|
Originally published In Press as doi:10.1074/jbc.M003845200 on July 25, 2000
J. Biol. Chem., Vol. 275, Issue 43, 33222-33230, October 27, 2000
A Microsomal GTPase Is Required for Glycopeptide Export from the
Mammalian Endoplasmic Reticulum*
Bassam R. S.
Ali ,
Agneta
Tjernberg§¶,
Brian T.
Chait§, and
Mark C.
Field
From the Wellcome Trust Laboratories for Molecular
Parasitology, Department of Biochemistry, Imperial College of
Science, Technology, and Medicine, Exhibition Road, London SW7 2AY,
United Kingdom and the § Laboratory of Biological Mass
Spectrometry and Gaseous Ion Chemistry, Rockefeller University,
New York, New York 10021
Received for publication, May 5, 2000, and in revised form, July 20, 2000
 |
ABSTRACT |
Bidirectional transport of proteins via the
Sec61p translocon across the endoplasmic reticulum (ER) membrane is a
recognized component of the ER quality control machinery. Following
translocation and engagement by the luminal quality control system,
misfolded and unassembled proteins are exported from the ER lumen back
to the cytosol for degradation by the proteasome. Additionally, other ER contents, including oligosaccharides, oligopeptides, and
glycopeptides, are efficiently exported from mammalian and yeast
systems, indicating that bidirectional transport across ER membranes is
a general eukaryotic phenomenon. Glycopeptide and protein export from
the ER in in vitro systems is both ATP- and
cytosol-dependent. Using a well established system to study
glycopeptide export and conventional liquid chromatography, we isolated
a single polypeptide species of 23 kDa from rat liver cytosol that was
capable of fully supporting glycopeptide export from rat microsomes in
the presence of an ATP-regenerating system. The protein was identified
by mass spectrometric sequence analysis as guanylate kinase (GK), a
housekeeping enzyme critical in the regulation of cellular GTP levels.
We confirmed the ability of GK to substitute for complete cytosol by
reconstitution of glycopeptide export from rat liver microsomes using
highly purified recombinant GK from Saccharomyces
cerevisiae. Most significantly, we found that the GK (and hence
the cytosolic component) requirement was fully bypassed by low
micromolar concentrations of GDP or GTP. Similarly, export was
inhibited by non-hydrolyzable analogues of GDP and GTP, indicating a
requirement for GTP hydrolysis. Membrane integrity was fully maintained
under assay conditions, as no ER luminal proteins were released.
Competence for glycopeptide export was abolished by very mild protease
treatment of microsomes, indicating the presence of an essential
protein on the cytosolic face of the ER membrane. These data
demonstrate that export of glycopeptide export is controlled by a
microsomal GTPase and is independent of cytosolic protein factors.
| |
INTRODUCTION |
In eukaryotic cells, the trafficking of secretory proteins and
glycoproteins is initiated upon translocation into the
ER1 lumen via the Sec61p
translocon complex. During translocation, various processes, including
removal of signal peptide, disulfide bond formation, and glycosylation,
are initiated; and the protein enters folding pathways assisted by a
number of molecular chaperones present in the ER (1, 2). As a result of
these processes, a number of byproducts are generated within the ER
lumen, including oligopeptides, free oligosaccharides, glycopeptides,
misfolded proteins, and unassembled subunits of protein complexes (1, 3-8). Allowing these byproducts and folding intermediates into the
Golgi complex or other post-ER secretory pathway compartments can lead
to competition with true secretory cargo for the various modification
and trafficking processes with deleterious consequences.
Eukaryotic cells have a stringent quality control machinery at the ER
level that ensures that only properly folded and fully assembled
proteins are allowed to exit by vesicular transport to the Golgi
complex. Misfolded and unassembled proteins are retained in the ER
lumen by molecular chaperones and then exported back to the cytosol,
where they are processed and degraded by the ubiquitin/proteasome systems (5, 6, 9-11). Interestingly, these proteins are exported via
the Sec61p translocon, the same channel through which they were
imported into the ER (12, 13).
Oligosaccharides that are generated during glycoprotein synthesis by
various mechanisms within the ER lumen (including the futile addition
of an N-linked carbohydrate precursor to water instead of a
protein) are exported to the cytosol for further trimming before being
imported into lysosomes for complete degradation (7, 8, 14, 15). The
bidirectional movement of macromolecules across the ER membrane is also
an integral part of antigen presentation by MHC class I molecules of
ER-targeted proteins and cytosol-derived oligopeptides. After
translocation into the ER lumen via TAP, oligopeptides are sampled by
the antigen presentation system, and the oligopeptides that do not fit
within the MHC class I molecule active site are recycled back to the
cytosol, by an unknown mechanism, for further degradation and trimming
by the proteasome. Some of the resulting oligopeptides are re-imported,
via TAP, into the ER lumen, where the suitable peptides
assemble with MHC class I (16, 17). It was also found that ER-targeted
proteins can provide a source of peptides for antigen presentation by
being exported out of the ER and then subsequently degraded by the
proteasome to generate peptides, which can ultimately be imported
via TAP (18).
Glycopeptide export is a powerful model system for the study of
retrograde ER transport in vitro (19, 20). The export can be
achieved using heterologous sources for the membrane and cytosol,
indicating the conservation of this process in eukaryotes and its
essential role (20). Glycopeptide export is monitored in a cell-free
system by introducing into the ER a hydrophobic iodinated tripeptide
(acetyl-NYT-NH2) that contains a canonical N-glycosylation sequon. Within the ER lumen, the
peptide is rapidly glycosylated by oligosaccharyltransferase, thereby
increasing both mass and polarity and preventing diffusion back across
the ER membrane. The addition of an ATP-regenerating system (21) and
cytosol and incubation at physiological temperature are required to
achieve export, which is easily quantitated by capture with concanavalin A.
Recently, we reported that this system is closely related to protein
export based on biochemical criteria allowing the differentiation of
the retrograde transport of glycopeptides from the export of free
oligosaccharides, hence suggesting that this system could allow
identification of factors common to both peptide and protein retrotranslocation mechanisms (22). As glycopeptide export from rat
liver microsomes is dependent upon cytosol, in common with protein
export in vitro systems, we exploited this system to
identify the cytosolic protein(s) required for retrograde
translocation. Surprisingly, we were able to demonstrate that GTP can
substitute for cytosol; and hence, glycopeptide export from mammalian
microsomes does not require cytosolic proteins.
| |
EXPERIMENTAL PROCEDURES |
Materials
Acetyl-NYT-NH2 was synthesized by Albachem Ltd.
ATP, GDP-mannose, GDP, GMP, cGMP, GDP S, GTP S, and
Canavilia lectin (concanavalin A)-Sepharose were from
Sigma. 125I (100 mCi/ml) was from Amersham Pharmacia
Biotech. Rat livers were from Harlan Sera Lab Ltd.
(Loughborough, UK). Creatine kinase and creatine phosphate were from
Roche Molecular Biochemicals. Other chemicals were obtained from
commercial sources and were of the highest purity available.
Peptide Iodination and Glycopeptide Export Assay of Rat Liver
Microsomes
The acetyl-NYT-NH2 peptide was iodinated with
125I using chloramine T as described by Wieland et
al. (23). The iodinated peptide was purified from unincorporated
125I by binding to a Sep-Pak C18 light
cartridge and then eluting with 60% acetonitrile in 0.1%
trifluoroacetic acid. The iodinated peptide was used within 1 month of iodination.
Glycopeptide Export Assay of Rat Liver Microsomes
The glycopeptide export assay was performed exactly as described
by Romisch and Ali (20) as follows.
Loading--
Crude rat liver membranes (100 µl;
A280 ~ 200) were suspended in 1 ml of ice-cold
B88 buffer (20 mM HEPES-KOH (pH 7.4) 150 mM
KOAc, 250 mM sorbitol, and 5 mM
Mg(OAc)2) containing 0.5 M KCl or NaCl and
placed on a rotator for 15 min at 4 °C. The membranes were then
sedimented in a cooled (4 °C) Eppendorf microcentrifuge at
20,000 × g for 5 min and washed with 1 ml of B88
buffer (pH 7.4). The membranes were resuspended in B88 buffer (200 µl); 125I-acetyl-NYT-NH2 was added at 1 × 107 cpm/100 µl; and the reaction mixture was incubated
at 10 °C for 20 min. This allowed the peptide to enter the ER
membranes and to be glycosylated by the endogenous
oligosaccharyltransferase. To remove the unglycosylated tripeptide,
2 × 1 ml of ice-cold B88 buffer was added, and the membranes were
then sedimented as described above and resuspended in 200 µl of B88 buffer.
Export--
Loaded membranes (5 µl of washed membranes;
A280 ~ 10) were placed in Eppendorf tubes on a
precooled ice-cold block, and the various components (as necessary)
were added to individual tubes in the following order. B88 buffer was
added to bring the final volume to 25 µl; 2.5 µl of partially
purified rat liver cytosol (5 µg of protein) was then added; and 2.5 µl of a 10× ATP-regenerating system was added last and mixed quickly
(final concentrations: 1 mM ATP, 40 mM creatine
phosphate, 0.2 mg/ml creatine kinase, and 50 µM
GDP-mannose). Export reactions were initiated by incubating the tubes
at 32 °C for a specified period of time, after which the membranes
were quickly sedimented at 4 °C in microcentrifuge, and the
supernatant (cytosol) was separated from the pellet (membranes). Very
little ongoing glycosylation was observed during the export stage of
the assay as evidenced by the constant amount of total glycopeptide
recovered in both the supernatant and membrane fractions and by the
observation that at least 90% of the total iodinated peptides
recovered after the loading and washing steps were glycosylated (22).
Quantitation of Glycopeptide Export--
To each supernatant or
pellet fraction was added 100 µl of 2% SDS, and samples were heated
immediately at 95 °C for 5 min. The samples were cooled, and 1 ml of
concanavalin A buffer (20 mM Tris-HCl (pH 7.5), 0.5 M NaCl, and 1% Triton X-100) was added, followed by 50 µl of 50% immobilized concanavalin A-Sepharose slurry; and the
mixture was placed on a rotator at room temperature for 2 h. The
beads were sedimented using a microcentrifuge and washed once (by
suspending and then sedimenting by brief centrifugation) with each of
the following: immunoprecipitation buffer (15 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, and 0.1%
SDS), urea buffer (100 mM Tris-HCl (pH 7.5), 200 mM NaCl, 1% Triton X-100, and 2 M urea), and
concanavalin A buffer. Radioactivity was measured by -counting using
an LKB 1275 mini--counter. Glycopeptide export was determined
as the percentage of radioactivity (counts/min) in the supernatant
relative to the total counts in both the supernatant and membranes.
Preparation of Rat Liver Microsomes and Purification of the
Glycopeptide Export Factor
Rat liver rough membranes and cytosol were prepared as described
previously (20). Cytosol was prepared from frozen rat livers purchased
from Harlan Sera Lab Ltd., and rough microsomes from fresh rat livers
were obtained from the CBS unit of the Imperial College.
Glycopeptide export activity (obtained from a 100,000 × g cytosolic fraction of 18 rat livers) was used for the
purification of the glycopeptide export factor as follows. The
clarified cytosol (300 ml) was subjected to ammonium sulfate
precipitation, and glycopeptide export activity was precipitated
between 40 and 60% ammonium sulfate. The protein pellet was dissolved
in 100 ml of 20 mM Tris-HCl (pH 8.0) containing 1 mM DTT and then dialyzed against the same buffer for
16 h. Insoluble material was removed by centrifugation at
15,000 × g, and the supernatant was loaded onto a
Q-Sepharose fast flow column (5 × 20 cm) at 5 ml/min. After washing the column extensively (1500 ml) with the same buffer, the
bound proteins were eluted with a KCl gradient (0-1.0 M)
over 1000 ml in the same buffer. Fractions (10 ml each) were collected and assayed for glycopeptide export activity, which showed that the
active proteins eluted between 300 and 500 mM KCl. The
active fractions were pooled (160 ml), and their protein contents were precipitated with 70% ammonium sulfate. The precipitated proteins were
dissolved in 40 ml of 25 mM Tris-HCl (pH 7.0) containing 1 mM DTT and then loaded in four portions (10 ml each) onto a Superdex G-75 26/60 HiLoad column. Elution was in the same buffer at 2 ml/min. Fractions (5 ml each) were collected and assayed for
glycopeptide export activity, which was recovered in fractions 36-44.
The fractions from four runs were pooled (150 ml) and then added to 50 ml of hydroxylapatite type I (Sigma) slurry in the above buffer and
mixed gently for 30 min. The hydroxylapatite was removed by
centrifugation, and the supernatant was further clarified by filtering
thorough a glass-fiber filter. Most of the activity was recovered in
the unbound fraction, brought to 1.5 M ammonium sulfate,
and then loaded onto a 5-ml phenyl-Sepharose fast flow column
pre-equilibrated with the same buffer. Bound proteins were eluted with
a decreasing ammonium sulfate gradient (1.5 to 0 M).
Fractions (2 ml each) were collected, and the active protein was found
in fractions 28-36 (0.6 to 0.3 M ammonium sulfate). Active
fractions (~20 ml) were pooled and ultra-filtered through a 10-kDa
cutoff membrane, and the buffer was exchanged with 20 mM
Tris-HCl (pH 8.0) containing 1 mM DTT and then
chromatographed on a Mono Q fast protein liquid chromatography column.
After loading the sample onto Mono Q, the column was washed with 5 ml
of loading buffer, followed by a KCl gradient (0-0.5 M)
over 20 ml. Fractions (1 ml each) were collected from the wash and
gradient and assayed for glycopeptide export activity, which was
recovered in fractions 13 and 14 (200-220 mM KCl).
SDS-PAGE analysis and silver staining indicated the presence of two
protein bands in fraction 14, one of which was present in the inactive
fraction (fraction 12), leaving the one at ~23 kDa as the
likely active protein.
Mass Spectrometry
Peptide sequence data were obtained by in-gel digestion with
trypsin and analysis of the products by both matrix-assisted laser
desorption/ionization time-of-flight mass spectrometry (MS) and liquid
chromatography electrospray ionization MS/MS using a quadrupole
ion-trap mass spectrometer as described (24). Peptide sequences were
used to identify the protein by searching the NCBI Nonredundant
Database with the programs PROFOUND and PEPFRAG (25).
Assessment of Microsomal Latency
After incubating the glycopeptide export reactions at the
specified temperatures and the specified period of times, tubes were
cooled on ice; 25 µl of ice-cold B88 buffer was added; and the
reactions were spun down at 20,000 × g for 10 min at
2 °C. The supernatant (50 µl) was removed, and the membrane pellet
was resuspended in 50 µl of B88 buffer. SDS was added from a stock solution to a final concentration of 2% to both fractions and heated
at 95 °C for 4 min. 15 µl of each fraction (supernatant and
pellet) was removed to a new tube for protein-disulfide isomerase (PDI)
analysis, and the rest was used for glycopeptide quantitation as
described above. SDS sample buffer was added to the samples designated
for PDI analysis, heated for an additional 3 min, and then
electrophoresed on 12.5% SDS-polyacrylamide gels. As a control, Triton
X-100 was used for the total release of ER luminal contents by membrane
solubilization prior to fractionation into soluble and
membrane-associated fractions. Proteins were transferred from the gels to HybondTM ECL nitrocellulose membranes (Amersham
Pharmacia Biotech) and probed with anti-PDI polyclonal antibodies.
Peroxidase (Sigma)-conjugated secondary antibodies were used along with
a chemiluminescence detection kit to visualize anti-PDI antibodies.
| |
RESULTS |
Purification of a Cytosolic Component That Supports Glycopeptide
Export--
A fully active glycopeptide export fraction was purified
from rat liver by ammonium sulfate precipitation, followed by
sequential chromatography on Q-Sepharose ion exchange, Superdex G-75
gel filtration, hydroxylapatite, phenyl-Sepharose hydrophobic
interaction, and Mono Q ion exchange columns (Fig.
1A). From 7 g of total
soluble hepatic proteins, we purified ~50 µg of the active export
factor. The elution profile of total protein
(A280) and glycopeptide export activity from the
final Mono Q chromatography step is shown in Fig. 1B. The
activity eluted at ~200 mM KCl and was collected as two
fractions (fractions 13 and 14) coinciding with an
A280 peak. Proteins from these and adjacent
fractions were analyzed by SDS-PAGE, followed by silver staining (Fig.
1C). The active fractions contained a single common unique
protein band with a molecular mass of 23 kDa (arrowhead);
fraction 14 contained two proteins, one at 23 kDa and a larger one at
27 kDa. This latter protein was also present in fraction 12; and as
this fraction was devoid of export activity, we concluded that the
export activity was most likely to be the 23-kDa protein, which we
designated p23. To confirm this assignment, we used partially purified
cytosol, corresponding to the active fractions from the first ion
exchange chromatography step of the purification, and resolved the
export activity by gel filtration on a Superdex G-75 high resolution gel permeation column in 20 mM Tris-HCl (pH 7.5)
containing 1 mM DTT and 150 mM NaCl. By
comparison with standard proteins, the activity eluted as a tight peak
at ~22.5 kDa (Fig. 1D). Because both the denatured and
native proteins have an identical apparent molecular mass, the export
factor is likely to be globular, soluble, and monomeric, typical of a
cytosolic protein.
View larger version (29K):
[in this window]
[in a new window]
|
Fig. 1.
Purification of the glycopeptide export
factor from rat cytosol. A, schematic representation of
the purification. The active fractions from each step (represented by a
bar) were pooled and loaded onto the next chromatography
column as described under "Experimental Procedures." The start of
the gradient (or fractions) for each chromatography step is shown on
the left, and the end is shown on the right. QFF,
Q-Sepharose fast flow. B, elution profile of the final
chromatography step of the purification (Mono Q HR5/5 fast protein
liquid chromatography). After loading, the column was washed with 5 ml
of buffer, followed by a KCl gradient (0-0.5 M) over 20 ml. 1-ml fractions were collected and assayed for glycopeptide export
activity (shaded) and protein content
(A280). Only fractions 13 and 14 were active for
glycopeptide export. C, SDS-PAGE analysis of proteins
present in fractions 9-15 of the Mono Q column. 15 µl from each
fraction was run on 15% SDS-polyacrylamide gel under denaturing
conditions and then silver-stained. Fractions 13 and 14 contained one
unique protein at ~23 kDa (arrowhead) that was not present
in fractions 12 and 15, which are inactive for glycopeptide export.
Fraction 15 was electrophoresed on the same gel as the other fractions,
but after the molecular standards lane, and therefore was manipulated
with Adobe Photoshop to place it adjacent to fraction 14 for
presentation. D, the glycopeptide (GP) export
factor migrates as a 23-kDa protein on a gel filtration column. 2 ml of
the 40-60% ammonium sulfate-precipitated fraction, which contained
the glycopeptide activity, was loaded onto a Superdex G-75 26/60 HiLoad
column pre-equilibrated with 25 mM Tris-HCl (pH 7.5)
containing 100 mM NaCl and 1 mM DTT and then
eluted in the same buffer at 30 ml/h. 5-ml fractions were collected,
and 2 µl from selected fractions was used in the glycopeptide export
assay. Protein gel filtration standards (Sigma MW-GF-70 kit) were also
chromatographed under the same conditions, and their elution positions
(in kilodaltons) are indicated by arrows. The active
glycopeptide protein eluted at ~23 kDa. Protein standards were bovine
serum albumin (66 kDa), carbonic anhydrase (29 kDa), cytochrome
c (12.4 kDa), and aprotinin (6.5 kDa).
|
|
Identification of p23 as Guanylate Kinase--
To identify p23, we
further purified ~25 µg of the export factor by preparative
SDS-PAGE. The protein in the 23-kDa band was subjected to in-gel
digestion with trypsin, yielding a mixture of proteolysis products that
was analyzed by both matrix-assisted laser desorption/ionization
time-of-flight MS and liquid chromatography electrospray ionization
MS/MS using a quadrupole ion-trap mass spectrometer (24). Data obtained
from the resulting mass spectra were used to identify the protein by
searching the NCBI Nonredundant Database with the programs PROFOUND and
PEPFRAG (25). Liquid chromatography electrospray ionization MS/MS
revealed the presence of four peptides with sequences identical to
portions of the known sequence of mouse guanylate kinase (Fig.
2A). The MS/MS spectrum of one
of these peptides is shown in Fig. 2B, which shows that this
spectrum corresponds to the amino-terminal peptide from which the
initiator methionine has been removed and in which the alanine at
position 2 has been acetylated. The MS analysis identified fully 44%
of the guanylate kinase sequence, providing unambiguous identification
of the protein as rat guanylate kinase (GK) (GMP kinase; EC 2.7.4.8).
Identification of GK was unexpected, as this activity was not predicted
to be involved in ER-to-cytosol export (26). GK catalyzes the
interconversion of GMP and GDP in the following equilibrium: GMP + ATP
 GDP + ADP. It is therefore a critical enzyme for dGTP and
GTP biosynthesis and hence might be involved in guanine
nucleotide-mediated signal transduction pathways (27). Therefore, we
sought to test the essential role of GK in the export system by several
criteria.
View larger version (31K):
[in this window]
[in a new window]
|
Fig. 2.
Identification of the active protein for
glycopeptide export as guanylate kinase. A, peptide
sequences determined from MS data are shown aligned with the sequence
of mouse GK. B, liquid chromatography electrospray
ionization MS/MS of a tryptic peptide from the 23-kDa protein band. The
molecular mass of the peptide was determined to be 1490.83 Da, which is
close to that calculated for the amino-terminally acetylated peptide
shown (1490.82 Da). The fragmentation nomenclature is as described
previously (37).
|
|
First, many nucleotide-binding enzymes, including GK, bind tightly to
dye affinity matrices via the nucleotide-binding site and can be eluted
specifically with nucleotides. We analyzed the interaction of partially
purified glycopeptide export factor by Cibacron blue-Sepharose
chromatography. Greater than 70% of the glycopeptide export activity
bound to the affinity matrix and was eluted with 1 mM ATP
(Fig. 3A), consistent with
both the properties of GK and the prediction of a nucleotide-binding
site in the active factor. GK behaved in a similar fashion, under the
conditions used in this study, on a Cibacron blue-Sepharose column,
which was a crucial step in the original purification to homogeneity from bovine tissue (28). Second, partially purified Sus
scrofa brain cytosolic GK also promoted glycopeptide export from
rat liver microsomes in the presence of ATP and an ATP-regenerating system under standard assay conditions (data not shown). Although it is
unlikely that this source would also contain a copurifying ER export
factor, we cannot rule this out based on these data alone. Hence,
third, highly purified recombinant Saccharomyces cerevisiae
guanylate kinase expressed in Escherichia coli (29) was
assayed and substituted fully for cytosol in the glycopeptide export
system when added with ATP and an ATP-regenerating system (Fig.
3B). Recombinant yeast GK elicited glycopeptide export at the same level as partially purified rat liver cytosolic factor (Fig.
3B, lanes 5-7 versus lane 3). GK,
when added alone, had no effect on export and hence did not compromise
membrane integrity (Fig. 3B, lane 4). These data
indicate that GK is functionally capable of supporting ER export, and
the reconstitution with GK from two totally independent sources
effectively rules out the presence of an undetectable moiety in our
preparation as the true export factor.
View larger version (13K):
[in this window]
[in a new window]
|
Fig. 3.
Reconstitution of glycopeptide export with
guanylate kinase. A, the glycopeptide (GP)
export cytosolic factor binds to Cibacron blue-Sepharose. 1 ml of
partially purified export factor (after gel filtration) was dialyzed
against 5 mM HEPES-KOH (pH 7.0) and loaded onto a 1-ml
Cibacron blue-Sepharose HiTrap column (Amersham Pharmacia Biotech) at
0.25 ml/min. The column was washed with 2 ml of loading buffer
containing 50 mM NaCl and 1 mM
MgCl2. The bound material was eluted with loading buffer
plus 1 mM ATP. Fractions (0.5 ml each) were collected
throughout the chromatography and assayed for glycopeptide export
activity. Total activity in the flow-through (F.T.), wash,
and eluted fractions was determined and shows that ~75% of the
activity was retained by the Cibacron blue-Sepharose and could
be eluted with ATP. B, recombinant yeast guanylate kinase
substitutes for rat liver cytosol in glycopeptide export from rat liver
microsomes. Purified wild-type S. cerevisiae GK expressed in
E. coli (29) was added to glycopeptide-loaded microsomes in
place of rat cytosol as described under "Experimental Procedures."
Inset, demonstration of purity of recombinant yeast
guanylate kinase by Coomassie staining of the SDS-polyacrylamide gel.
Lane 1, ~10 µg of yeast GK; lane 2, Amersham
Pharmacia Biotech molecular mass standards.
|
|
GTP and ATP Alone Support Microsomal Glycopeptide
Export--
The data above strongly suggest involvement of guanine
nucleotides in the control of ER export. In common with most other in vitro transport systems, our assay contains an
ATP-regenerating system to provide a constant supply of nucleotide
triphosphate (21). This system is also capable of generating GTP from
GDP; and hence, it is likely that the export system requires GTP,
indicating that the true role of GK in this system would be to recycle
guanine nucleotides into the regenerating system. We directly tested
this possibility by attempting to support export by adding nucleotides in the absence of any cytosolic proteins. GMP and cGMP at up to 1 mM failed to support glycopeptide export when added
together with ATP (data not shown). By contrast, GDP and GTP
substituted fully for the cytosolic factor (Fig.
4, A and B,
respectively). In the presence of ATP and an ATP-regenerating system, 1 µM GDP or GTP was sufficient to bypass the requirement
for cytosol, and maximal transport was achieved at 1-5
µM nucleotide. However, neither GDP nor GTP could support
export in the absence of ATP. This result demonstrated a requirement
for both nucleotides and importantly ruled out the possibility that the
guanine nucleotides were impure and providing a source of ATP rather
than being directly required. GDP supports export because it can be
converted to GTP by the regeneration system, whereas GMP, in the
absence of GK, cannot be converted to GTP. This interpretation is
supported by the observation that non-hydrolyzable analogues of GDP and
GTP (GDPS and GTPS, respectively) inhibited export, unambiguously confirming an absolute requirement for nucleotide hydrolysis (Fig. 4,
A and B).
View larger version (24K):
[in this window]
[in a new window]
|
Fig. 4.
GTP hydrolysis is required for glycopeptide
export from rat liver microsomes. A, glycopeptide
(GP) export assays were carried out in the absence of GDP (white bars) or in
the presence of 0.25, 1, 5, and 25 µM GDP
(light-gray to black bars) or 100 µM GDPS (far right bars). All
reactions were performed at 32 °C for 15 min in duplicate in the
presence of cytosol, ATP, or both as indicated. B,
glycopeptide export assays were carried out in the absence of GTP
(white bars) or in the presence of 0.25, 1, 5, and 25 µM GTP (light-gray to black bars)
or 100 µM GTPS (striped bars). Conditions
were as described for A. C, shown is a time
course of glycopeptide export from rat liver microsomes in the absence
(open symbols) or presence (closed symbols) of 10 µM GDP. Glycopeptide-loaded microsomes were incubated in
the presence of cytosol only (squares), ATP only
(triangles), or both cytosol and ATP
(circles). The assays were carried out in duplicate at
32 °C.
|
|
To demonstrate that GTP is the required component and to rule out
the possibility that cytosol has an additional role, we compared the
kinetics of export using cytosol and guanine nucleotides. A time course
of glycopeptide export in the absence or presence of 10 µM GDP is shown in Fig. 4C. Export reactions
with 10 µM GTP were very similar (data not shown). Export
in the presence of guanine nucleotide was more rapid than under other
conditions. In full reactions containing ATP and cytosol (open
circles), but without guanine nucleotide, the time required for
export of 50% of the total loaded peptide was ~11 min, whereas in
the presence of 10 µM GDP (closed circles),
the corresponding period was ~2.5 min. The measured time required for
50% export under standard assay conditions in the presence of ATP,
cytosol, and 10 µM GTP was ~2.3 min (data not shown).
The complete absence of additive export activity when cytosol was added
to GTP-containing export reactions effectively rules out a role for
additional cytosolic factors. The more rapid export with GDP/GTP
compared with cytosol alone is most likely due to increased local GTP
concentrations achieved by supplementing reactions with the guanine
nucleotide rather than by generating GTP indirectly via GK. Overall,
the data above indicate that glycopeptide may be exported from rat liver microsomes in the absence of cytosolic factors. GK is able to
supply hydrolyzable guanine nucleotide to the system, but the presence
of the enzyme per se is not a requirement.
The Source of Guanine Nucleotide in the Assay System Is
GDP-mannose--
The apparent dependence of glycopeptide export on GK
indicated the presence of a guanine nucleotide source and specifically GMP. Specifically, both GDP and GTP can substitute for cytosol/GK, but
GMP cannot within the assay system. Hence, we reasoned that GK is
required to convert GMP to GDP. GDP is then converted to GTP by the
ATP-regenerating system. A clear source of guanine nucleotide in the
assay is GDP-mannose, present at 50 µM as part of the
original ATP-regenerating system formulation (21). To test if GDP-Man
is a source of GMP, we performed export assays in the presence or
absence of GDP-Man. The nucleotide sugar was not essential for maximal
glycosylation of the peptide, consistent with the large quantity of
pre-existing dolichol-linked oligosaccharide donor present in mammalian
microsomes (data not shown). When GDP-Man was omitted from the
ATP-regenerating system (Fig. 5,
ATP*), no glycopeptide export above background was detected
in the presence of either cytosol or GK (lanes 5 and
7). However, export was restored by the addition of GMP
(Fig. 5, lanes 8 and 9) or GTP (data not shown),
demonstrating that GDP-Man is a source of GMP.
View larger version (16K):
[in this window]
[in a new window]
|
Fig. 5.
Guanine nucleotide in the assay is derived
from GDP-mannose. Glycopeptide (GP) export from
mammalian microsomes was performed using either the regular
ATP-regenerating system (ATP; see "Experimental
Procedures" for detailed composition) or the same system but omitting
GDP-man (ATP*). The export reactions were performed at
32 °C for 10 min. Adding rat liver cytosol and a regular
ATP-regenerating system (lane 3) resulted in 40-50%
release. However, when the GDP-Man-free ATP-regenerating system was
used, no export was observed (lane 5). Adding 10 µM GMP to this reaction restored export (lane
8). Purified yeast GK substituted fully for rat liver cytosol
(lane 9). Control reactions (GMP or ATP* alone or their
combination) had no effect (lanes 4, 6, and
7, respectively).
|
|
Maintenance of Luminal Latency--
An additional concern with the
ability of GTP to reconstitute glycopeptide export was the possibility
that latency of luminal components may be compromised. The fusion of
microsomes to produce networks and large structures has been reported
on several occasions (30), including an increased permeability to low
molecular mass compounds, specifically monosaccharides (31). The
importance of this fusion behavior to normal ER function is not known;
but to exclude this phenomenon as accounting for the GTP-mediated glycopeptide export detected here, we analyzed soluble and pellet fractions in an export assay for release of a luminal marker
protein, PDI. No significant release of PDI was observed under the
conditions used for glycopeptide export. Incubation (32 °C, 10 min)
of glycopeptide-loaded microsomes with the ATP-regenerating system and
either partially purified rat liver export fraction or recombinant
yeast GK resulted in ~50% export of glycopeptides (Fig.
6A, reactions 5 and
8). On the other hand, PDI was predominantly present in the
membrane fractions, and no significant release was detected under these conditions (Fig. 6B, reactions 5 and
8). Glycopeptide and PDI localization was determined using
the same reactions (see legend to Fig. 6). Control reactions showed
only background release of glycopeptide and no release of PDI (Fig. 6,
A and B, reactions 1-4, 6,
and 7). We also confirmed that GTP at low micromolar
concentrations (1 and 5 µM) had no effect on PDI
compartmentalization (Fig. 6, C and D). No
detectable amounts of PDI were released into the incubation
supernatants under the conditions where ~70% glycopeptide was
exported (Fig. 6, C and D, compare
reactions 3 (for 1 µM GTP) and 6 (for 5 µM GTP)). When Triton X-100 was added to the
reactions prior to fractionation into cytosol and membranes, PDI was
quantitatively released into the soluble fraction (Fig. 6E).
We conclude that under the conditions that allowed the export of
glycopeptides, the ER luminal soluble contents were not released,
indicating that the integrity of these membranes was not compromised,
confirming the physical relevance of our observations.
View larger version (19K):
[in this window]
[in a new window]
|
Fig. 6.
Membrane integrity is maintained during
glycopeptide export. Quantitation of both glycopeptide and PDI
present in the membrane and supernatant fractions was performed for
each reaction as described under "Experimental Procedures."
A, glycopeptide (GP) export using partially
purified rat liver cytosolic factor and yeast GK. Reactions
5 and 8 contained the ATP-regenerating system and
either rat cytosol or GK, respectively, and were incubated for 10 min
at 32 °C. Other reactions (reactions 1-4, 6,
and 7) are controls that show only background release and
confirm that glycopeptide export requires ATP, cytosol, or GK and
incubation at physiological temperature (compare the controls
versus Reactions 5 and 8). Reactions
were performed in duplicate, and data are presented as means ± S.E. B, compartmentalization of the ER luminal protein PDI
in the reactions in A. No significant release of PDI into
the supernatant (designated S and P for soluble
and pellet, respectively) was observed in any of the reactions,
including those (reactions 5 and 8) where
glycopeptide export was ~50%, indicating that the integrity of the
membranes was not compromised under the glycopeptide export conditions.
C, glycopeptide export in the presence of the
ATP-regenerating system and 1 or 5 µM GTP with no
cytosolic proteins added. GTP at 1 or 5 µM and in the
presence of an ATP-regenerating system and incubation at 32 °C for
10 min resulted in 45 and 70% glycopeptide export (reactions
3 and 6, respectively). GTP on its own
(reactions 1 and 4) or GTP and ATP
incubated on ice for 10 min (reactions 2 and
5) show background release of glycopeptide. Reactions were
performed in duplicate, and data are presented as means ± S.E.
D, compartmentalization of the ER luminal protein PDI in the
reactions in C. No release of PDI was observed under
conditions that allowed 45 and >70% export (reactions
3 and 6, respectively). The experiment was
performed done twice, with essentially identical results. E,
confirmation that PDI is released into the medium once membrane
integrity has been compromised by treating the microsomes with Triton
X-100. In the control sample (0%), PDI was exclusively localized to
the membrane fraction (P); however, when Triton X-100 was
added (0.5 and 1%), the protein was quantitatively released into the
medium (S).
|
|
Requirement for a Microsomal Cytosolically Oriented Protein in
Glycopeptide Export--
The microsomes used in the assay were
routinely stripped of peripheral membrane proteins with 0.5 M KCl or NaCl. Therefore, we suspected an integral membrane
GTPase as being the regulator of export activity and hence the site of
GTP requirement. To test this hypothesis, we used a method developed
for analysis of the signal recognition particle receptor and treated
microsomes with very small amounts of trypsin to selectively inactivate
cytosolically oriented proteins before assaying for export activity
(32). Treatment with >1 µg/ml trypsin for 10 min on ice prior to
loading with the iodinated tripeptide was sufficient to abolish export activity (Fig. 7), whereas protease
treatment under these conditions did not affect peptide import or
subsequent glycosylation (data not shown). SDS-PAGE analysis indicated
that several protein species were released, but Western blot analysis
of the microsomes indicated that the signal recognition particle
receptor  subunit was still present on the microsomes even following
exposure to trypsin treatment that fully inactivated glycopeptide
export competence (data not shown). Hence, a component of the
glycopeptide export system is exquisitely sensitive to protease.
View larger version (40K):
[in this window]
[in a new window]
|
Fig. 7.
Glycopeptide export from rat liver microsomes
is sensitive to trypsin treatment. Microsomes were stripped with
salt and incubated without (white bars) or with 1, 2.5, or
10 µg/ml trypsin (light-gray to black bars) for
10 min on ice. The membranes were then washed twice with buffer and
assayed for glycopeptide export competence in the presence of 10 µM GTP and cytosol, ATP, or both as indicated. The
experiment was performed in duplicate.
|
|
| |
DISCUSSION |
There is increasing evidence that movement of macromolecules
across ER membranes is bidirectional and considerably more complex than
appreciated as recent as 5 years ago. At least four classes of
byproduct of the ER synthetic machinery can be re-exported from the ER
lumen into the cytosol: proteins, peptides, glycopeptides, and
oligosaccharides. In addition, maintaining the relative oxidative environment of the ER lumen, an important property for protein folding,
requires translocation of both GSH and GSSG across the ER membranes in
both directions (33). Biochemically, there are clear distinctions
between the export reactions for each of these classes, which implies
the involvement of a large number of protein factors and possibly
several distinct routes out of the ER. In addition, some of the
exported molecules may require cytosolic factors for their export, in
particular Hsp90, recently implicated in the degradation of mutant
forms of the insulin receptor (34). This system is also an important
part of the immunological surveillance mechanism for sampling of
peptides for presentation to MHC class I, as oligopeptides are known to
be exported from the ER for processing by the proteasome prior to
reentry through the TAP transporter for interaction with nascent class
I molecules (5, 16, 17).
The Sec61p translocon complex has been implicated in the export and
degradation of misfolded proteins, but the exact mechanisms for these
processes remain largely undefined (12). This pathway is an important
component of the quality control system, with the major function of
delivering proteins to the ubiquitin/proteasome degradation machinery.
Oligosaccharide export is also most likely a quality control process
and functions to remove glycans that have been generated from
dolichol-linked precursors transferred to water rather than to a
polypeptide chain. Important features of this system are a requirement
for luminal calcium, deglucosylation of the glycan, and sensitivity to
mannosides, suggesting involvement of an ER luminal lectin (7, 8, 35).
By contrast, glycopeptide export is unaffected by manipulation of ER
calcium levels, is not affected by mannosides, and does not require
removal of glucose residues, consistent with the hypothesis that the
calnexin system is not involved in the export of this class of
substrate (22). However, both oligosaccharide and glycopeptide
retranslocation systems require ATP, whereas the translocon itself has
not been identified for these substrates.
The observations in this study demonstrate that glycopeptide export
does not require cytosolic protein factors. We have been able to
reconstitute efficient glycopeptide release by the addition of either
GDP or GTP. However, we take the ability of GDP to bypass the cytosol
requirement to be due to efficient conversion to GTP by the
ATP-regenerating system included in the assay. Guanine nucleotide is
provided by GDP-Man present in the ATP-regenerating system (21). The
guanine nucleotide cannot be GDP generated directly from GDP-Man by
glycosyltransferase activities on the cytosolic face of the ER
producing dolichol-P-Man and lipid-linked oligosaccharides
because the ATP-regenerating system cannot support export unless GTP,
GDP, or, more importantly, GK is added. The ability of GK to bypass the
GDP/GTP requirement strongly indicates that GMP is generated. Our data
indicate that GDP-Man or GMP can support export in the context of an
ATP-regenerating system. This is analogous to the situation in S. cerevisiae, where GDP-Man is imported into Golgi membranes for
mannosylation of N- and O-linked glycans. The GDP
product is dephosphorylated to GMP by Golgi luminal guanosine
diphosphatase before re-export back to the cytosol (36).
Most significantly, non-hydrolyzable GTP analogues do not support
export. Hence, we conclude that GTP hydrolysis is required; and thus, a
GTPase activity is involved in regulating glycopeptide export. Clearly,
this suggests that a GTPase is important in the control of the
translocation system and potentially may serve to gate the translocon
itself. Most significantly, the GTPase activity is microsomal and hence
may potentially interact directly with the glycopeptide export channel.
It is worth noting here that a large number of studies have been
performed using this ATP-regenerating system (i.e.
containing GDP-Man) in in vitro systems to study transport
across membranes. Hence, the probability of generating GTP upon
addition to membranes must be considered under these conditions.
The kinetics of glycopeptide export from rat liver microsomes in the
presence of ATP and GTP are rapid, with a half-life for glycopeptide
exported of ~3 min. The export half-life of the MHC class I heavy
chain from the ER to the cytosol in cells expressing the human
cytomegalovirus genes U2 and US11 is also in the range of 2-3 min and
is therefore consistent with those of other substrates (13).
Additionally, efflux of oligopeptides from microsomes during antigen
presentation is independent of TAP, requires ATP, and follows
relatively rapid kinetics (16, 17). On the other hand, misfolded
protein degradation and oligosaccharide export are slower than for
glycopeptides (7, 11, 12). This is presumably because misfolded
proteins enter folding and quality control pathways distinct from
simple molecules like glycopeptides, and they may also require
unfolding before being transported back to the cytosol.
We have also demonstrated that a trypsin-sensitive component on the
microsomal membrane is essential for export of glycopeptides. This
second export factor could be the same as the GTPase; but alternatively, these two factors could be distinct. The present data do
not allow us to distinguish between the two possibilities. The latter
interpretation gives rise to the interesting possibility that the
GTPase is a regulatory factor, whereas the protease-sensitive molecule
may be a channel or other structural component. Sensitivity of export
to trypsin digestion indicates that at least part of the system is
located on the cytosolic face of the ER membrane, and this may be the
cytoplasmically orientated loops of a channel protein. Very recent data
from studies on S. cerevisiae indicate that glycopeptide
export is mediated by Sec61p (38). Hence, a possible role for GTP is in
regulating opening of the Sec61p translocon, but the identity of this
GTPase is currently unknown. Our data suggest that it is unlikely to be
signal recognition particle receptor , although we cannot rule out a
very small, but critical proteolytic inactivation that may not have
been detected by our analysis. In addition, the role of the
transmembrane subunit has not been addressed; but as the mammalian
ER membrane contains numerous GTPases, there are many other candidates
for this role. It is worth noting that GTP did not substitute for cytosol in glycopeptide export from S. cerevisiae
semi-intact cells (data not shown), indicating that at least this
aspect of the pathway is likely restricted to metazoans. The
identification of both the microsomal GTPase identified here and the
glycopeptide translocon, presumed to be Sec61p in mammalian systems, is
clearly a vital requirement for complete characterization of
glycopeptide export, for addressing the relationship of this process to
the better characterized Sec61p-mediated protein retranslocation
pathway, and finally for understanding the role this mechanism plays in antigen presentation by MHC class I.
| |
ACKNOWLEDGEMENTS |
We thank Dr. Honggao Yan (Michigan State
University) for recombinant yeast guanylate kinase and Dr. Peter Walter
(University of California at San Francisco) for anti-signal recognition
particle receptor antibodies. Preliminary studies on fractionation
of cytosolic factors were performed in the laboratory of Dr. Karin Romisch (Laboratory of Molecular Cell Biology-University College London). We also thank Dr. Debbie Smith for comments on the manuscript and members of the Field laboratory for discussions.
| |
FOOTNOTES |
*
This work was supported by Biotechnology and
Biological Sciences Research Council Project Grant 28/C09893 (to
M. C. F.) and National Institutes of Health Grant RR00862 (to
B. T. C.).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.
¶
Present address: Dept. of Structural Chemistry,
Pharmacia & Upjohn, S-11287 Stockholm, Sweden.
To whom correspondence should be addressed. Tel.:
44-171-5945277; Fax: 44-171-5945207; E-mail: mfield@ic.ac.uk.
Published, JBC Papers in Press, July 25, 2000, DOI 10.1074/jbc.M003845200
| |
ABBREVIATIONS |
The abbreviations used are:
ER, endoplasmic reticulum;
MHC, major histocompatibility complex;
TAP, transporters associated with antigen processing;
GDPS, guanosine 5'-O-3-(thio)diphosphate;
GTPS, guanosine 5'-3-O-(thio)triphosphate;
DTT, dithiothreitol;
PAGE, polyacrylamide gel electrophoresis;
MS, mass spectrometry;
MS/MS, tandem mass spectrometry;
PDI, protein-disulfide isomerase;
GK, guanylate kinase.
| |
REFERENCES |
| 1.
|
Hurtley, S. M.,
and Helenius, A.
(1989)
Annu. Rev. Cell Biol.
5,
277-307
|
| 2.
|
Gaut, J. R.,
and Hendershot, L. M.
(1993)
Curr. Opin. Cell Biol.
5,
589-595
|
| 3.
|
Hammond, C.,
and Helenius, A.
(1995)
Curr. Biol.
7,
523-529
|
| 4.
|
Ellgaard, L.,
Molinari, M.,
and Helenius, A.
(1999)
Science
286,
1882-1888
|
| 5.
|
Bonifacino, S. J.,
and Weissman, A. M.
(1998)
Annu. Rev. Cell Dev. Biol.
14,
19-57
|
| 6.
|
Suzuki, T.,
Yan, Q.,
and Lennarz, W. J.
(1998)
J. Biol. Chem.
273,
10083-10086
|
| 7.
|
Moore, S. E. H.,
Bauvy, C.,
and Codogno, P.
(1995)
EMBO J.
14,
6034-6042
|
| 8.
|
Moore, S. E. H.
(1999)
Trends Cell Biol.
9,
441-446
|
| 9.
|
Brodsky, J. L.,
and McCracken, A. A.
(1997)
Trends Cell Biol.
7,
151-156
|
| 10.
|
Kopito, R. R.
(1997)
Cell
88,
427-430
|
| 11.
|
Wilbourn, B.,
Nesbeth, D. N.,
Wainwright, L. J.,
and Field, M. C.
(1998)
Biochem. J.
332,
111-118
|
| 12.
|
Pilon, M.,
Schekman, R.,
and Romisch, K.
(1997)
EMBO J.
16,
4540-4548
|
| 13.
|
Wiertz, E. J. H. J.,
Tortorella, D.,
Bogyo, M., Yu, J.,
Mothes, W.,
Jones, T. R.,
Rapoport, T. A.,
and Ploegh, H. L.
(1996)
Nature
384,
432-438
|
| 14.
|
Saint-Pol, A.,
Bauvy, C.,
Codogno, P.,
and Moore, S. E. H.
(1997)
J. Cell Biol.
136,
45-59
|
| 15.
|
Cacan, R.,
and Verbert, A.
(2000)
Glycobiology
10,
645-648
|
| 16.
|
Roelse, J.,
Gromme, M.,
Momburg, F.,
Hammerling, G.,
and Neefjes, J.
(1994)
J. Exp. Med.
180,
1591-1597
|
| 17.
|
Schumacher, T. N. M.,
Kantesaria, D. V.,
Heemels, M.-T.,
Ashton-Rickardt, P. G.,
Shepherd, J. C.,
Fruh, K.,
Yang, Y.,
Peterson, P. A.,
Tonegawa, S.,
and Ploegh, H. L.
(1994)
J. Exp. Med.
179,
533-540
|
| 18.
|
Bacik, I.,
Snyder, H. L.,
Anton, L. C.,
Russ, G.,
Chen, W.,
Rennink, J. R.,
Urge, L.,
Otvos, L.,
Dudkowska, B.,
Eisenlohr, L.,
and Yewdell, J. W.
(1997)
J. Exp. Med.
186,
479-487
|
| 19.
|
Romisch, K.,
and Schekman, R.
(1992)
Proc. Natl. Acad. Sci. U. S. A.
89,
7227-7231
|
| 20.
|
Romisch, K.,
and Ali, B. R. S.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
6730-6734
|
| 21.
|
Baker, D.,
Hicke, L.,
Rexach, M.,
Schleyer, M.,
and Schekman, R.
(1988)
Cell
54,
335-344
|
| 22.
|
Ali, B. R. S.,
and Field, M. C.
(2000)
Glycobiology
10,
383-391
|
| 23.
|
Wieland, F. T.,
Gleason, M. L.,
Serafini, T. A.,
and Rothman, J. E.
(1987)
Cell
50,
289-300
|
| 24.
|
Qin, J.,
Herringand, C. J.,
and Zhang, X.
(1998)
Rapid Commun. Mass Spectrom.
12,
209-216
|
| 25.
|
Fenyo, D.,
Qin, J.,
and Chait, B. T.
(1998)
Electrophoresis
19,
998-1005
|
| 26.
|
Brady, W. A.,
Kokoris, M. S.,
Fitzgibbon, M.,
and Black, M. E.
(1996)
J. Biol. Chem.
271,
16734-16740
|
| 27.
|
Woods, D. F.,
and Byant, P. J.
(1991)
Cell
66,
451-464
|
| 28.
|
Hall, S. W.,
and Kuhn, H.
(1986)
Eur. J. Biochem.
161,
551-556
|
| 29.
|
Li, Y.,
Zhang, Y.,
and Yan, H.
(1996)
J. Biol. Chem.
271,
28038-28044
|
| 30.
|
Lavoie, C.,
Lanoix, J.,
Kan, F. W. K.,
and Paiement, J.
(1996)
J. Cell Sci.
109,
1415-1425
|
| 31.
|
Paiement, J.,
and Bergeron, J. J. M.
(1983)
J. Cell Biol.
96,
1791-1796
|
| 32.
|
Walter, P.,
Jackson, R. C.,
Marcus, M. M.,
Lingappa, V. R.,
and Blobel, G.
(1979)
Proc. Natl. Acad. Sci. U. S. A.
76,
1795-1799
|
| 33.
|
Bánhegyi, G.,
Lusini, L.,
Puskás, F.,
Rossi, R.,
Fulceri, R.,
Braun, L.,
Mile, V.,
Simplicio, P.,
Mandl, J.,
and Benedetti, A.
(1999)
J. Biol. Chem.
274,
12213-12216
|
| 34.
|
Imamura, T.,
Haruta, T.,
Takata, Y.,
Usui, I.,
Iwata, M.,
Ishihara, H.,
Ishiki, M.,
Ishibashi, O.,
Ueno, E.,
Sasaokaand, T.,
and Kobayashi, M.
(1998)
J. Biol. Chem.
273,
11183-11188
|
| 35.
|
Moore, S. E. H.
(1998)
Glycobiology
8,
373-381
|
| 36.
|
Berninsone, P.,
Miret, J. J.,
and Hirschberg, C. B.
(1994)
J. Biol. Chem.
269,
207-211
|
| 37.
|
Biemann, K.
(1992)
Annu. Rev. Biochem.
61,
977-1010
|
| 38.
|
Gillece, P.,
Pilon, M.,
and Romisch, K.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
4609-4614
|
Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
 CiteULike  Complore  Connotea  Del.icio.us  Digg  Reddit  Technorati What's this?
This article has been cited by other articles:
|
|
|
|
 
T. Suzuki and W. J. Lennarz
Glycopeptide export from the endoplasmic reticulum into cytosol is mediated by a mechanism distinct from that for export of misfolded glycoprotein
Glycobiology,
December 1, 2002;
12(12):
803 - 811.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. S. Nair, C. J. DaFonseca, A. Tjernberg, W. Sun, J. E. Darnell Jr., B. T. Chait, and J. J. Zhang
Requirement of Ca2+ and CaMKII for Stat1 Ser-727 phosphorylation in response to IFN-gamma
PNAS,
April 30, 2002;
99(9):
5971 - 5976.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
L.-M. Chen, M. L. Skinner, S. W. Kauffman, J. Chao, L. Chao, C. D. Thaler, and K. X. Chai
Prostasin Is a Glycosylphosphatidylinositol-anchored Active Serine Protease
J. Biol. Chem.,
June 8, 2001;
276(24):
21434 - 21442.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. S. Nair, C. J. DaFonseca, A. Tjernberg, W. Sun, J. E. Darnell Jr., B. T. Chait, and J. J. Zhang
Requirement of Ca2+ and CaMKII for Stat1 Ser-727 phosphorylation in response to IFN-gamma
PNAS,
April 30, 2002;
99(9):
5971 - 5976.
[Abstract]
[Full Text]
[PDF]
|
|
|
Copyright © 2000 by the American Society for Biochemistry and Molecular Biology.
|
Advertisement
Advertisement
|
TOP
ABSTRACT
INTRODUCTION