Signal Peptidase and Oligosaccharyltransferase Interact in a Sequential and Dependent Manner within the Endoplasmic Reticulum*

Xuemin Chen, Clint VanValkenburghDagger, Haobo Liang, Hong Fang, and Neil Green§

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



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We demonstrate that the signal peptides of prepro-alpha -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

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-alpha -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

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-Delta 200 trp1-Delta 901 suc2-Delta 9 (from Todd Graham, Vanderbilt University), and strain CVY9 is MATalpha wbp1-1 ura3-52.

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-alpha -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).

In Vitro Signal Peptidase Assay-- To generate substrate for the assay, 3 A600 cell equivalents of strains CMYD1 (sec11)/pXC4 (encoding prepro-alpha -factor-HA minus sequon 4) and CMYD1 (sec11)/pXC5 (encoding prepro-alpha -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.

Other Biochemical Assays-- Pulse-labeling methods have been described (9). Immunoprecipitation of proteins under denaturing and nondenaturing conditions has been described (10).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Efficient Glycosylation of the alpha  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 alpha  factor, a yeast mating pheromone. Prepro-alpha -factor is translocated across the ER membrane and cleaved by the SPC, generating pro-alpha -factor (13). Pro-alpha -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-alpha -factor (13) and preinvertase (14) are shown. Sequon 4, the first Asn-linked acceptor site in prepro-alpha -factor and preinvertase, is illustrated using shaded boxes.

A temperature-sensitive sec11 mutant expressing prepro-alpha -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-alpha -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-alpha -factor (Fig. 2A, lane 4), because almost no alpha  factor precursor could be detected in wild-type (wt) MATalpha 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-alpha -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-alpha -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-alpha -factor-HA or prepro-alpha -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.

Two major forms of the alpha  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-alpha -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.

Because of its proximity to the signal peptide, we reasoned that sequon 4 of prepro-alpha -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-alpha -factor but not preproCPY.

The second approach to determine whether glycosylation was dependent on signal peptidase function was to construct mutations that changed Ala to Asn at the -1 position and Ala to Gln at the -3 position in prepro-alpha -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-alpha -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.

The pulse-labeling analysis presented in Fig. 2C shows that two differentially glycosylated forms of the alpha -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-alpha -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-alpha -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-alpha -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-alpha -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-alpha -factor, which caused a defect in its glycosylation.

Glycosylation of Sequon 4 Is Dependent on Cleavage of the Signal Peptides of Prepro-alpha -factor and a Preinvertase Fragment-- The data presented thus far suggest that the presence of an uncleaved signal peptide inhibited glycosylation of prepro-alpha -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.

Consistent with the results shown above, prepro-alpha -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-alpha -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-alpha -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-alpha -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-alpha -factor. Strain CMYD1 (sec11) bearing plasmids encoding either prepro-alpha -factor-HA or prepro-alpha -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.

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.



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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.

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-alpha -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-alpha -factor-HA minus sequon 38 and prepro-alpha -factor-HA minus sequon 4, because we could easily resolve their cleaved and uncleaved forms on SDS-polyacrylamide gel electrophoresis gels.

Upon mixing enzyme and substrate, we observed that the signal peptide of prepro-alpha -factor-HA minus sequon 4 was cleaved by the SPC, yielding ~30% pro-alpha -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-alpha -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-alpha -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-alpha -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-alpha -factor. Prepro-alpha -factor minus sequon 4 (lanes 1-4) and prepro-alpha -factor minus sequon 38 (lanes 6-9) were incubated with the SPC for the times indicated at the top of the figure. Pro-alpha -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."

Prepro-alpha -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-alpha -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-alpha -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

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 alpha  factor and invertase (Fig. 1). Prepro-alpha -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.

Our data show that addition of carbohydrate to sequon 4, the amino-terminal acceptor site in both prepro-alpha -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-alpha -factor and the preinvertase fragment are glycosylated independently of cleavage. However, all three sites in prepro-alpha -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.

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.



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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-alpha -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-alpha -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-alpha -factor can be found in the water-soluble and carbonate-extracted fractions.2 It is therefore plausible that partitioning of prepro-alpha -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-alpha -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.


    ACKNOWLEDGEMENT

We acknowledge Todd Graham (Vanderbilt University) for providing antibodies and yeast strains.


    FOOTNOTES

* 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.

Dagger 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.


    ABBREVIATIONS

The abbreviations used are: ER, endoplasmic reticulum; SPC, signal peptidase complex; OST, oligosaccharyltransferase; HA, hemagglutinin; wt, wild type; CPY, carboxypeptidase Y.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


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