From the Department of Microbiology and Immunology, School of Medicine, Vanderbilt University, Nashville, Tennessee 37232-2363
Received for publication, August 23, 2000, and in revised form, October 30, 2000
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ABSTRACT |
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We demonstrate that the signal peptides of
prepro- Most of the glycoproteins secreted from eukaryotic cells enter the
secretory pathway attached to amino-terminal signal peptides that serve
to target newly synthesized polypeptide chains to the endoplasmic
reticulum (ER)1 membrane.
Signal peptides exhibit three distinct regions: an amino-terminally
charged region, a hydrophobic core, and a polar carboxyl-terminal region containing the cleavage site (1-3). Upon entering the ER lumen a glycoprotein must undergo a series of
maturation and folding events before it can be transported through the secretory pathway and, eventually, out of the cell. Two
early events in the maturation process are the cleavage of signal
peptides by the signal peptidase complex (SPC) and the addition of core
oligosaccharides to Asn-X-Ser/Thr acceptor tripeptides by
oligosaccharyltransferase (OST) (4-6).
The SPC and OST are integral proteins of the ER membrane. The SPC
active site is probably located at the lumenal surface of the membrane,
based on the crystal structure of Escherichia coli leader
peptidase, a single subunit enzyme that is functionally related to the
SPC (7). In contrast, the active site of the OST is positioned within
the lumen, 30-40 Å above the lipid bilayer. This estimate comes from
an in vitro study showing that the minimal distance between
an Asn-X-Ser/Thr acceptor site and the end of a
transmembrane segment of an integral membrane protein required for
half-maximal glycosylation is ~10 residues (8).
The secretory proteins prepro- Yeast Strains--
Genotypes of yeast strains CMYD1, RSY427,
PBY408A, HFY402 (9), and CVY1 (10) have been described previously. The
genotype of strain TBY103 is MATa sec23-1 leu2-3,112 ura3-52
his3- Plasmids, Site-directed Mutations, and Antibodies--
A
description of the plasmid constructs and oligonucleotides used to make
site-directed mutations is available upon request. Antibodies were used
in the following amounts: anti-HA antibodies (2 µg/3
A600 equivalents of yeast cells),
anti- In Vitro Signal Peptidase Assay--
To generate substrate for
the assay, 3 A600 cell equivalents of strains
CMYD1 (sec11)/pXC4 (encoding prepro- Other Biochemical Assays--
Pulse-labeling methods have been
described (9). Immunoprecipitation of proteins under denaturing and
nondenaturing conditions has been described (10).
Efficient Glycosylation of the
A temperature-sensitive sec11 mutant expressing
prepro-
Two major forms of the
Because of its proximity to the signal peptide, we reasoned that sequon
4 of prepro-
The second approach to determine whether glycosylation was dependent on
signal peptidase function was to construct mutations that changed Ala
to Asn at the
The pulse-labeling analysis presented in Fig. 2C shows that
two differentially glycosylated forms of the Glycosylation of Sequon 4 Is Dependent on Cleavage of the Signal
Peptides of Prepro-
Consistent with the results shown above, prepro-
A protein smear, whose apparent molecular mass was 1-2 kDa greater
than the major protein species, was evident above some of the bands
depicted in Fig. 3. As discussed below, this heterogeneity was probably
due to a slow transport of this protein through the Golgi stacks,
concomitant with modification of the attached carbohydrate.
In searching the Saccharomyces genome data base, we found
that preinvertase contained a glycosylation site also positioned four
amino acids downstream of the signal peptide (Fig. 1). Invertase has a
total of 13 Asn-X-Ser/Thr sequons. Therefore, to reduce the
complexity of our analysis, we prepared a truncated DNA fragment encoding the signal peptide and the first 93 residues of the mature portion of invertase. The truncated protein contained three acceptor sites located 4, 45, and 78 amino acids downstream of the signal peptide. The truncation also contained a carboxyl-terminal HA epitope, and transcription was placed under control of the
ADH1 promoter.
We prepared a series of site-directed mutations, T6A, T47A, and S80A,
that eliminated each of the glycosylation sites in the invertase
fragment. These constructs were introduced into sec23 and
sec11 mutant strains, and log phase cells were analyzed by pulse labeling. The data presented in Fig.
4 demonstrate that efficient
glycosylation of sequon 4 of the preinvertase fragment was dependent on
cleavage of the signal peptide.
Attachment of Carbohydrate Four Amino Acids Downstream of the
Signal Peptide Does Not Block Cleavage--
Our data have thus far
revealed a sequential interaction between the SPC and OST. What is the
functional significance of this interaction? We reasoned that reversing
the order of action of the SPC and OST may cause a dead end in the
maturation pathway. That is, attachment of carbohydrate only four amino
acids from the signal peptide may produce a molecule whose attached
glycan sterically inhibits cleavage. To test this, we devised an
in vitro assay that used uncleaved prepro-
Upon mixing enzyme and substrate, we observed that the signal peptide
of prepro-
Prepro- In this paper we have investigated the functional relationship
between two enzymes that operate early in the secretory pathway, the
SPC, which cleaves amino-terminal signal peptides, and OST, which
attaches the preassembled oligosaccharide
Glc3Man9GlcNAc2 to asparagines in
Asn-X-Ser/Thr tripeptides. We have examined two
secretory proteins that have naturally occurring glycosylation sites
located only four amino acids downstream of their signal peptides, the
precursors to Our data show that addition of carbohydrate to sequon 4, the
amino-terminal acceptor site in both prepro- The probable positioning of the SPC active site near the surface of the
ER membrane (7) and the OST active site 30-40 Å above the lipid
bilayer (8) provides a likely explanation for the observed sequential
interaction between the SPC and OST. As illustrated in Fig.
6, a hydrophobic signal peptide is
probably able to pull a nearby Asn-X-Ser/Thr tripeptide
toward the surface of the ER membrane and away from the OST active
site. Cleavage of the signal peptide is thus a prerequisite for
efficient glycosylation of an acceptor tripeptide located close to a
signal peptide.
-factor and preinvertase must be cleaved before
Asn-X-Ser/Thr acceptor tripeptides located near the signal
peptides of these precursors can be efficiently glycosylated within the
endoplasmic reticulum of the yeast Saccharomyces
cerevisiae. The data support a model whereby the interaction of a
signal peptide with the membrane prevents an acceptor tripeptide
juxtaposed to the signal peptide from accessing the
oligosaccharyltransferase active site until the signal peptide is cleaved.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-factor and preinvertase expressed by
the yeast Saccharomyces cerevisiae are examples of
glycoproteins that contain cleavable signal peptides. Interestingly,
both precursors have an acceptor tripeptide located only four amino
acids from the signal peptide. Because signal peptides typically
contain a hydrophobic core that could possibly pull an acceptor
tripeptide close to the surface of the ER membrane and thus away from
the OST active site, we used these precursors to test the idea that a
signal peptide must be cleaved before a nearby acceptor tripeptide can
be glycosylated. The data presented in this paper support this
hypothesis, demonstrating that a sequential and dependent interaction
exists between the SPC and OST.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
200 trp1-
901 suc2-
9 (from Todd Graham, Vanderbilt University), and strain CVY9
is MAT
wbp1-1 ura3-52.
-factor antibodies (1 µl/1 A600
equivalent of yeast cells) (from Todd Graham), and anti-CPY antibodies
(0.5 µl/1 A600 equivalent of yeast cells) (from Todd Graham). Anti-FLAG antibodies (1 µg/1
A600 equivalent of yeast cells) and protein
G-agarose were used instead of agarose-conjugated anti-FLAG antibodies
for native immunoprecipitation of FLAG-Sec11p (10).
-factor-HA minus sequon 4) and CMYD1 (sec11)/pXC5 (encoding
prepro-
-factor-HA minus sequon 38) were grown to log phase at
23 °C. Cells were shifted to 37 °C for 30 min to inactivate the
SPC and then were subjected to a 15-min pulse labeling. Substrate was
immunoprecipitated under denaturing conditions using anti-HA
antibodies, and the antibodies were captured using protein G-agarose.
30 µl of SDS (0.1%) was added to the protein G pellet. 3 µl of the
suspended protein G pellet was added to each reaction. To generate
enzyme, the SPC was immunoprecipitated using anti-FLAG antibodies under nondenaturing conditions from 1000 A600 log
phase cell equivalents of strain CVY1-bearing plasmids pCV101
(FLAG-Sec11p) and pCV102 (HA-Spc3p) (10). Immunoprecipitated SPC bound
to protein G-agarose beads was resuspended in 300 µl of a solution
containing 75 mM KOAc, pH 8, 0.1% Triton X-100, 0.5 mg/ml
phosphatidylcholine, 50 mM triethanolamine, pH 8, 1 mM dithiothreitol (11). 27 µl of a protein G suspension
containing the SPC was used in each reaction. The reactions were
performed for up to 2 h at 16 °C and terminated by boiling in a
solution containing 8 µl of SDS-polyacrylamide gel
electrophoresis sample buffer (4×). One-half of each reaction was
loaded onto an SDS-polyacrylamide gel electrophoresis gel. The
substrate and enzyme suspensions were stable for at least 3 days on ice.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Factor Precursor Is Dependent on
Cleavage of Its Signal Peptide--
We took two approaches to
determine whether glycosylation was dependent on signal peptidase
function. The first was to inhibit the SPC using the sec11
mutation (12) and then ask whether glycosylation was, in turn,
inhibited in the yeast S. cerevisiae. The substrate employed
was the precursor to
factor, a yeast mating pheromone. Prepro-
-factor is translocated across the ER membrane and cleaved by
the SPC, generating pro-
-factor (13). Pro-
-factor has a total of
three Asn-linked glycosylation sites. The first site, sequon 4, is
placed only four amino acids downstream of the signal peptide (Fig.
1).
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Fig. 1.
Sequences of typical signal peptides.
Sequences of the signal peptides of prepro- -factor (13) and
preinvertase (14) are shown. Sequon 4, the first Asn-linked acceptor
site in prepro-
-factor and preinvertase, is illustrated using
shaded boxes.
-factor was grown to log phase at the permissive temperature
(23 °C) and shifted to 30 °C for 15 min to inactivate Sec11p, the
catalytic subunit of the SPC (10). Cells were pulse-labeled for 5 min, and proteins were precipitated from cell extracts using anti-
-factor antibodies. Yeast strains and their genotypes are listed in the figure
legends and under "Experimental Procedures." Controls, which were
treated identically, included a wbp1 mutant containing a
temperature-sensitive defect in the Wbp1p subunit of the OST protein
complex (15) and a sec23 mutant containing a
temperature-sensitive defect in Sec23p that is required for vesicular
transport to the Golgi apparatus (16). The sec23 mutant was
needed to visualize fully glycosylated pro-
-factor (Fig.
2A, lane 4),
because almost no
factor precursor could be detected in wild-type
(wt) MAT
cells (lane 1) because of the
efficient transport and proteolytic processing of this protein in the
Golgi stacks.
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Fig. 2.
Mutations inhibiting signal peptide cleavage
inhibit glycosylation of prepro- -factor but
not preproCPY. A, strains HFY402 (wt), CVY9
(wbp1), and PBY408A (sec11) were grown to log
phase at 23 °C, shifted to 30 °C for 15 min, and pulse labeled
for 5 min. Strain RSY427 (sec23) was examined similarly
except that the temperature shift and pulse were performed at 37 °C.
Proteins were precipitated from cell extracts using anti-
-factor
antibodies. The number of Asn-linked glycans added to each protein is
indicated. B, strains RSY427 (sec23), CVY9
(wbp1), and PBY408A (sec11) were examined as
described in A, except that proteins were precipitated from
cell extracts using anti-CPY antibodies. The positions of fully
glycosylated preproCPY (ppCPY) and proCPY (pCPY),
along with a series of underglycosylated species (*), are indicated.
C, strains CVY1 (wt) and TBY103 (sec23) bearing
plasmids encoding either prepro-
-factor-HA or prepro-
-factor-HA
containing the A
1N and A
3Q mutations were grown at 23 °C,
shifted to 35 °C for 15 min, and pulse labeled for 10 min. Proteins
were precipitated from cell extracts using anti-HA antibodies.
factor precursor were present in the
sec11 mutant (Fig. 2A, lane 3),
one having all three of its acceptor sites modified and one containing
only two Asn-linked glycans. The presence of this latter species
suggested that the sec11 mutation inhibited glycosylation of
prepro-
-factor. Two additional underglycosylated polypeptides
appeared in wbp1 cells (Fig. 2A,
lane 2), one containing a single Asn-linked glycan and one
lacking carbohydrate. Because these latter two forms were absent in
sec11 cells (Fig. 2A, lane 3),
the data suggest that addition of carbohydrate to only one acceptor
site was inhibited by the sec11 mutation.
-factor may be glycosylated inefficiently in the
sec11 mutant. We therefore repeated our analysis using a
protein lacking an acceptor site next to the signal peptide. The
precursor to carboxypeptidase Y (CPY), a vacuolar protease, contains
four Asn-X-Ser/Thr tripeptides (17). Importantly, the first
is separated from the signal peptide by 104 residues. When this protein
was examined by pulse labeling, the fully glycosylated species, and no
apparent underglycosylated species, was present in the sec11
mutant (Fig. 2B). Thus, the sec11 mutation
appeared to inhibit glycosylation of prepro-
-factor but not preproCPY.
1 position and Ala to Gln at the
3 position in
prepro-
-factor (Fig. 1). These positions in signal peptides usually
contain small uncharged amino acids and are important for signal
peptide cleavage (1-3). For convenience, we introduced a sequence
encoding the HA epitope (18) at the carboxyl terminus of
prepro-
-factor, and we placed the construct under control of the
ADH1 promoter (19), which produced levels of protein that
were ~50% of that synthesized by the natural promoter (data not
shown). The HA-tagged constructs containing and lacking the A
1N and
A
3Q mutations were introduced into wt and sec23 mutant strains.
-factor-HA precursor were present in wt cells when signal peptide cleavage was inhibited by
the A
1N and A
3Q mutations (lane 3). As expected,
pro-
-factor-HA that lacked the A
1N and A
3Q mutations was present
primarily in the form containing three Asn-linked glycans in
sec23 mutant cells (Fig. 2C, lane
2), and low levels of pro-
-factor-HA were present in wt cells
(lane 1). Evidence that the A
1N and A
3Q mutations
inhibited cleavage came from the fact that prepro-
-factor-HA containing these mutations was retained in the ER (Fig.
2C, compare lanes 1 and 3).
Further evidence was that the protein present in Fig.
2C, lane 2 migrated more slowly on
SDS-polyacrylamide gel electrophoresis gels than the corresponding
protein in lane 3. We have reported previously that
prepro-
-factor migrates faster than its signal peptide cleaved form
in our gel system (9). The data in Fig. 2C thus demonstrate
that the introduced signal peptide mutations inhibited cleavage of
prepro-
-factor, which caused a defect in its glycosylation.
-factor and a Preinvertase Fragment--
The
data presented thus far suggest that the presence of an uncleaved
signal peptide inhibited glycosylation of prepro-
-factor, probably
at only one of its three acceptor sites. To identify the site affected,
we made three constructs, each containing one of the following
site-directed mutations, T6A, T40A, and T50A. These mutations disrupted
sequon 4, sequon 38, and sequon 48, respectively. The constructs were
expressed in the sec11 mutant, and cells were subjected to
pulse labeling.
-factor-HA lacking
an acceptor site mutation exhibited two glycosylated forms in
the sec11 mutant, one containing 3 glycans and one
containing 2 glycans (Fig. 3, lane
1). Prepro-
-factor-HA minus sequon 4 exhibited primarily the
form containing two Asn-linked glycans, which was fully glycosylated
for this construct (Fig. 3, lane 2). Eliminating either sequon 38 (Fig. 3, lane
3) or sequon 48 (lane 4) from prepro-
-factor-HA
resulted in two proteins appearing, one containing two Asn-linked
glycans and one containing only one glycan. These data therefore
demonstrated that efficient glycosylation of sequon 4 in
prepro-
-factor-HA was dependent on cleavage of the signal
peptide.
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Fig. 3.
The sec11 mutation inhibits
glycosylation of sequon 4 in
prepro- -factor. Strain CMYD1
(sec11) bearing plasmids encoding either
prepro-
-factor-HA or prepro-
-factor-HA minus sequon 4, 38, or 48 was grown to log phase at 23 °C, shifted to 32 °C for 15 min, and
pulse labeled for 10 min. Proteins were precipitated from cell extracts
using anti-HA antibodies.
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Fig. 4.
The sec11 mutation inhibits
glycosylation of sequon 4 in preinvertase. Strains TBY103
(sec23) and CMYD1 (sec11) bearing plasmids
encoding either the preinvertase fragment (preinv-f-HA) or
the preinvertase fragment lacking sequon 4, 45, or 78 were pulse
labeled as described in Fig. 2A (sec23) and Fig.
3 (sec11). Proteins were precipitated using anti-HA
antibodies.
-factor-HA
immunoprecipitated from the sec11 mutant and the SPC
immunoprecipitated from wild-type yeast overexpressing the SPC (see
"Experimental Procedures"). The specific substrates employed were
prepro-
-factor-HA minus sequon 38 and prepro-
-factor-HA minus
sequon 4, because we could easily resolve their cleaved and uncleaved
forms on SDS-polyacrylamide gel electrophoresis gels.
-factor-HA minus sequon 4 was cleaved by the SPC, yielding
~30% pro-
-factor-HA after 2 h at 16 °C (Fig. 5, lanes 1-4). This protein
lacked an amino-terminal glycosylation site and therefore served as a
positive control. Prepro-
-factor-HA minus sequon 38 exhibited two
differentially glycosylated forms because it was immunoprecipitated
from the sec11 mutant (Fig. 5, lane 6).
The higher molecular weight form resulted from carbohydrate attachment
to sequon 4 and sequon 48, whereas the lower molecular weight form was
modified only at sequon 48. Not surprisingly, the form containing only
one glycan (prepro-
-factor-HA + 1 glycan) was cleaved with an
efficiency similar to that detected for the above-mentioned positive
control (Fig. 5, lanes 6-9). Interestingly, the signal
peptide was similarly cleaved from the twice-modified form
(prepro-
-factor-HA + 2 glycans) (Fig. 5, lanes 6-9),
indicating that attachment of carbohydrate to the Asn residue of sequon
4 (Fig. 1) did not prevent cleavage.
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Fig. 5.
In vitro cleavage of the signal
peptide of prepro- -factor.
Prepro-
-factor minus sequon 4 (lanes 1-4) and
prepro-
-factor minus sequon 38 (lanes 6-9) were
incubated with the SPC for the times indicated at the top of
the figure. Pro-
-factor-HA lacking sequon 4 (lane 5) or
sequon 38 (lane 10) was immunoprecipitated from cell
extracts of strain RSY427 (sec23) that had been shifted to
37 °C for 15 min and pulse labeled for 10 min. The position of a
proteolytic fragment is indicated by a star. The assay
conditions are described under "Experimental Procedures."
-factor-HA + 1 glycan from the sec11 mutant
migrated as a series of closely spaced bands (see Fig. 5, lane
9), resolved on this gel because of the absence of sodium
salicylate treatment (compare Fig. 5 to Fig. 3). In contrast, a small
amount of pro-
-factor + 1 glycan was present in the sec23
mutant, and this protein was represented by a single species (Fig. 5,
lane 10). Because this protein comigrated with the fastest
migrating form of pro-
-factor + 1 glycan in the sec11
mutant (Fig. 5, lane 9), we believe the multiple forms
present in the sec11 mutant represent a series of
carbohydrate modifications occurring because of a slow transport of
this abnormal protein through the Golgi stacks. We also detected a
small amount of a second cleavage product (Fig. 5, lanes 3, 4, 8, and 9; indicated by a
star). This product appears to represent cleavage by the SPC
at an uncharacterized site downstream of the signal peptide.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
factor and invertase (Fig. 1). Prepro-
-factor contains three Asn-X-Ser/Thr sequons. Preinvertase contains
13 Asn-X-Ser/Thr tripeptides; however, to simplify our
analysis, the gene encoding preinvertase was truncated to encode a
protein containing only the first three acceptor tripeptides.
-factor and the preinvertase fragment, is partially inhibited when cleavage of the
signal peptide is inhibited within cells of the yeast S. cerevisiae (Figs. 2, A and C, 3, and 4). The
remaining acceptor sites in prepro-
-factor and the preinvertase
fragment are glycosylated independently of cleavage. However, all three
sites in prepro-
-factor and the preinvertase fragment can be
glycosylated when the signal peptide is cleaved. These results support
the hypothesis of Nilsson and von Heijne (8) that glycosylation of an
Asn-X-Ser/Thr acceptor site located close to a signal
peptide may be dependent on cleavage of the signal peptide.
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Fig. 6.
Model for the sequential interaction between
the SPC and OST. At the top of the figure
(I), a precursor polypeptide containing three carbohydrate
acceptor sites (closed diamonds) is shown being translocated
across the ER membrane in an amino (N) to carboxyl
(C) orientation through a protein channel called the Sec61
complex (20). When signal peptidase activity is present
(left-hand side), the signal peptide is cleaved during or
immediately after translocation (II). The cleavage event
releases the translocating polypeptide from the membrane, and the first
(amino-terminal) acceptor site is then able to receive an Asn-linked
glycan (indicated using a Y) from the OST (III).
Signal peptide cleavage thus precedes glycosylation of the first
acceptor site; however, acceptor sites distal to the signal peptide are
glycosylated independently of cleavage. This diagram arbitrarily shows
the second acceptor site being glycosylated before signal peptide
cleavage (II). In the absence of signal peptidase activity
(right-hand side), the signal peptide cannot be cleaved
(II). The precursor is anchored to the membrane by its
hydrophobic signal peptide (III). Thus, the amino-terminal
acceptor site cannot reach the OST and is not glycosylated. Acceptor
sites located downstream of the amino terminus can be glycosylated,
even in the absence of signal peptide cleavage.
A similar situation has been found to exist in integral membrane
proteins. Asn-X-Ser/Thr tripeptides are ~50% glycosylated if placed 10 residues from a transmembrane segment as monitored by an
in vitro assay (8) or ~5 residues from a transmembrane segment in Xenopus oocytes (21). In our study, ~50% of
prepro--factor (Fig. 3) and ~75% of the preinvertase fragment
(Fig. 4) were glycosylated at sequon 4 in the absence of signal peptide
cleavage. Based on the integral membrane protein model, one
interpretation of this result is that the partial glycosylation seen
here reflects the relative distances between sequon 4 and the
membrane-spanning portions of the signal peptides of prepro-
-factor
and the preinvertase fragment. However, most signal peptides exhibit
less hydrophobic character than bona fide transmembrane
segments. Indeed, using a standard cell fractionation analysis, we have
discovered that 60-70% of prepro-
-factor can be found in the
water-soluble and carbonate-extracted
fractions.2 It is therefore
plausible that partitioning of prepro-
-factor and the preinvertase
fragment out of the membrane could contribute to the partial
glycosylation seen. We do not believe, however, that an abundance of
uncleaved precursors leave the ER membrane normally, because most
signal peptides are processed during or immediately after translocation
in cells expressing signal peptidase activity.
At present, the functional significance for the observed sequential
interaction between the SPC and OST is unclear. One idea that we had
was that addition of carbohydrate to an Asn residue located only four
amino acids from the signal peptide may prevent cleavage because of a
steric effect. We have shown, however, that glycosylation of sequon 4 prior to cleavage does not prevent cleavage of the signal peptide of
prepro--factor, based on an in vitro assay (Fig. 5). To
further assess the functional significance of the sequential
interaction between the SPC and OST, a key issue is whether
polypeptides entering the ER begin the maturation process before their
signal peptides are cleaved. If cleavage is the first event following
translocation, then a sequential interaction between the SPC and OST
may be fortuitous and have no bearing on the maturation process. If
other maturation events precede cleavage, then the maturation process
could be dramatically influenced by a functional interaction between
the SPC and OST. For example, if acceptor sites distal to the signal
peptide can be glycosylated before cleavage (see Fig. 6), then our
observed interaction between the SPC and OST could affect the order in
which acceptor sites are glycosylated and, thus, the overall folding pathway.
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ACKNOWLEDGEMENT |
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We acknowledge Todd Graham (Vanderbilt University) for providing antibodies and yeast strains.
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FOOTNOTES |
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* This work was supported by the Department of Health and Human Services Training Grant 2 T32 CA09385-11 (to C. V.), by grants from the National Science Foundation (to H. F. and N. G.) and American Heart Association (to N. G.), and by a CAREER Award from the National Science Foundation (to H. F.).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.
Current address: Dept. of Molecular Microbiology, Washington
University School of Medicine, St. Louis, MO 63110-1093.
§ To whom correspondence should be addressed. Tel.: 615-343-0453; Fax: 615-343-7392; E-mail: neil.green@mcmail.vanderbilt.edu.
Published, JBC Papers in Press, October 31, 2000, DOI 10.1074/jbc.M007723200
2 N. Green, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: ER, endoplasmic reticulum; SPC, signal peptidase complex; OST, oligosaccharyltransferase; HA, hemagglutinin; wt, wild type; CPY, carboxypeptidase Y.
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