pX-1 and pX-2) or PmlI (pX-3
and pX-4) and BamHI and then ligated to the plasmid pSG5rGH
that had been cut with Eco47III and BamHI. The
NH-terminal side oligonucleotides (oligonucleotides 1, 2,
3, and 4) and the COOH-terminal side oligonucleotide (oligonucleotide
C) are: oligonucleotide 1 for pX-1, ATTCCGGCAGCTGTT; oligonucleotide 2
for pX-2, GATGAGCTCTGCCGGCTGTACCTCGGGGTCGTGGC; oligonucleotide 3 for
pX-3, GATGAGCTCGCACGTGTCATCCACCTCATCACGGG; oligonucleotide 4 for pX-4,
TGATGAGCTCGCACGTGCTGTCATCTTCCTCATTGG; oligonucleotide C,
CCATGGCTCGAGCTTAAG.
Dog Pancreas Microsomes (Referred to as
``Microsomes'' in This Paper Unless
Specified)
Microsomes were prepared from canine pancreas as
described by Walter and Blobel(1983). Occasionally microsomal membranes
were obtained from Promega Corp.
In Vitro Transcription
The plasmids pN-1, pN-2,
pN-3, and pN-4 and pX-1, pX-2, pX-3, and pX-4 were cut with
SmaI and BamHI, respectively, and the linearized DNAs
were purified with a Geneclean kit. A 45 µl of transcription
reaction mixture contains 5 µg of linearized plasmid DNA, 80
mM HEPES-KOH (pH 7.5), 16 mM MgCl, 2
mM spermidine, 40 mM DTT, 1 unit/µl RNasin, 25
µg/ml bovine serum albumin, 0.5 µM cap analog
(m7G(5`)ppp(5`)G), and 50 units of SP6 RNA polymerase (for pN-1, pN-2,
pN-3, and pN-4) or T7 RNA polymerase (for pX-1, pX-2, pX-3, and pX-4).
After the mixture was incubated for 10 min at 40 °C, 5 µl of
nucleotide mixture (0.5 mM each of ATP, CTP, UTP, and GTP) was
added and incubated again at 40 °C for 1 h. Then, in order to
obtain the maximal amount of the transcripts, 50 units of RNA
polymerase were added, and the reaction mixture was incubated at 40
°C for an additional hour. The transcripts were then purified with
phenol/chloroform extraction, followed by ethanol precipitation, and
the pellets were dissolved in 50 µl of diethyl
pyrocarbonate-treated water.
In Vitro Translation
The translation reaction was
carried out as described in the Promega's technical manual.
Briefly, a 20-µl translation mixture contains 14 µl of
reticulocyte lysate, 1.6 µl of 10 mCi/ml
[S]methionine, 20 µM each of the
other 19 amino acids, 20 units of RNasin, and 68 mM KOAc
(endogenous amount of KOAc, 78 mM, contained in the lysate was
not included). The reaction mixture was incubated at 30 °C for 90
min in the presence or absence of microsomal membranes (5
A
units/ml of translation reaction).
Isolation of Microsomal Membranes from the Translation
Mixture
Immediately after the incubation was over, the
translation mixture was chilled on ice and diluted with high salt
buffer (500 mM KOAc, 3 mM Mg(OAc), 50
mM triethanolamine (pH 7.5), and 1 mM DTT) to 100
µl. The diluted sample was incubated on ice for an additional 10
min and overlaid onto a 100-µl cushion containing 0.5 M
sucrose, 500 mM KOAc, 50 mM triethanolamine, 3
mM Mg(OAc)
, and 1 mM DDT. The step
gradient was centrifuged at 60,000 rpm for 150 min in the 100.3 rotor
of the Beckman centrifuge TL-100. The top 125 µl was collected as
the supernatant fraction, the rest of the sucrose cushion was
discarded, and the pellet (microsomal fraction) was gently washed once
with high salt buffer.
Removal of Globin from the Translation Reaction
Mixture
The supernatant fractions (see the previous paragraph)
or the translation products synthesized in the absence of membranes
were mixed with 1.7 volume of saturated ammonium sulfate solution.
Trasylol was added to a final concentration of 2 µg/ml. The mixture
was incubated on ice for 30 min and centrifuged at 4 °C for 4 min.
The resultant pellets were suspended in a small volume of 5%
trichloroacetic acid and centrifuged at 4 °C for 4 min in an
Eppendorf centrifuge. The pellets were suspended in a small volume of
0.1 M Tris-HCl (pH 9.2), 2% SDS, and 50 mM DTT. The
suspension was incubated for 30 min at 55 °C and then analyzed in
12.5% SDS-PAGE.
Endoglycosidase H (Endo H) Treatment
The
microsomal membranes isolated from the translation mixture as described
earlier were dissolved in a 30 µl 1% SDS solution containing 50
mM DTT, incubated for 3 min in boiling water, followed by
addition of an equal volume of 0.2 M sodium citrate buffer (pH
5.5) containing 0.2 µg/ml Trasylol. The samples were then equally
divided into two parts: one part was supplemented with 3 µl of 1
mU/µl Endo H and to the other 3 µl of water was added. Both
samples were incubated at 37 °C overnight. After addition of 33
µl of the sample loading buffer (0.1 M Tris-HCl pH 9.2, 50
mM DTT and 10% glycerol) the samples were incubated in boiling
water for 3 min and analyzed in 12.5% SDS-PAGE.
Immunoprecipitation
Microsomal membranes isolated
from the translation mixture were suspended in 40 µl of dilution
buffer (2.5% Triton X-100, 190 mM NaCl, 6 mM EDTA, 50
mM Tris-HCl (pH 7.4)) containing 0.1 µg/ml Trasylol. 10
µl of 20% SDS solution was added, and the sample was incubated in
boiling water for 4 min. After the sample was chilled to about 4
°C, 4 volumes of cold dilution buffer and then 4 µl of
antiserum against CAT were added. The samples were processed as
described by Anderson and Blobel(1983). For immunoprecipitation of the
translation products synthesized in the absence of microsomal
membranes, immunoprecipitation was conducted after the endogenous
globins were removed by addition of saturated ammonium sulfate, as
described before.
Protease Digestion
Translation mixture was diluted
with 3 volumes of 50 mM Tris-HCl (pH 7.5), 100 mM
NaCl, and incubated in the presence of 3 mM tetracaine
hydrochloride at room temperature for 10 min to stabilize the
microsomal membranes. Freshly prepared stock solutions of trypsin and
chymotrypsin were added to a final concentration of 100 µg/ml each
in the presence or absence of 1% Triton X-100. Proteolytic digestion
was carried out at room temperature for 30 min and terminated by the
addition of Trasylol (at a final concentration of 2 µg/ml) and
phenylmethylsulfonyl fluoride (at a final concentration of 3
mM). The reaction mixture was then supplemented with KOAc to a
final concentration of 400 mM and overlaid onto 100 µl of
0.5 M sucrose/0.5 M KOAc cushion. The step gradient
was centrifuged at 60,000 rpm for 15 min in the 100.3 rotor of the
Beckman centrifuge TL-100. The membrane pellets were washed once with
50 µl of high salt buffer solution and analyzed in SDS-PAGE after
incubation in boiling water for 5 min in the presence of sample loading
buffer.
RESULTS
As shown in the hydropathy plot (Fig. 1), the
NH-terminal third of the Na,K-ATPase
subunit contains
four hydrophobic segments. They are located at residues
Leu
-Ile
(H1),
Leu
-Gln
(H2),
Phe
-Leu
(H3), and
Ala
-Ala
(H4). The topogenic nature of
these hydrophobic segments was examined using in vitro insertion into endoplasmic reticulum (ER) of chimeras that were
expressed by the cDNA constructs pN-1, pN-2, pN-3 and pN-4. These
constructs contained one or more hydrophobic segments sequentially in
their native context. As described in the Introduction, two criteria,
occurrence of N-linked glycosylation of the reporter protein
and membrane protection of the reporter protein from proteolysis, were
used as markers for the luminal location of the ligation site.
Figure 1:
Amino acid
sequence (NH-terminal portion) deduced from the nucleotide
sequence of rat brain Na,K-ATPase
-1 cDNA 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 (Hara et al., 1987). Hydrophobic segments
(H1-H4) are boldface and are also indicated by a
broken line. The sites at which truncated
subunits are
linked in frame with COOH-terminal reporter CAT protein at cDNA level
are indicated by an upward arrow. Hydropathy plot was based on
the Kyte and Doolittle scale. Hydrophobic regions are above the x axis and hydrophilic regions are below the
axis.
Hydrophobic Segment H1 Functions as a Signal/Anchor Type
II
When the mRNA transcribed from pN-1 was translated in a
rabbit reticulocyte lysate in the absence of microsomal membranes, the
immunoprecipitates with anti-CAT antibody contained a single band with
molecular mass of 39 kDa, which corresponds to the size of the primary
translation product of the fusion protein N-1 (Fig. 2, lane
1). Translation in the presence of microsomal membranes yielded
two major proteins with molecular masses of about 42 and 39 kDa
(Fig. 2, lane 2). This 39-kDa protein was slightly
larger than the primary translation product in lane 1, which
suggested that the 39-kDa protein was not the primary translation
product but the glycosylated fusion protein N-1 that was initiated from
an internal methionine, Met. This is supported by Endo H
digestion results discussed below. The 42-kDa protein comprised about
60% of the total immunoprecipitates. The immunoprecipitates also
contained a smaller protein (about 36 kDa) as a minor product. Since an
internal methionine residue is located at the 32nd amino acid from the
NH
terminus (see Fig. 1), the minor protein may be
the unglycosylated fusion protein N-1 that was initiated by the
internal methionine. When the immunoprecipitates shown in lane 2 were digested with Endo H, the two major proteins shifted
quantitatively from 42 and 39 kDa to 39 and 36 kDa, respectively
(lane 3). This 36-kDa protein migrated exactly at the same
speed as the minor protein in lane 2, strongly supporting that
the 39-kDa protein in lane 2 was the glycosylated fusion
protein N-2 initiated by Met
Therefore, the glycosylated
form comprised more than 80% of the newly synthesized fusion proteins.
This high efficiency of translocation of the fusion protein N-1
initiated by the hydrophobic segment H1 allowed us to examine the
topogenic properties of its downstream hydrophobic segments in an
in vitro translation/translocation system.
Figure 2:
In
vitro membrane insertion of chimeras N-1, N-2, N-3, and N-4.
Top, in vitro transcripts of pN-1, pN-2, pN-3, and
pN-4 were translated in a reticulocyte lysate in the absence (lanes
1, 4, 6, and 9) and presence (lanes 2, 3, 5, 7,
8, and 10) of microsomes. After translation, all samples
were subjected to immunoprecipitation with anti-CAT antibody, followed
by SDS-PAGE analysis as described under ``Experimental
Procedures.'' Mb and Endo H represent microsomes
and endoglycosidase H, respectively. Open and solid
circles indicate glycosylated and unglycosylated forms,
respectively. Bottom, schematic drawing shows the orientation
of the inserted chimeras, based on the above results. Solid hearts and diamonds indicate glycosylated forms and potential
N-linked glycosylation sites, respectively. N and
C represent NH and COOH termini,
respectively.
To examine
whether the hydrophobic segment H1 functions as an anchor signal, the
fusion protein N-1, which was synthesized in the presence of
microsomes, was digested with trypsin and chymotrypsin in the presence
or absence of Triton X-100. The membrane-protected fragments were
recovered by immunoprecipitation with anti-CAT antibody. In this
experiment, if H1 functioned as an anchor signal, 95
NH-terminal flanking residues of H1 would remain
untranslocated in the cytoplasmic side of the membrane and would be
cleaved by proteases in the absence of Triton X-100. Therefore, the
size of a peptide that is protected by membranes from proteolysis will
be smaller by about 10 kDa than the control fusion protein N-1. As
shown in Fig. 3, lane 2, one fragment whose molecular
mass was about 30 kDa was recovered by immunoprecipitation when
digested in the absence of Triton X-100. As no fragments were recovered
upon digesting in the presence of Triton X-100 (Fig. 3, lane
3), it is reasonable to conclude that the fragment recovered was a
membrane protected one. These results clearly demonstrated that the
hydrophobic segment H1 functioned as an insertion signal as well as an
anchor of the inserted signal sequence and also confirmed that the
NH
-terminal flanking region of the H1 is located in the
cytoplasmic side of the membrane.
Figure 3:
Sensitivity to proteolysis of the inserted
chimeras N-1, N-2, N-3, and N-4. Top, mRNAs derived from pN-1,
pN-2, pN-3, and pN-4 were translated in a reticulocyte lysate in the
presence of microsomes. One-third of the translation mixture was used
as a control (lanes 1, 4, 7, and 10), one-third was
digested with trypsin and chymotrypsin in the absence of Triton X-100
(lanes 2, 5, 8, and 11), and one-third was digested
with trypsin and chymotrypsin in the presence of Triton X-100
(lanes 3, 6, 9, and 12). All samples were subjected
to immunoprecipitation with anti-CAT antibody, followed by SDS-PAGE
analysis as described under ``Experimental Procedures.''
Mb, Trypsin, and Triton represent
microsomes, trypsin and chymotrypsin, and Triton X-100, respectively.
Open and solid circles indicate glycosylated and
unglycosylated forms, respectively. Bottom, schematic drawing
that shows the membrane orientation of the fragments protected by
microsomal membranes from proteolysis. Solid hearts indicate
glycosylated forms.
Hydrophobic Segment H2 Functions as a Halt Transfer
Signal
Upon translation of the plasmid pN-2 transcript in a
reticulocyte lysate without addition of microsomes, a major protein
with molecular mass of about 51 kDa was recovered by
immunoprecipitation with anti-CAT antibody (Fig. 2, lane
4). This size corresponds to that of the unglycosylated fusion
protein N-2. A minor band (about 48 kDa) was also recovered. This
protein was probably an unglycosylated fusion protein N-2 that was
initiated by the internal Met. The molecular mass
difference between the major and minor proteins was about 3 kDa, which
corresponds to the size of the peptide between Met
and Met
. When the pN-2 transcripts were translated
in the presence of microsomes, a 51-kDa protein was recovered as a
major product (Fig. 2, lane 5). This product had the
same apparent molecular mass as those obtained in the absence of
microsomes (compare the sizes of the major bands in lanes 4 and 5), which strongly indicated that the fusion protein
N-2 was not glycosylated. As predicted, the apparent molecular mass of
this band did not shift down by Endo H treatment (the data are not
shown here). Since the CAT protein was linked to an amino acid residue
in the COOH-terminal flanking region of H2, the result showed that the
translocation which was initiated by H1 was halted by H2, and
consequently the CAT protein remained in the cytoplasmic side of
membranes as illustrated in Fig. 2, bottom. These
results were further supported by proteolysis experiments.
Fig. 3
, lane 5, showed that no membrane-protected
fragments were recovered by immunoprecipitation upon digestion with
proteases.
The Third Hydrophobic Segment H3 Functions as a
Signal/Anchor Type II
The immunoprecipitates from the
translation of pN-3 transcript in the absence of microsomes contained a
major band with molecular mass of about 59 kDa (Fig. 2, lane
6), which is the size of an unglycosylated fusion protein N-3. A
minor band (about 56 kDa) might be an unglycosylated fusion protein
that was initiated by an internal methionine. Translation in the
presence of microsomes yielded an additional band with molecular mass
of about 62 kDa (Fig. 2, lane 7). The larger band,
however, shifted its molecular mass to 59 kDa by Endo H treatment
(Fig. 2, lane 8), which confirmed that the shift was due
to deglycosylation of the N-linked sugar moiety. Since
hydrophobic segment H3 was linked to an amino acid residue in the
COOH-terminal flanking region of H3, the results demonstrated that the
second round of translocation of the N-3 peptide was initiated by H3,
and the following CAT protein was translocated into the ER lumen where
it was glycosylated.
The Fourth Hydrophobic Segment H4 Functions as a Halt
Transfer Signal
Translation of the plasmid pN-4 transcript
carried out in a reticulocyte lysate without addition of microsomal
membranes produced a major band with molecular mass of about 70 kDa
(Fig. 2, lane 9), which corresponds to the
unglycosylated form of the fusion protein N-4. Translation performed in
the presence of microsomes produced also a 70-kDa protein
(Fig. 2, lane 10). This result indicated that the H4
halted the translocation of its downstream peptide containing CAT
protein, and therefore, the CAT protein remained in the cytoplasmic
side of the membranes.
Hydrophobic Segments H1 and H3 Are Also Capable of
Halting Translocation of Their Downstream Peptide
Fig. 2
and Fig. 3have shown that hydrophobic segments H2
and H4 function as halt transfer signal in the native context. In order
to examine whether the topogenic nature of H2 and H4 is intrinsic and
whether H1 and H3 are also capable of halting translocation, we chose a
rat growth hormone signal sequence and constructed plasmids pX-1, pX-2,
pX-3, and pX-4. These plasmids encoded fusion proteins consisting of
the NH-terminal 66 amino acid residues (including a
cleavable signal sequence) of rat growth hormone and one of the
hydrophobic segments (H1, H2, H3, and H4) linked at its COOH-terminal
flanking region to the CAT protein. As a control, plasmid pX-c was
constructed. This plasmid encoded a chimera consisting of
NH
-terminal 66 amino acid residues of rat growth hormone
and CAT protein only. In this experiment, the topogenic nature of each
hydrophobic segment was characterized by occurrence of
N-linked glycosylation of the reporter protein and complete
protection by membranes from proteolysis, as shown schematically in
Fig. 4B.
Figure 4:
A, topogenic properties of the four
hydrophobic segments examined individually (all function as halt
transfer signals). mRNAs derived from the plasmids pX-c, pX-1, pX-2,
pX-3, and pX-4 were translated in a reticulocyte lysate in the absence
(lane 1) or presence (lanes 2-5) of microsomes.
The latter translation mixtures were divided into four aliquots. The
first aliquot was used as a control (lane 2), the second was
treated with Endo H (lane 3), the third was digested with
trypsin and chymotrypsin in the absence of Triton X-100 (lane
4), and the last aliquot was digested with trypsin and
chymotrypsin in the presence of Triton X-100 (lane 5). After
treatment, all samples were subjected to immunoprecipitation with
anti-CAT antibody, followed by SDS-PAGE analysis as described under
``Experimental Procedures.'' Note: the results of the fifth
aliquot are shown only for the sample X-c. Mb represents
microsomes. B, schematic drawings show the orientation of the
chimeras inserted into microsomal membranes before and after protease
digestion (indicated by +P). Solid hearts and
diamonds indicate glycosylated forms and potential
N-linked glycosylation sites, respectively. Cleavage sites by
signal peptidase are indicated by arrows. sGH and
GH represent the signal sequence of rat growth hormone.
H, N, and C represent hydrophobic segment,
NH termini, and COOH termini,
respectively.
The transcripts of plasmids pX-c, pX-1,
pX-2, pX-3, and pX-4 were translated in a rabbit reticulocyte lysate in
the presence and absence of microsomes. An aliquot of the translation
mixture was immunoprecipitated with anti-CAT antibody, and the
resulting precipitates were digested with Endo H. Other aliquots were
digested with trypsin and chymotrypsin in the presence or absence of
Triton X-100, and membrane-protected fragments were recovered by
immunoprecipitation with anti-CAT antibody. The results are summarized
in Fig. 4A.
-terminal third of
subunit have halt transfer
signal function when they are in a different context and therefore
their nature seems to be intrinsic. In addition, these experiments
further confirmed that hydrophobic segments H2 and H4 are transmembrane
domains.
DISCUSSION
We report here the topogenesis of the NH-terminal
third of the
subunit examined by using in vitro ER
membrane insertion of two different groups of chimeras. The first group
of chimeras encoded fusion proteins consisting of different lengths of
truncated
subunit with one or more hydrophobic segments located
sequentially in the native context and CAT protein as a reporter
protein. The second group encoded fusion proteins consisting of
NH
-terminal 66 amino acid residues (including a cleavable
signal sequence) of rat growth hormone, one of the H1, H2, H3, or H4
sequences, and CAT protein. Our data demonstrated that the membrane
insertion of the NH
-terminal third of
subunit was
initiated by H1 which functioned as a signal/anchor type II and was
achieved by the downstream hydrophobic segments, which acted
alternately as halt transfer (H2), insertion (H3), and halt transfer
(H4) signals. The data also demonstrated that H1 and H3 were capable of
halting translocation of their downstream peptides and that halt
transfer function shown by all four hydrophobic segments seemed to be
intrinsic.
subunits that contain two
adjacent hydrophobic segments such as H1 and H2 or H3 and H4 to examine
which of the hydrophobic segments function as an insertion signal.
Since the truncated polypeptides that were inserted into microsomal
membranes in an N-ethylmaleimide-sensitive fashion were
resistant to alkali extraction, Homareda et al.(1989) have
suggested that at least two insertion signals were present in the
NH
-terminal third of the
subunit. The present data
confirm the existence of two insertion signals in the
NH
-terminal third and furthermore demonstrate that H1 and
H3 function as a signal/anchor type II.
-terminal third of
subunit
established here is in agreement with those reported previously
(Jorgensen et al., 1982; Nicholas, 1984; Farley et
al., 1986; Ovchinnikov et al., 1987; Capasso et
al., 1992). Orientation of each hydrophobic segment in the lipid
bilayer had been predicted as H1 (N
and C
),
H2 (N
and C
), H3 (N
and
C
), and H4 (N
and C
) based on
the sidedness of the NH
terminus (Farley et al.,
1986; Ovchinnikov et al., 1987; Ball and Loftice, 1987;
Felsenfeld and Sweadner, 1988), the sidedness of the H1-H2 loop
(Arystarkhova et al., 1992; Mohraz et al., 1994), the
sidedness of the H2-H3 loop (Farley et al., 1986), the
sidedness of the H3-H4 loop (Kano et al., 1990), the sidedness
of the long COOH-terminal flanking region of H4 (Jorgensen and Collins,
1986; Jorgensen and Andersen, 1988; Farley et al., 1986;
Ovchinnikov et al., 1987; see reviews by Vasilets and
Schwarz(1993); Lingrel and Kuntzweiler, 1994), and the number of
transmembrane domains predicted by hydropathy plot. The present data
have confirmed the orientation of H1 and H3 by the occurrence of
N-linked glycosylation of the reporter protein at the
COOH-terminal flanking region of the hydrophobic segments and
sensitivity to protease digestion of the NH
-terminal
flanking region of the segments.
-terminal
flanking region of H2 and H4. The conclusion was drawn from the data
together with the following circumstantial observations: (a)
the COOH-terminal flanking regions of H1 and H3 are located in the ER
lumen, (b) the loops between H1 and H2 and particularly
between H3 and H4 are too short to span the lipid bilayer, and
(c) both loops are highly charged. In order to obtain
conclusive results on the orientation, we constructed plasmids encoding
chimeras in which one of hydrophobic segments was engineered into a
passenger protein that had a cleavable signal sequence with high
translocation efficiency. This allowed us to directly examine if these
hydrophobic segments function as a halt transfer signal. In this
approach, cleavage of an insertion signal was used as a marker for the
luminal location of the NH
-terminal flanking region of each
hydrophobic segment, and digestion of the reporter protein by proteases
was used as a marker for the cytoplasmic location of the COOH-terminal
flanking region of the segment. As seen in Fig. 4, the control
chimera X-c was translocated very efficiently by the cleavable signal
sequence of rat growth hormone and almost all of the translocated
protein was glycosylated. The apparent molecular mass of the
glycosylated protein became smaller by about 3 kDa when digested with
Endo H (Fig. 4, X-c: lanes 1 and 3), indicating
occurrence of the signal sequence cleavage as expected. Under the same
translation/translocation conditions, the signal sequence of the
chimeras X-2 and X-4 were cleaved, but the inserted chimeras were not
glycosylated. In addition, they were digested by proteases into
fragments which did not immunoprecipitate with anti-CAT antibody (see
Fig. 4A). These experiments have conclusively confirmed
the orientation of H2 and H4. The data have also showed that H1 and H3
are not only a signal/anchor type II but also capable of halting
translocation, if employed, in a different context.
subunit are currently in
progress.
-1 subunit cDNA and J. Culkin and F. Forcino for
their excellent photographic work. We are very grateful to Dr. Manijeh
Mohraz for critical reading of the manuscript and helpful discussion.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.