From the Istituto Nazionale di Ricerca per gli
Alimenti e la Nutrizione, Via Ardeatina 546, 00178 Rome, Italy and the
§ Department of Cell Biology, New York University, School of
Medicine, New York, New York 10016
Received for publication, July 28, 2000, and in revised form, January 22, 2001
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Retinol transport and metabolism have been well
characterized in mammals; however, very little is known in fish. To
study the mechanism by which fish retinol-binding protein (RBP) is able to remain in plasma besides its small molecular size, we isolated RBP
cDNA from a carp liver cDNA library. Comparison of the deduced amino acid sequence with that of known vertebrate RBPs showed that carp
RBP has high homology to the other cloned vertebrate RBPs, but it lacks
the COOH-terminal tetrapeptide, RNL(S)L, which is most likely involved
in the interaction with transthyretin in mammalian RBPs. In addition,
the primary structure of carp RBP contains two consensus
N-linked glycosylation sites that represent a unique
feature. We have obtained experimental evidence, by in vitro and in vivo expression experiments, that both
sites are indeed glycosylated. We have also characterized the protein
as a complex type N-linked glycoprotein by lectin binding
assay, neuraminidase and endoglycosidase H and F digestion. Inhibition of glycosylation by tunicamycin treatment of transfected cells caused a
great reduction of RBP secretion. Since kidney filtration of anionic
proteins is less than half that of neutral protein of the same size,
this finding strongly suggests that the amount of carp RBP filtration
through kidney glomeruli may be reduced by a
glycosylation-dependent increase in the molecular size and negative charge of the protein. A second unique feature of carp RBP as
secretory protein is the presence of a nonconserved
NH2-terminal hydrophobic domain, which functions as an
insertion signal but is not cleaved cotranslationally and remains in
the secreted RBP.
Retinol binding protein
(RBP,1 molecular mass of
about 20 kDa) is the specific blood carrier of vitamin A (retinol) that
transports retinol from its storage site, the liver, to the various
vitamin A-dependent tissues, where it is internalized and
metabolized to its active form, retinoic acid. Retinoic acid is an
important transcription modulator involved in the regulation of
proliferation and differentiation of many cell types, as well as in
fetal morphogenesis (1).
RBP is synthesized mainly by hepatocytes and secreted when bound to
retinol (molar ratio of 1:1) and to a 55-kDa homotetrameric protein,
the transthyretin (TTR) (2-4). The association of RBP with TTR
stabilizes the complex and at the same time increases the size of the
RBP-retinol complex, thus lengthening the time of RBP-retinol
circulation in plasma, by preventing easy filtration of the relatively
small RBP molecule through the kidney (5).
RBP has been isolated and characterized from different species: human
(6), rat (7), rabbit (8), chicken (9), Xenopus (10), and
others (11, 12). In some species, the structure has been resolved by
x-ray diffraction (13, 14). These studies show that RBP is an extremely
conserved protein, sharing strong homology among the species studied,
particularly in the regions involved in retinol binding, interaction
with TTR and tertiary structure organization.
The structure and function of TTR have also been analyzed in detail
(14, 15). These studies revealed that in the circulating complex one
RBP molecule binds to a TTR tetramer and that the affinity of the
interaction is much higher when RBP is in the "holo" form, charged
with retinol. Furthermore, a putative interaction domain with TTR has
been identified in human RBP (15).
RBP was purified by column chromatography from rainbow trout plasma,
and its primary and three-dimensional structures were analyzed, as well
as its binding affinity for human TTR (16, 17). The amino acid sequence
and the tertiary structure are highly conserved, but the in
vitro affinity of trout RBP for human TTR appears to be much lower
than that of mammalian RBP. Moreover, no circulating RBP·TTR complex
was found in trout plasma by gel filtration techniques. A thyroid
hormone-binding protein displaying amino acid sequence homology with
higher vertebrate TTR was recently isolated from salmon serum (18).
Cloning of sea bream TTR provided experimental evidence that TTR is
expressed in fish (19), in contrast to previous findings obtained by
other investigators through radiolabeled thyroxine binding assays (20).
These observations indicate that, in fish, RBP transports retinol
without forming a complex with TTR, raising the question on how such a
small molecular mass RBP-retinol complex can be retained in plasma and
escape kidney filtration.
The elucidation of the mechanisms by which vitamin A is distributed to
target tissues in fish is extremely important to optimize aquaculture
conditions. In fact, different fish species grown in aquaculture
exhibit a high rate of skeletal malformations during embryonic
development, and this phenomenon might be related to an incorrect
supply of some nutrients in the diet. In particular, vitamin A or, more
exactly, its active metabolite retinoic acid plays an essential role in
modulating some step of antero-posterior axis formation during
embryogenesis by regulating Hox gene expression. Moreover,
since it regulates proliferation and differentiation of various cell
types, including chondrocytes, it is directly involved in skeleton
development (21-24).
As a first step in the study of the possible mechanisms by which
RBP-retinol is retained in fish plasma without forming a complex with
TTR, a cDNA clone for RBP was isolated from a carp liver cDNA
library and characterized using in vivo and in
vitro expression systems.
We present in this paper the experimental evidence that the protein has
two unique features; one is the presence of two N-linked sugar moieties, and the second is the presence of a nonconserved uncleaved NH2-terminal signal peptide. Such features may
play an important physiological role in the proper intracellular
transport of the protein and in decreasing its plasma clearance through the kidney.
Materials--
Monoclonal antibody against c-Myc epitope tag and
all chemicals unless specified otherwise were obtained from Sigma
Italia (Milan, Italy). Nick translation kit, nylon membranes, pGEX
glutathione S-transferase fusion vector, and protein
G-agarose were obtained from Amersham Pharmacia Biotech (Rainham,
United Kingdom). Plaque lifts (Nytran filters) and nitrocellulose
filters were from Schleicher & Schuell GmbH (Dassel, Germany). The carp
liver cDNA library (Uni-ZAPTM XR Vector) and ExAssist/SOL-R system
were from Stratagene (Heidelberg, Germany), and Dynabeads,
oligo(dT)25 were from Dynal (Oslo, Norway). SuperSignal
Substrate was from Pierce Europe B.V. (Oud Beijerland, The
Netherlands). Restriction enzymes and T3 RNA polymerase were from
Promega Corp. (Madison, WI). Endoglycosidase H (EC 3.2.1.96),
N-glycosidase F kit (EC 3.5.1.52), neuraminidase from
Vibrio cholerae (EC 3.2.1.18), and DIG glycan
differentiation kit, RNA inhibitor, dog pancreas microsomes, and
Fugene6 were obtained from Roche Diagnostic S.p.a. (Monza, Italia).
QIAQUICK DNA purification/extraction kit was from Qiagen GmbH (Hilden, Germany). [35S]Methionine-cysteine
(35S-Protein Labeling Mix, specific activity >1000
Ci/mmol) was from PerkinElmer Life Sciences (Les Ulis, France).
Culture media, fetal calf serum, antibiotics, glutamine, and amino
acids were from HyClone (Logan, UT). Plasmid pCMV/Myc/ER was obtained
from Invitrogen (Carlsbad, CA). RI-100 rainbow trout liver cells were
obtained from Deutsche Sammlung von Mikroorganismen und Zellkulturen
GmbH (Braunschweig, Germany). 10-20% acrylamide gradient
precast gels were purchased from Novex (Carlsbad, CA).
cDNA Library Screening--
1 × 105
plaques were screened with a human RBP cDNA, as probe, labeled with
[ Plasmid Construction--
Carp RBP cDNA was amplified by PCR
using two oligonucleotides that contained NheI upstream of
the initiator methionine codon and NotI just before the
termination codon. After digestion with NheI and
NotI, the cDNA was cloned into pClneo-Myc (modified from pClneo) to construct pClneo-carpRBPmyc. pEGFP-carp putative signal peptide, defined as "chimeric protein" or "carp signal green
fluorescent protein (GFP)," was constructed by ligating to pEGFP-N3
vector the carp RBP NH2-terminal region (26 amino acids)
amplified by PCR using two oligonucleotides containing NheI
upstream and SalI downstream. Plasmid pGEX-carpRBP was
constructed by ligating to pGEX vector the carp RBP sequence amplified
by PCR using two oligonucleotides containing BamHI upstream
and EcoRI downstream. The resulting fusion protein is formed
by glutathione S-transferase followed by carp RBP.
RNA Preparation and Northern Blot Analysis--
For Northern
blot analysis, total RNA was extracted as described by Chirgwin
et al. (26) from different carp tissues (liver, intestine,
brain, kidney, and female gonads). Poly(A)+ RNA was then
isolated from total RNA on Dynabeads, oligo(dT)25. Poly(A)+ RNA samples were resolved by electrophoresis in
agarose, 1.85 M formaldehyde gel and transferred onto nylon
membranes, which were then hybridized with 32P-labeled carp
RBP cDNA and with rat actin cDNA for normalization under
standard conditions (27).
Cell Culture, Transfection, and Metabolic Labeling--
Cos-1
cells were grown in Dulbecco's modified Eagle's medium (DMEM)
supplemented with 4 mM glutamine, 100 units/ml
penicillin-streptomycin, 1% nonessential amino acids, and 10% fetal
calf serum. R1-100 rainbow trout liver cells were grown in Medium 199 with 10% fetal calf serum.
Transfection was performed on cells at 80% confluence in 35-mm plates.
3 µl of Fugene6 was mixed with 97 µl of serum-free medium in an
Eppendorf tube, 1 µg of DNA was added, and the mixture was incubated
for 15 min at room temperature. The mixture was added to each plate in
2 ml of complete medium, and the plates were incubated at 37 °C for
24 h.
Cells were washed with phosphate-buffered saline (PBS), supplied with
methionine-cysteine-free DMEM and incubated at 37 °C for 30 min for
starvation of the two amino acids. After this period, 100 µC/ml
35S-protein labeling mix was added to fresh
methionine-cysteine-free DMEM (300 µl/plate) and the cells were
incubated for the times indicated in the figure legends.
Antibody Preparation and Immunoprecipitation--
Antibody
against purified trout RBP was raised in mice. This antibody has been
proven to recognize carp RBP by Western blotting but not by direct
immunoprecipitation. Antibody against carp RBP was therefore raised in
rabbit by injecting the fusion protein glutathione
S-transferase-carpRBP; the fusion protein, expressed in
Escherichia coli cells, was extracted from inclusion bodies (27) by boiling in sample buffer (50 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 100 µg/ml bromphenol blue, 10 mM
Labeled cells were harvested in 1 ml of cold radioimmunoprotein assay
buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl,
0.1% SDS, 1% sodium deoxycholate, 1% Triton X-100), and samples were
subjected to immunoprecipitation as previously described (3).
Immunoprecipitates were dissolved in the desired amount of sample
buffer (50 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 100 µg/ml bromphenol blue, 10 mM Western Blotting--
Proteins fractionated on SDS-PAGE as
described above were transferred on a nitrocellulose filter using 25 mM Tris, 192 mM glycine, pH 8.3, buffer
containing 20% methanol. The blot was stained with Ponceau S to check
the extent of transfer, destained with PBS, and immunostained as
described (3). Finally, the blot was incubated with SuperSignal
substrate (Pierce) and exposed to x-ray films for appropriate time.
In Vitro Transcription, Translation, and Treatment of the
Products with Endo H and Proteases--
Plasmid encoding carp RBP was
linearized and then purified using a QIAQUICK DNA
purification/extraction kit. Transcription and translation were carried
out as described in the Promega technical manual. Immediately after the
incubation, the translation mixture was chilled on ice, diluted to 100 µl with a buffer containing 0.25 M sucrose, 50 mM Hepes/KOH, pH 7.5, 140 mM KCl, and 3 mM MgCl2 and then overlaid onto a 200 µl
cushion containing 0.5 M sucrose, 50 mM
Hepes/KOH, pH 7.5, 500 mM KCl, and 5 mM
MgCl2. The step gradient was centrifuged at 65,000 rpm at
4 °C for 60 min in the 100.3 rotor of the Beckman centrifuge TL-100.
The pellet was resuspended in 20 µl of 0.25 M sucrose
buffer and divided in two samples; one was incubated at 37 °C with
endoglycosidase H (Endo H, 2.5 milliunits (specific activity 125 units/mg of enzyme protein)) overnight, and the other was used as
untreated control. In the protease protection experiment, the pellet
was treated as described previously (29).
In Vitro Deglycosylation of Serum RBP and Neuroaminidase
Treatment--
Two carp serum samples (2.5 µl each) were mixed with
2.5 µl of Dilution Buffer and boiled for 3 min in the presence of 1%
Treatment with neuraminidase from V. cholerae and
subsequent analysis were carried out as follows. The immunoprecipitates by anti-Myc/protein G from culture media of Cos-1 cells, which have
been transiently transfected with pClneo-carpRBPmyc and metabolically labeled for 6 h, were suspended in a small volume of 20 mM Hepes, pH 7.3, 1% Triton X-100. After incubating in
boiling water for 5 min, the suspension was centrifuged and the
supernatant was adjusted to 1 mg/ml bovine serum albumin, 150 mM NaCl, and 5 mM CaCl2. Each
sample received either 5 milliunits of enzyme or buffer alone as
control and was incubated at 37 °C for 60 min. Digested samples were
analyzed in 10-20% native acrylamide gel (pre-cast by Novex). In
parallel 2 µg of purified human transferrin was treated in the same
way and analyzed by the same methods as positive control.
Lectins Binding Assay--
Six carp serum samples (4 µl each)
were immunoprecipitated with anti-carp RBP antibody. The six
immunoprecipitates were individually suspended in gel loading buffer,
incubated in boiling water for 5 min, and centrifuged. The supernatant
and 2 µl of carp serum, which was mixed with gel loading buffer and
incubated in boiling water for 5 min, were fractionated on 13%
SDS-PAGE and transferred to nitrocellulose filter as described earlier.
The filter was then cut into seven strips and stained with lectins as
described in the legend to Fig. 4A.
Isolation of Carp RBP cDNA and Amino Acid Sequence Comparison
with Other Species--
The cDNA encoding carp RBP was cloned from
a liver cDNA library by hybridization, using a human RBP cDNA
as probe under low stringency conditions. Three positive candidate
clones were isolated. The largest, of about 800 base pairs, contained
an open reading frame corresponding to 213 amino acid residues. To
confirm that the cDNA encodes RBP, the deduced amino acid sequence
was compared with human (6), rat (7), rabbit (8) chicken (9)
Xenopus (10), and trout I and II (16, 17) RBPs. As it is
evident from Fig. 1, the primary
structure of carp RBP has high homology with all six species. All
cysteine residues that are involved in disulfide bond formation
(indicated by closed rectangles), as well as the
amino acid residues that are predicted to form the "retinol pocket"
(indicated by underlines) (17), are evolutionarily conserved. As expected from such high degree of conservation, the Expression of RBP mRNA in Carp Tissues--
To examine whether
carp RBP is expressed in specific tissues like mammalian RBP,
poly(A)+ RNA was purified from total RNA prepared from
adult carp liver, kidney, intestine, brain, and female gonads and was
subjected to Northern blot analysis using carp RBP cDNA as a probe
and actin cDNA for normalizing the amount of mRNA. The results
(Fig. 2) show that, like in mammals, carp
RBP is highly expressed in liver (lane 3),
whereas, unlike in mammals, a significant amount of mRNA is also
expressed in intestine. RBP transcription is barely detectable in
kidney and brain and absent in female gonads.
Two Consensus N-Linked Glycosylation Sites Are Cotranslationally
Glycosylated in Vitro--
As seen in Fig. 1, carp RBP contains two
consensus N-linked glycosylation sites in the
NH2-terminal region. Since none of the vertebrate RBPs
sequenced to date have N-linked glycosylation sites, we
examined in vitro glycosylation of the carp protein. The
cDNA was transcribed in vitro, and the transcript was
translated in a rabbit reticulocyte lysate cell-free system. SDS-PAGE
analysis of the primary translation product shows migration at a
position corresponding to about 24 kDa (Fig.
3A, lane 1,
indicated by an asterisk). When the transcript was
translated in the presence of dog pancreas microsomes, two additional
bands were detected at 27 and 30 kDa (lane 2),
corresponding to the expected molecular mass of single and double
glycosylated products, respectively. However, when microsomes were
added to the translation mixture 90 min following initiation of
translation, only one product with a molecular mass of 24 kDa was
obtained (data not shown). As the band with the smallest molecular mass
migrated at the same rate as the primary translation product in
lane 1, it may be either untranslocated or
translocated but unglycosylated RBP. To discern between the two
possibilities, we performed a digestion with exogenously added
proteases.
When the products translated in the presence of microsomes were
digested with proteases in the absence of detergent, all three bands
were protected (lane 4), but none of the band was
protected when digested in the presence of detergent (lane
5). This result indicates that all three forms of the
protein are cotranslationally translocated into the endoplasmic
reticulum (ER) lumen, where they are protected from digestion by
exogenously added proteases. This result suggests the interesting
possibility that the NH2-terminal hydrophobic domain
(predicted signal peptide) of carp RBP (see Fig. 1) may not be cleaved
during cotranslational translocation because the fastest migrating band
in lane 4 (indicated by an asterisk)
has the same mobility as the primary translation product indicated by
an asterisk in lane 1.
To confirm that the two high molecular weight bands represent
N-linked glycosylated forms of carp RBP, the products
translated in the presence of microsomes were digested with Endo H. This enzyme specifically removes, by cutting between the two
N-acetylglucosamine residues, the "core" carbohydrates
added to N-linked glycoprotein in the ER, but does not
remove the carbohydrates of complex and hybrid type that are further
modified in the Golgi apparatus. As shown in Fig. 3B
(lanes 1 and 2), following digestion
the two bands with higher molecular mass shifted to the position of the smallest molecular mass (24 kDa, deglycosylated RBP, indicated by an
asterisk). These results clearly demonstrate that carp RBP is cotranslationally glycosylated. It is apparent from comparison between panels A (lanes 3 and 4) and B (lanes 1 and
2) of Fig. 3 that the protease-resistant, fastest migrating
band (indicated by an asterisk in Fig. 3A,
lane 4) migrated at the same rate as the
deglycosylated RBP (indicated by an asterisk in Fig.
3B, lane 2). Since the fastest
migrating band in Fig. 3A (lane 4)
comigrated with the primary translation product (indicated by
asterisks in Fig. 3A, lane
1), the deglycosylated form shown in Fig. 3B also appears to have the same molecular mass as the primary translation product. In other words, newly in vitro synthesized carp RBP
was cotranslationally translocated in the ER lumen where it was
glycosylated, but the NH2-terminal hydrophobic domain was
not cotranslationally cleaved.
NH2-terminal Hydrophobic Domain Is Not Cleaved during
Its Synthesis in Carp Liver--
To examine whether the
NH2-terminal hydrophobic domain of carp RBP is cleaved
during translocation in vivo, total proteins from carp
plasma were digested with the deglycosylating enzyme Endo F (Fig.
3B), which removes the core carbohydrate of all types of
N-linked glycoprotein by cutting between asparagine residue and N-acetylglucosamine. The size of the Endo F-digested RBP
was then compared with that of the deglycosylated, microsomal RBP synthesized in vitro (Fig. 3B, lane
2). Western blot analysis of the circulating form of carp
RBP was performed using an anti-trout RBP antibody. The results show
that undigested carp plasma RBP migrates as a single band of about 32 kDa. However, upon Endo F digestion, the protein appears as a single
band that migrates at the same rate as the deglycosylated, microsomal
RBP. These data confirm that the NH2-terminal hydrophobic
domain of carp RBP is not cleaved cotranslationally, but remains in the
secreted RBP.
It should be pointed out here that the molecular mass of the in
vivo synthesized carp RBP is slightly larger than the microsomal form translated in vitro. This can be attributed to
post-translational oligosaccharide processing that is carried out in
the Golgi apparatus. To examine this possibility, additional analysis
was carried out to characterize the terminal carbohydrate of serum carp
RBP using the "DIG glycan differentiation kit," as described in the
legend to Fig. 4. This kit contains five
different lectins: Galanthus nivalis agglutinin
(specific to terminal mannose), Sambucus nigra agglutin (specific to Neu5Ac NH2-terminal Hydrophobic Domain Translocates the
Cytosolic Protein GFP to the ER--
The presence of an uncleavable
signal peptide is quite unusual in secretory proteins. Comparison of
the hydrophobicity profile (30) of carp RBP with that of the two
examples found in the literature, i.e. ovalbumin (31, 32)
and plasminogen activator inhibitor (PAI-2) (33), shows that the two
proteins contain an internal hydrophobic domain that functions as
uncleaved signal peptide, while in carp RBP the strongest hydrophobic
domain is present in the NH2-terminal region, which
consists of 17 amino acid residues (Fig. 1), and displays the typical
features of an insertion signal with respect to amino acid composition
and its arrangement, which is very similar to that of mammalian RBPs.
To test whether the NH2-terminal hydrophobic domain of carp
RBP functions as an insertion signal, we constructed a plasmid encoding
a chimeric protein the NH2-terminal 26 amino acids,
including the first N-linked glycosylation site of carp RBP,
in frame with the cytosolic protein GFP, and this construct was
transiently expressed in fish tissue culture cells (RI-100), as well as
in mammalian Cos-1 cells. A control protein containing a signal
sequence and a KDEL sequence in-frame with GFP (pCMV/Myc/ER: termed
"ER-GFP") was used as ER-specific marker. The fluorescence
micrographs in Fig. 5 show that the
chimeric protein localized in the perinuclear Golgi region and in the
ER network spreading to the plasma membrane, both in fish (Fig.
5a) and in mammalian cells (Fig. 5d), while GFP
alone remained in the cytosol (Fig. 5c). The chimeric
protein was also detected in small cytosolic granules, some of which
located very close to the plasma membrane, probably representing
secretory granules. When cells expressing the chimeric protein were
treated for 2 h with brefeldin A, which reversibly disassembles
the Golgi apparatus, a redistribution of the chimeric protein in the ER was detected (Fig. 5e), with a fluorescence pattern similar
to that of the ER marker protein (Fig. 5, b and
f). These results show that the NH2-terminal
hydrophobic domain of carp RBP is able to translocate a cytosolic
protein into the ER, functioning therefore as an insertion signal.
To confirm that the chimeric protein is secreted without cleavage of
the NH2-terminal hydrophobic domain, cell lysates and culture media of Cos-1 cells expressing RBP-GFP (unglycosylated form,
30 kDa; glycosylated form, 33 kDa), GFP (26 kDa), and ER-GFP (29 kDa)
were analyzed by Western blotting using an anti-GFP antibody, and the
size of the products was compared by migration in SDS-PAGE. The results
(Fig. 6) demonstrate that only the
glycosylated chimeric protein is secreted (33 kDa, indicated by a
closed arrowhead in lane
5), thus confirming that the NH2-terminal
hydrophobic domain definitely functions as an insertion signal.
Furthermore, the results clearly demonstrate that the
NH2-terminal hydrophobic domain remained uncleaved in the
secreted chimeric protein, because the molecular masses of the slower
migrating band in lane 2 (indicated by an
open arrowhead) and that of the secreted protein (indicated by a closed arrowhead in lane
5) correspond to the unglycosylated (30 kDa) and
glycosylated (33 kDa) chimera, respectively. The high amount of
cytosolic GFP found in cells expressing the chimeric protein (thick,
faster migrating band in lane 2) can be explained by a much more efficient translation from the internal GFP initiation codon. Interestingly, unglycosylated chimeric protein seemed to be
retained in cells (lane 2, slowly migrating band
indicated by an open arrowhead), suggesting
that N-linked glycosylation may be important for
intracellular transport of carp RBP.
Secretion of Carp RBP Is Dependent on N-Linked Glycosylation in
Transiently Transfected Cos-1 Cells--
To examine this hypothesis,
Myc-tagged construct of carp RBP was transiently expressed in Cos-1
cells and metabolically labeled for 4 h in the presence and
absence of the core glycosylation inhibitor tunicamycin. The amount of
RBP secreted in the two experimental conditions was then compared. As
negative control, untransfected Cos-1 cells were treated in the same
way (lanes 1 and 2); as positive control, human RBP was transiently expressed in Cos-1 cells. The results in Fig. 7 show that, in the
absence of tunicamycin, carp RBP was secreted with almost the same
efficiency as human RBP expressed in Cos-1 cells (compare thick bands
in lanes 3 and 4 with those in
lanes 9 and 10). However, in the
presence of tunicamycin, carp RBP was secreted with much less
efficiency (compare the thick band indicated by
an asterisk in lane 7 and the
corresponding faint band in lane 8), suggesting
that N-linked glycosylation is important for intracellular
transport at the proper speed. As predicted, in brefeldin A-treated
cells, the glycosylated protein was immunoprecipitated only from cell
lysates (lanes 5 and 6), because brefeldin A
blocks transport of newly synthesized proteins from the ER to the Golgi
apparatus. Two thick bands can be recognized in lane
3. The faster migrating band migrated at the same rate as
the thick band in lane 5 (both are indicated by
open circles), and the slower migrating band,
indicated by a closed circle, migrated faster
than secreted RBP in lane 4. Such migration
difference of newly synthesized RBPs may reflect the different extents
of glycosylation.
Mammalian RBPs circulate in plasma bound to the transthyretin
tetramer. Since the molecular mass of the complex is about 80 kDa,
formation of this complex is able to decrease the extent of glomerular
filtration in the kidney (5), whose size limitation is about 50 kDa.
However, no RBP·TTR complex was found in trout plasma (16, 17), thus
leaving an open question as to the mechanism of plasma retention. To
examine whether this is a common feature in fish RBP, we have isolated
a candidate cDNA clone for RBP from a carp liver cDNA library
and characterized the corresponding protein. The amino acid sequence of
carp RBP shows strong homology with that of the other known vertebrate
RBPs. All structural and functional domains are highly conserved, with
exception of the putative signal peptide sequence and of the
COOH-terminal tetrapeptide RNL(S)L, that is predicted to stabilize the
RBP·TTR complex (15).
In addition, we have found two unique sequence features within the
amino-terminal region of carp RBP: the presence of two consensus
N-linked glycosylation sites and of an uncleaved signal peptide.
The results of in vivo and in vitro
expression experiments have demonstrated that, unlike all other
vertebrate RBPs, carp RBP is indeed glycosylated at both sites (Figs.
3, 4, and 7). Lectin binding assays (Fig. 4A), various
glycosidase digestions (Figs. 3 and 4B) and synthesis of RBP
in the presence of tunicamycin (Fig. 7) reveal that carp RBP is a
complex type N-linked glycoprotein. The physiological
significance of N-linked glycosylation of carp RBP might be
related to a different mechanism for plasma retention in this species.
Studies on the effect of electrical charge on the fractional clearance
of dextran molecules of various sizes in rats have shown that the
negative charges in the glomerular membrane retard the passage of
negatively charged molecules and facilitate the passage of positively
charged molecules. As one example, filtration of anionic substances 4 nm in diameter is less than half that of neutral substances of the same
size (34, 35). Since two N-linked glycosylations increase
molecular mass as well as negative charge due to sialylation (as shown
in Fig. 4), glomerular filtration of carp RBP is likely to be greatly reduced even without forming a complex with TTR. This possibility will
be further examined by measuring the clearance of unglycosylated and
glycosylated RBP after intravenous injection.
Recently we have found that sea bream RBP is also a N-linked
glycoprotein.2 It is known
that both carp and sea bream have evolved in a different direction than
trout, whose RBP is not a glycoprotein, and that the structure of carp
kidney is quite different from that of trout. The fact that two out of
three fish RBPs examined up to date are N-linked
glycoproteins strongly suggests that this might be a common feature in
fish RBP, and, more importantly, that N-linked glycosylation
may play an important role in fish RBP function.
The results of the present study (Fig. 7) show that N-linked
glycosylation may directly be involved in intracellular transport of
carp RBP. We have shown that secretion of carp RBP is greatly reduced
by glycosylation inhibitors (compare the amount of secreted RBP in
lane 4 with that in lane
6), and four possible explanations can be provided for these
results. First, unglycosylated RBP could be captured by chaperone(s) as
a malformed molecule and retained in the ER lumen. Second, the
core sugar of RBP may function as a signal for the exit from the ER
to the Golgi compartment as is the case for secretory glycoproteins
(36), and the unglycosylated form would be retained in the ER lumen.
Third, the core sugar of RBP might play an important role in retinol
binding and therefore in the conversion of newly synthesized apo-RBP to
the holo form. If this were the case, newly synthesized RBP
would be held onto the ER membrane through the interaction between the
Glc1Man9GlcNAc2 of RBP and the
lectin site of calnexin-like lectin (37, 38) until apo-RBP binds
retinol to become holo-RBP. This conversion would be the critical step
for the exit of RBP from the ER (39-41) and will be further
examined by testing whether unglycosylated RBP is present as apo or
holo form. Fourth, N-linked glycosylation may be essential
for translocating the signal peptide from the lipid bilayer of the ER
membrane into the ER lumen. Therefore, if not glycosylated, newly
synthesized RBP may hang in the lumen through the signal peptide,
anchored in the lipid bilayer, thus remaining in the ER. If this were
the case, newly synthesized unglycosylated RBP would be resistant to
exogenously added proteases (Fig. 3A, lanes
3-5) and also resistant to alkaline extraction. In
conclusion, the present observations suggest that glycosylation of carp
RBP may be important both for intracellular transport along the
secretory pathway and for decreasing RBP clearance from plasma.
Another unique feature of carp RBP is that the NH2-terminal
hydrophobic domain (residues 1-17) functions as an insertion signal, but is not cleaved cotranslationally and remains in secreted RBP. The
role of the NH2-terminal hydrophobic domain as an insertion signal has been shown in Figs. 5 and 6 using a chimeric protein consisting of GFP and the NH2-terminal 26 amino acid
residues of RBP, including the NH2-terminal hydrophobic
domain and the first N-linked glycosylation site. When this
chimeric protein was transiently expressed in Cos-1 cells, it was
properly secreted while a control GFP remained in the cytosol. Since a
difference of 2 kDa between two proteins with molecular mass in the
range of 20-30 kDa can be clearly discriminated on a 13%
SDS-polyacrylamide gel, we have demonstrated in the experiments shown
in Figs. 3 and 6 that the NH2-terminal hydrophobic domain
is not removed cotranslationally and remains in the secreted protein.
Two examples of uncleavable signal peptides have been reported up to
date. One is ovalbumin (31, 32), and the other is PAI-2 (32). However,
the nature of their uncleavable signal peptides seems to be different
from that of carp RBP. First, the position within the primary structure
is different. In both cases, the signal peptide is located internally
(ovalbumin, amino acid residues 22-41; PAI-2, amino acid residues
25-46). Furthermore, the amino acid composition of the signal peptides
is different. The signal peptide of PAI-2 contains 11 low hydropathy
index amino acids such as Thr, Ser, Gly, Pro, Gln, and Asn (42) within
a stretch of 22 amino acids, while that of carp RBP contains only two
low hydropathy index amino acids at both ends, the remaining residues
showing very high hydropathy index, similarly to those present in
cleavable signal peptides. Based on such differences, it is conceivable
that the mechanism by which the signal peptide of PAI-2 is translocated
into the ER lumen is different from that of carp RBP. It was observed
that hydrophobic domains predicted by hydropathy analysis as
transmembrane domains in polytopic membrane proteins are occasionally
translocated into the ER lumen when their average hydropathy index was
low (29). However, the index of the signal peptide of carp
RBP is not low enough to allow for this mechanism to occur (its value
being 1.83, including the NH2-terminal charged amino acids,
while those of PAI-2 and ovalbumin are 0.33 and 0.94, respectively). Therefore, in the case of carp RBP, a conformational
change of the nascent chain, induced by glycosylation, may generate a
physical force that pulls out the signal peptide from the ER lipid
bilayer into the lumen. This interpretation is supported by the
observations that both glycosylation sites are located very close to
the predicted COOH terminus of the signal peptide and that
N-linked glycosylation is generally carried out cotranslationally.
In conclusion, the results reported in the present paper uncovered the
existence of novel structure-function relationships in carp RBP. On the
basis of these results, it will be very interesting to further
investigate which families of fish express N-linked glycosylated RBP and if a more general role can be established for
N-linked glycosylation in RBP function.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]CTP using a nick translation kit. Plaque lifts
(Nytran filters) were hybridized overnight at 58 °C and washed twice
with 2× SSC (1× SSC: 150 mM NaCl, 15 mM
sodium citrate, pH 7.0), 0.1% SDS, plus twice with 1× SSC, 0.1% SDS.
Filters were subjected to autoradiography. Positive clones were
isolated and excised from phage DNA with ExAssist/SOL-R system; the
inserted DNA fragments were sequenced by a conventional
dideoxynucleotide chain termination method (25) using the universal
primers (T3, T7) of pBlueScript, located downstream and upstream of the
cloned cDNA and several synthetic oligonucleotides as primers.
-mercaptoethanol) and separated by 13% SDS-PAGE. The corresponding
gel band was then cut and eluted overnight in 10 mM
Tris-HCl, pH 8.
-mercaptoethanol),
incubated in boiling water for 5 min, and then analyzed on 13%
SDS-PAGE, followed by sodium salicylate fluorography (28).
-mercaptoethanol. After addition of 5 µl of Reaction Buffer to all
samples, one of the two samples received 5 µl of Reaction Buffer as
control, and the other received 5 µl of Endo F (5 milliunits). All
the samples were incubated for 1 h at 37 °C, mixed with 5 µl
of SDS gel loading buffer, boiled for 5 min, and then analyzed by
SDS-PAGE, followed by immunostaining as described previously (3).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
helix and
sheet distribution in carp RBP is almost identical to
that of human RBP (data not shown). The primary structure also shows
that there is a hydrophobic domain at the NH2 terminus, which contains 13 consecutive hydrophobic amino acid residues resembling signal peptides of Xenopus, chicken, and
mammalian RBPs. Moreover, the primary sequence of this putative signal
is not evolutionarily conserved. Additional features of carp RBP are:
(i) the absence of the COOH-terminal tetrapeptide, RNL(S)L, that is
presumed to stabilize the interaction with TTR in mammalian RBPs
(indicated by open rectangle) (15), and (ii) the
presence of two consensus N-linked glycosylation sites
(indicated by waved underlines) near the
NH2 terminus (residues 25 and 46), which are absent in all
the available RBP sequences from other species, including trout.
View larger version (94K):
[in a new window]
Fig. 1.
Amino acid sequence alignment. Alignment
of carp RBP with trout, Xenopus, chicken, rabbit, rat, and
human RBPs is shown. The putative carp RBP signal sequence at the
NH2 terminus and N-glycosylation sites are
indicated by double line and waved
line, respectively. The six cysteines involved in disulfide
bonds are indicated by closed rectangles. The
amino acids predicted to form the retinol pocket are
underlined, and the COOH-terminal tetrapeptides that are
predicted to stabilize RBP·TTR complex are indicated by an
open rectangle. Amino acid residues that are
identical to those of carp RBP are indicated by bold
letters. Cleavage site of the signal peptide of
Xenopus, chicken, and mammalian RBPs is shown by an
arrow.
View larger version (66K):
[in a new window]
Fig. 2.
Tissue expression analysis of carp RBP.
Poly(A)+ RNA (3 µg/lane) obtained from a pool of three
carps' intestine (lane 1), female gonads
(lane 2), liver (lane 3),
kidney (lane 4), and brain (lane
5) were separated electrophoretically, transferred to a
nylon membrane and probed with 32P-labeled carp RBP
cDNA (upper panel) or 32P-labeled
rat actin (lower panel) as described under
"Experimental Procedures."
View larger version (25K):
[in a new window]
Fig. 3.
A, in vitro
translation of carp RBP. Plasmid encoding carp RBP cDNA was
linearized by enzymatic digestion and transcribed in vitro.
The transcript was used for in vitro translation in a rabbit
reticulocyte lysate system, in the absence (lane
1) or in the presence (lane 2) of dog
pancreas microsomes. For the protease protection assay, three samples
were translated in the presence of microsomes, after which one was put
on ice (lane 3), one was digested with proteases
in the absence of detergent (lane 4), and one was
digested in the presence of detergent (lane 5) as
described under "Experimental Procedures." All samples were
analyzed by 13% SDS-PAGE. Asterisks indicate molecular mass
of the primary translation product. B, deglycosylation assay
of carp RBP. Carp RBP was translated in the presence of microsomes.
Microsomes were then recovered from the mixture and resuspended as
described under "Experimental Procedures." Half of the sample was
used as control (lane 1), while the rest was
subjected to Endo H digestion (lane 2). Two carp
serum samples were prepared and one used as control (lane
3), while the other was subjected to Endo F digestion
(lane 4) as described under "Experimental
Procedures." All four samples were run in parallel on 13% SDS-PAGE
and transferred to nitrocellulose filter. Then the filter was cut into
two parts; one part (lanes 1 and 2)
containing radioactive samples was exposed to x-ray film, and the other
part (lanes 3 and 4) was immunostained
with anti-trout RBP antibody and visualized by chemioluminescence.
Alignment of the two parts of the filter was based on molecular weight
markers loaded in a central lane. Open and closed
circles indicate high mannose type (ER form) and serum carp
RBPs, respectively, and asterisks indicate the
deglycosylated form.
2-6Gal and Neu5Ac
2-6GalNAc),
Maakia amurensis agglutinin (specific to sialic
acid
2-3Gal), peanut agglutinin (specific to Gal
1-3GalNAc), and
Datura stramonium agglutinin (specific to
Gal
1-4GlcNAc). The results (Fig. 4A) show that, among
five lectins, only MAA and DSA bound to serum RBP, indicating that the
terminal sugar moiety is composed of sialic acid (
2-3) and
galactose (
1-4), but not mannose. The presence of sialic acid in
the secreted RBP was also examined by comparing the migration rate of
neuraminidase treated and undigested RBP in native PAGE, because the
migration rate of the sialylated protein becomes slower upon digestion
with neuraminidase, due to the decrease in negative charge. For this
experiment Cos-1 cells were transiently transfected with a Myc-tagged
expression construct of carp RBP, and secreted RBP was purified from
the culture medium by immunoprecipitation with anti-Myc antibody and digested with neuraminidase from V. cholerae. As
positive control, transferrin was digested with neuraminidase and
analyzed with the same procedure. Fig. 4B clearly shows that
neuraminidase digested RBP (lane 2) migrated
slower than undigested RBP (lane 1) as in the
case of digested (lane 4) and undigested
(lane 3) transferrin. These results led us to
conclude that carp RBP is a complex type of N-linked
glycoprotein.
View larger version (30K):
[in a new window]
Fig. 4.
A, lectin staining assay of carp RBP. 2 µl of carp serum (lane 1) or 5 µl each of
carp serum subjected to immunoprecipitation with anti-carp RBP antibody
(lanes 2-7) were separated by 13% SDS-PAGE and
transferred on nitrocellulose by Western blotting as described under
"Experimental Procedures." The nitrocellulose filter was then cut
into seven strips. As molecular weight marker, the carp serum sample
and one immunoprecipitated carp serum sample were immunostained with
anti-carp RBP (lanes 1 and 2,
respectively). The remaining five strips were subjected to five
different lectin binding assays according to the manufacturer's
manual. These include: G. nivalis agglutinin
(lane 3), S. nigra agglutinin
(lane 4), M. amurensis agglutinin
(lane 5), peanut agglutinin (lane
6), and D. stramonium agglutinin (lane
7). B, neuraminidase treatment of serum carp RBP.
The anti-Myc antibody immunoprecipitates from the culture medium of
Cos-1 cells transiently transfected with pClneo-carpRBPmyc
(lanes 1 and 2), and 2 µg of
purified human transferrin as the positive control (lanes
3 and 4) were digested or not with neuraminidase
and analyzed by native polyacrylamide gel electrophoresis as described
under "Experimental Procedures."
View larger version (75K):
[in a new window]
Fig. 5.
NH2-terminal hydrophobic domain
of carp RBP targets cytosolic protein GFP to ER in
vivo. RI-100 fish cells and Cos-1 mammalian
cells were transiently transfected with chimeric protein
(NH2-terminal hydrophobic domain of carp RBP-GFP)
(a, d, and e), ER-GFP as ER marker
(b and f), and GFP as cytosol marker
(c). Cells were washed with PBS, fixed with 2.5%
paraformaldehyde for 20 min at room temperature, washed again with PBS,
and then observed by a Zeiss Axioskop 2 fluorescent microscope.
View larger version (51K):
[in a new window]
Fig. 6.
Chimeric protein is secreted in the culture
medium without cleavage of the NH2-terminal hydrophobic
domain (putative signal peptide). Cos-1 cells were transiently
transfected with GFP (lanes 1 and 4),
chimeric protein (lanes 2 and 5) and
ER-GFP (lanes 3 and 6). Cell lysates
(lanes 1-3) and media (lanes
4-6) were fractionated on 13% SDS-PAGE, transferred on
nitrocellulose filter, and immunostained with anti-GFP antibody as
described under "Experimental Procedures." The unglycosylated and
glycosylated forms of the chimeric protein are indicated by
open and closed arrowheads,
respectively.
View larger version (47K):
[in a new window]
Fig. 7.
Secretion of carp RBP expressed transiently
in Cos-1 cells is dependent on N-linked
glycosylation. Cos-1 cells were transiently transfected with
pClneo-carpRBPmyc (lanes 3-8) and pClneo-human
RBP (lanes 9 and 10), metabolically
labeled for 4 h with [35S]methionine-cysteine in the
presence or absence of tunicamycin. Cell lysates (C;
lanes 1, 3, 5,
7, and 9) and media (M;
lanes 2, 4, 6,
8, and 10) were subjected to immunoprecipitation
with anti-c-Myc (lanes 1-8) or anti-human RBP
(lanes 9 and 10) antibodies and then
samples were fractionated on 13% SDS-PAGE, followed by autoradiography
as described under "Experimental Procedures." As negative control
for immunoprecipitation, nontransfected Cos-1 cells were treated in the
same way (lanes 1 and 2);
additionally, as negative control for secretion,
pClneo-carpRBPmyc-transfected cells were treated with brefeldin A
during metabolic labeling (lanes 5 and
6). Open and closed circles
indicate, respectively, partially and fully glycosylated carp RBPs; an
asterisk indicates unglycosylated carp RBP.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
![]() |
ACKNOWLEDGEMENTS |
---|
We are grateful to Rodolfo Berni (Department of Biochemistry, University of Parma, Parma, Italy) for the kind gift of purified trout RBP and to Flavia Ferrero (Department of Aquaculture, University of Tor Vergata, Rome, Italy) for kindly providing us with carp and advice. We thank Franca Serafini-Cessi (Department of Biochemistry, University of Bologna) for expert advice in the characterization of RBP carbohydrates.
![]() |
FOOTNOTES |
---|
* This work was supported by a grant from the Italian Ministry of Agriculture, Department of Aquaculture.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AJ277123.
¶ To whom correspondence should be addressed. Tel.: 39-06-51957069; Fax: 39-06-5031592; E-mail: gaetani@inn.ingrm.it.
Published, JBC Papers in Press, January 23, 2001, DOI 10.1074/jbc.M006779200
2 D. Bellovino, T. Morimoto, and S. Gaetani, unpublished observation.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: RBP, retinol-binding protein; GFP, green fluorescent protein; PBS, phosphate-buffered saline; Endo, N-glycosidase; ER, endoplasmic reticulum; PAI-2, plasminogen activator inhibitor-2; PAGE, polyacrylamide gel electrophoresis; DMEM, Dulbecco's modified Eagle's medium; TTR, transthyretin; PCR, polymerase chain reaction.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Blomhoff, R. (ed) (1994) Vitamin A in Health and Disease , pp. 1-35, Marcel Dekker, New York |
2. | Bellovino, D., Morimoto, T., Tosetti, F., and Gaetani, S. (1996) Exp. Cell Res. 222, 77-83[CrossRef][Medline] [Order article via Infotrieve] |
3. | Bellovino, D., Lanyau, Y., Garaguso, I., Amicone, L., Cavallari, C., Tripodi, M., and Gaetani, S. (1999) J. Cell. Physiol. 181, 24-32[CrossRef][Medline] [Order article via Infotrieve] |
4. | Soprano, D. R., Soprano, K. J., and Goodman, D. S. (1986) J. Lipid Res. 27, 166-171[Abstract] |
5. | Soprano, D. R., and Blaner, W. S. (1994) in The Retinoids: Biology, Chemistry and Medicine (Sporn, M. B. , Roberts, A. B. , and Goodman, D. S., eds), 2nd Ed. , pp. 257-282, Raven Press, New York |
6. | Colantuoni, V., Romano, V., Bensi, G., Santoro, C., Costanzo, F., Raugei, G., and Cortese, R. (1983) Nucleic Acids Res. 11, 7769-7776[Abstract] |
7. |
Laurent, B. C.,
Nilsson, M. H. L.,
Bavik, C. O.,
Jones, T. A.,
Sundelin, J.,
and Peterson, P. A.
(1985)
J. Biol. Chem.
260,
11476-11480 |
8. | Lee, S. Y., Ubels, J., and Soprano, D. R. (1992) Exp. Eye Res. 55, 163-171[Medline] [Order article via Infotrieve] |
9. | Vieira, A. V., Kuchler, K., and Schneider, W. J. (1995) DNA Cell Biol. 14, 403-410[Medline] [Order article via Infotrieve] |
10. |
McKearin, D. M.,
Barton, M. C.,
Keller, M. J.,
and Shapiro, D. J.
(1987)
J. Biol. Chem.
262,
4939-4942 |
11. | Stallings-Mann, M. L., Trout, W. E., and Roberts, R. M. (1993) Biol. Reprod. 48, 998-1005[Abstract] |
12. | Ong, D. E., Davis, J. T., O'Day, W. T., and Bok, D. (1994) Biochemistry 33, 1835-1842[Medline] [Order article via Infotrieve] |
13. |
Zanotti, G.,
Marcello, M.,
Malpeli, G.,
Folli, C.,
Sartori, G.,
and Berni, R.
(1994)
J. Biol. Chem.
269,
29613-29620 |
14. | Monaco, H. L., Rizzi, M., and Coda, A. (1995) Science 268, 1039-1041[Medline] [Order article via Infotrieve] |
15. | Naylor, H. N., and Newcomer, M. E. (1999) Biochemistry 38, 2647-2653[CrossRef][Medline] [Order article via Infotrieve] |
16. | Berni, R., Stoppini, M., and Zapponi, M. C. (1992) Eur. J. Biochem. 204, 99-106[Abstract] |
17. | Zapponi, M. C., Zanotti, G., Stoppini, M., and Berni, R. (1992) Eur. J. Biochem. 210, 937-943[Abstract] |
18. |
Yamauchi, K.,
Nakajima, J.,
Hayashi, H.,
Hara, A.,
and Yamauchi, K.
(1999)
Eur. J. Biochem.
265,
944-949 |
19. |
Santos, C. R. A.,
and Power, D. M.
(1999)
Endocrinology
140,
2430-2433 |
20. |
Shidoji, Y.,
and Muto, Y.
(1977)
J. Lipid Res.
18,
679-691 |
21. | Stainier, D. Y. R., and Fishman, M. C. (1992) Dev. Biol. 153, 91-101[Medline] [Order article via Infotrieve] |
22. | Hofmann, C., and Eichele, G. (1994) in The Retinoids: Biology, Chemistry and Medicine (Sporn, M. B. , Roberts, A. B. , and Goodman, D. S., eds), 2nd Ed , pp. 387-441, Raven Press, New York |
23. | Morriss-Kay, G. M., and Ward, J. (1999) Int. Rev. Cytol. 188, 73-131[Medline] [Order article via Infotrieve] |
24. | Hill, J., Clarke, J. D. W., Vargesson, N., Jowet, T., and Holder, N. (1995) Mech. Dev. 50, 3-16[CrossRef][Medline] [Order article via Infotrieve] |
25. | Sanger, F., Nicken, S., and Coulson, A. R. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 5463-5467[Abstract] |
26. | Chirgwin, J. M., Przybyla, A. E., MacDonald, R. J., and Rutter, W. I. (1979) Biochemistry 18, 5294-5299[Medline] [Order article via Infotrieve] |
27. | Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual , 2nd Ed. , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY |
28. | Chamberlain, J. P. (1979) Anal. Biochem. 268, 5182-5192 |
29. |
Xie, Y.,
Langhans-Rajasekaran, S. A.,
Bellovino, D.,
and Morimoto, T.
(1996)
J. Biol. Chem.
271,
2563-2573 |
30. | Hopp, T. P., and Woods, K. R. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 3824-3828[Abstract] |
31. | Lingappa, V. R., Lingappa, J. R., and Blobel, G. (1979) Nature 281, 117-121[Medline] [Order article via Infotrieve] |
32. | Tabe, L., Krieg, P., Strachan, R., Jackson, D., Wallis, E., and Colman, A. (1984) J. Mol. Biol. 180, 645-666[Medline] [Order article via Infotrieve] |
33. |
Ye, R. D.,
Wun, T. C.,
and Sadler, J. E.
(1988)
J. Biol. Chem.
263,
4869-4875 |
34. | Ganong, W. F. (1999) in Review of Medical Physiology (Ganong, W. F., ed), 19th Ed. , pp. 673-676, Appleton and Lange, Stanford, CT |
35. | Brebbirm, B. M., and Beeuwkes, R., III (1978) Hosp. Pract. 13, 35-46 |
36. |
Hauri, H.-P,
Kappeler, P.,
Anderson, H.,
and Appenzeller, C.
(2000)
J. Cell Sci.
113,
587-596 |
37. |
Ware, F. E.,
Vassilakos, A.,
Peterson, P. A.,
Jackson, M. R.,
Lehrman, M. A.,
and Villiams, D. B.
(1995)
J. Biol. Chem.
270,
4697-4704 |
38. | Hevert, D. N., Foellmer, B., and Helenius, A. (1995) Cell 81, 425-453[Medline] [Order article via Infotrieve] |
39. | Fries, E., Gustafsson, L., and Peterson, P. A. (1984) EMBO J. 3, 147-152[Abstract] |
40. | Ronne, H., Ocklind, C., Wiman, K., Obrink, R. B., and Peterson, P. A. (1983) J. Cell Biol. 96, 907-910[Abstract] |
41. | Rask, L., Valterson, C., Annundi, H., Kvist, S., Eriksson, U., Dallner, G., and Peterson, P. A. (1993) Exp. Cell Res. 143, 91-102[CrossRef] |
42. | Kyte, J. K., and Doolittle, R. F. (1982) J. Mol. Biol. 157, 105-132[Medline] [Order article via Infotrieve] |