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Originally published In Press as doi:10.1074/jbc.M410969200 on October 26, 2004

J. Biol. Chem., Vol. 280, Issue 3, 1864-1871, January 21, 2005
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Studies on the N-Glycosylation of the Subunits of Oligosaccharyl Transferase in Saccharomyces cerevisiae*

Guangtao Li{ddagger}, Qi Yan{ddagger}§, Aleksandra Nita-Lazar, Robert S. Haltiwanger, and William J. Lennarz

From the Department of Biochemistry and Cell Biology and Institute for Cell and Developmental Biology, State University of New York, Stony Brook, New York 11794-5215

Received for publication, September 23, 2004 , and in revised form, October 18, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In Saccharomyces cerevisiae, oligosaccharyl transferase (OT) consists of nine different subunits. Three of the essential gene products, Ost1p, Wbp1p, and Stt3p, are N-linked glycoproteins. To study the function of the N-glycosylation of these proteins, we prepared single or multiple N-glycosylation site mutations in each of them. We established that the four potential N-glycosylation sites in Ost1p and the two potential N-glycosylation sites in Wbp1p were occupied in the mature proteins. Interestingly, none of the N-glycosylation sites in these two proteins was conserved, and no defect in growth or OT activity was observed when the N-glycosylation sites were mutated to block N-glycosylation in either subunit. However, in the third glycosylated subunit, Stt3p, there are two adjacent potential N-glycosylation sites (N535NTWN539NT) that, in contrast to the other subunits, are highly conserved in eukaryotic organisms. Mass spectrometric analysis of a tryptic digest of Stt3p showed that the peptide containing the two adjacent N-glycosylation sites was N-glycosylated at one site. Furthermore, the glycan chain identified as Man8GlcNAc2 is found linked only to Asn539. Mutation experiments were carried out at these two sites. Four single amino acid mutations blocking either N-glycosylation site (N535Q, T537A, N539Q, and T541A) resulted in strains that were either lethal or extremely temperature sensitive. However, other mutations in the two N-glycosylation sites N535NTWN539NT (N536Q, T537S, N540Q, and T541S), did not exhibit growth defects. Based on these studies, we conclude that N-glycosylation of Stt3p at Asn539 is essential for its function in the OT complex.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Oligosaccharyl transferase (OT)1 catalyzes the N-glycosylation of most secretory and membrane-bound proteins. In Saccharomyces cerevisiae, OT consists of nine different subunits, of which five are essential gene products (1). At present, the detailed structural organization of the OT complex, as well as the function of each of the OT subunits, is unclear. Growing evidence supports the proposal that one of the essential OT subunits, Stt3p, is involved directly in the substrate recognition process (2–4). In addition, Stt3p has been shown to be involved in cell wall {beta}-1,6-glycan biosynthesis in S. cerevisiae and to interact with protein kinase cascade components via its N-terminal domain, which is oriented toward the cytosol. This function may be independent of its role in the recognition process of substrates, which involves the C-terminal domain that is oriented toward the lumen (5, 6).

The nine subunits of yeast OT have been hypothesized to comprise three subcomplexes, Ost1p-Ost5p, Ost2p-Swp1p-Wbp1p, and Stt3p-Ost4p-Ost3p (1), although recent data are not completely consistent with this model (7). It is of interest that each of these three subcomplexes contains one of the three glycoproteins, Ost1p, Wbp1p, or Stt3p (1). The importance of the N-glycosylation of OT subunits with respect to their function is unknown. In the current study, we have addressed this question by site-directed mutation of potential N-glycosylation sites on the Ost1p, Wbp1p, and Stt3p subunits. In addition, because our knowledge of the minimal spacing required for efficient N-glycosylation at two adjacent sites was limited, we also addressed this question in the current study.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Strains and Plasmids—The haploid strains QYY500 (MAT a ade2 can1 his3 leu2 trp1 ura3 {Delta}ost1::his5+ (Schizosaccharomyces pombe) pRS316-OST1), L2 (MAT a ade2 can1 his3 leu2 trp1 ura3 {Delta}wbp1::his5+ (S. pombe) pRS316-WBP1), and QYY700 (MAT a ade2 can1 his3 leu2 trp1 ura3 {Delta}stt3::his5+ (S. pombe) YEP352-STT3) were used in the Ost1p, Wbp1p, and Stt3p mutation assays, respectively. Plasmids pRS314-OST1HA, pRS314-WBP1HA and pRS314-STT3HA were constructed as described previously (2, 8, 9). The haploid strain AYY7 (7) was used to check the N-glycosylation of Ost6p.

PCR Mutagenesis for Block and Point Mutants—PCR mutagenesis was performed using a site-directed mutagenesis kit following the manufacturer's protocol (Stratagene). For all of the block and point mutations mentioned in this study, pRS314-OST1HA, pRS314-WBP1HA, and pRS314-STT3HA were used as the PCR templates for Ost1p, Wbp1p, and Stt3p mutagenesis, respectively. Mutagenized plasmids were sequenced, and those with the expected sequence were transformed into QYY500, L2, and QYY700. The transformants were selected for Trp and Ura prototrophy and further selected on plates containing 5-fluoroorotic acid (5-FOA) (Sigma) for growth.

Spotting Assay for Growth—To determine the growth difference between yeast transformants carrying either Ost1p or Wbp1p mutations and wild type control, an equal number of cells were collected after the strains grew to early log phase in –Trp medium at 25 °C. Then 7 µl of 1:10 serial dilutions of the cells were spotted onto –Trp plates, incubated at 25, 30, or 37 °C for 2 days, and examined for growth. For Stt3p wild type or mutants, cells were grown initially in the –Trp–Ura medium and then spotted on –Trp–Ura and –Trp+FOA plates.

Determination of OT Activity—The oligosaccharyl transferase activity was carried out as described previously (9). The glycosylation activity was expressed as the amount of labeled glycopeptide formed (in cpm)/unit of protein/unit of time.

Immunoprecipitation and Co-immunoprecipitation—QYY700 carrying wild type Stt3pHA or mutants (N60Q, N535Q, T537A, N539Q, T541A, and T537A/N539Q) was used for co-immunoprecipitation. Co-immunoprecipitation under a mild detergent condition was carried out as described previously (9). Immunoprecipitation of Stt3HAp and mutants was performed in harsh detergent buffer. The spheroplasts were suspended in 1% Triton X-100, 0.2% SDS, 150 mM NaCl, 10 mM Hepes (pH 7.5), 5 mM MgCl2, and 1 mM phenylmethylsulfonyl fluoride. The mixture was centrifuged for 20 min at 55,000 x g, and the clarified supernatant was used for immunoprecipitation using monoclonal anti-HA antibody (Covance). Recombinant protein G-agarose beads (Invitrogen) were added to recover the antibody. Samples were eluted from the agarose beads using 0.1 M glycine (pH 3.0) or SDS sample buffer.

Endo H and PNGase F Digestion—Endo H and PNGase F digestion was performed according to the manufacturer's protocol (New England Biolabs). Cell lysates of the strains with epitope-tagged Ost1p and Wbp1p were used directly for Endo H and PNGase F digestion. For Stt3HAp wild type and mutants, immunoprecipitates were eluted from agarose beads using 0.1 M glycine (pH 3.0) and then digested with Endo H.

Mass Spectrometry—Microsomes from 2000 A660 units of Stt3HAp wild type cells were solubilized and immunoprecipitated as described above. To confirm the identity of the purified protein and to obtain evidence for its glycosylation, analysis by SDS-PAGE and peptide mapping was performed. The protein on the beads was reduced in the SDS sample buffer using Tris(2-carboxyethyl)phosphine hydrochloride (Pierce) and was then carboxyamidomethylated prior to loading onto a SDS-polyacrylamide gel. The band corresponding to Stt3HAp was identified by zinc acetate staining (Bio-Rad) gel, cut out, and digested with trypsin (Promega), essentially as described previously (10). The resulting peptides were desalted on a C18 ZipTip microcolumn (Millipore). The peptides were then fractionated by reversed phase liquid chromatography electrospray ionization mass spectrometry (MS) using a Zorbax C18 capillary column (0.3 mm diameter) (Agilent) with a 60 min/linear gradient from 0 to 95% Buffer B (in which Buffer A consisted of 0.1% formic acid, and Buffer B was 95% acetonitrile in 0.1% formic acid) at 5 µl/min. The column effluent was sprayed into an XCT (Agilent) ion trap mass spectrometer. MS/MS of the two major ions in each spectrum was performed automatically to identify peptides, and MS/MS/MS was performed on the glycosylated peptide to confirm its sequence and determine the site of the modification. The data were analyzed using data analysis software (Agilent) combined with online Mascot and Global Protein Machine data base searching programs.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Potential Glycosylation Sites in Ost1p, Wbp1p, and Stt3p—In the OT enzyme complex of S. cerevisiae, three of the nine subunits have been reported to be N-linked glycoproteins: Ost1p, Wbp1p, and Stt3p (1). Based on the PROSITE program, four subunits had potential N-glycosylation sites: Ost1p, Wbp1p, Ost6p, and Stt3p (Fig. 1). Among them, Ost1p had six predicted N-glycosylation sites (N18VS, N99ST, N217ET, N336FT, N400NS, and N473VT). However, the first predicted site (N18VS) was located in the signal peptide region (1–22 amino acid), and the last site (N473VT) was located in the cytosolic tail where N-glycosylation is not likely to occur. Wbp1p has two predicted N-glycosylation sites (N60ST and N332DS) that are located in the ER luminal domain and have the potential to be N-glycosylated. Ost6p has only one N-glycosylation site (N175IS). Based on the TMHMM (a method for predicting transmembrane helices based on a hidden Markov model) program, this site is located in the ER lumen and is a potential N-glycosylation site. Stt3p has three predicted N-glycosylation sites (N60NS, N535NT, and N539NT). All of them are located in the ER lumen and clearly are candidates for N-glycosylation.2 Interestingly, only one amino acid, Trp538, separates the second and third prospective N-glycosylation sites in Stt3p.



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  		FIG. 1.
Schematic diagram of the potential N-glycosylation sites of Ost1p, Wbp1p, Stt3p, and Ost6p. The sites where N-glycosylation occurs in wild type proteins are shown. Those that are circled are not glycosylated in the wild type proteins.

 

To determine whether the N-glycosylation sites in these subunits were conserved, alignment of the homologues of these proteins across eukaryotic species was carried out. In a confirmation of earlier studies (1, 11), we found that none of the N-glycosylation sites in Ost1p, Wbp1p, and Ost6p was conserved. In contrast, among the three predicted N-glycosylation sites of Stt3p, the second and third N-glycosylation sites (N535NT and N539NT) as well as the tryptophan residue separating them are highly conserved across eukaryotic species, ranging from mammals to yeast (Fig. 2). As also shown in Fig. 2, the sequences flanking the N-glycosylation sites are identical or similar across the eukaryotic species. This is also true of the highly conserved predicted catalytic or glycosylation site recognition domain of Stt3p (W516WDYG).



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  		FIG. 2.
Alignment of Stt3p shows that the predicted second and third N-linked glycosylation sites of S. cerevisiae are highly conserved across a wide variety of eukaryotic species. The alignment was carried out using the yeast proteome site. The two N-glycosylation sites are in boldface, and the highly conserved putative catalytic and/or N-glycosylation recognition site is in boldface and boxed.

 
Growth Phenotype and Analysis of N-Glycosylation Site Occupancy—To test the importance of N-glycosylation for the functionality of these proteins, we prepared single amino acid mutations in each, changing the Asn to the similar amino acid Gln at each N-glycosylation site (because OST6 is not an essential gene, we did not mutate its N-glycosylation site). The site and results with all the mutants are summarized in Table I. In Ost1p, in addition to the single amino acid mutations (N99Q, N217Q, N336Q, and N400Q), we prepared a double mutant (N99Q/N217Q) in which the first two N-glycosylation sites were mutated, and we also prepared a strain (N/Q) that was mutated at all four sites. In Wbp1p, in addition to single mutants N60Q and N332Q, a mutant was prepared in which both N-glycosylation sites were mutated (N/Q). In Stt3p, the Asn residues were mutated to Gln (N60Q, N535Q, and N539Q), and the Thr residues in the +2 position relative to Asn were also mutated to Ala at the adjacent N-glycosylation sites (T537A and T541A). A double mutation (T537A/N539Q) was also prepared to block both of the adjacent N-glycosylation sites of Stt3p.


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  		TABLE I
Growth phenotype of the glycosylation sites mutation on S. cerevisiae OT subunits

 
First, we tested the growth phenotype of all the mutants. Cells carrying the mutant plasmid (which contains the Trp gene) and wild type gene plasmid (which contains the Ura gene) were patched on –Trp–Ura medium plates and grown for 2 days at 25 °C and then replicated to –Trp+FOA plates to deplete the wild type gene. After incubation at 25 or 37 °C for 2 days, respectively, cell growth was assessed. As summarized in Table I, we found that all the strains bearing the ost1p, wbp1p mutants and the N60Q mutant of Stt3p grew at both temperatures. In further spotting assays, we did not detect any difference in growth between these mutants and the wild type strain (Fig. 3A). In contrast, mutations at the adjacent N-glycosylation sites, N535NT and N539NT of Stt3p, displayed a significant defect; cells carrying mutations N535Q, T537A, and N539Q did not grow at any temperature tested. Cells carrying mutation T541A displayed a severe temperature sensitivity; they barely grew at 25 °C and did not grow at all at 37 °C. This result was confirmed by further spotting assays (Fig. 3B).



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  		FIG. 3.
Comparison of the growth rate of the N-glycosylation site mutants of Ost1p, Wbp1p, and Stt3p. A, cells carrying the Ost1p and Wbp1p mutants were spotted on two –Trp plates and incubated at 25 °C or 37 °C for 2 days. B, cells carrying wild type (WT) or Stt3p mutant were spotted on one –Trp–Ura plate and two –Trp+FOA plates. The –Trp–Ura plate and one of the –Trp+FOA plates were incubated at 37 °C, and the other –Trp+FOA plate was incubated at 25 °C for 2 days.

 
Next, the effect of these mutations on N-linked glycosylation of the individual subunits was determined by either Endo H or PNGase F digestion followed by Western blot analysis. The yeast cells carrying the ost1p, wbp1p mutants or wild type gene were harvested and lysed, and Endo H or PNGase F digestion was carried out with the wild type proteins (Fig. 4, A and B). The cell lysates with and without digestion were analyzed on a SDS-polyacrylamide gel and blotted with monoclonal anti-HA antibody. As shown in Fig 4A, the wild type Ost1p and all four point mutants exhibit two bands. The double mutant of Ost1p also has two bands and shows lower molecular mass than the highest band of the four single mutants. The four-site mutant (N/Q) of Ost1p migrates the fastest. Similar results are observed with Wbp1p (Fig. 4B) in which the wild type Wbp1p exhibits three bands, and the single mutants (N60Q and N332Q) exhibit lower molecular mass than the wild type. The double mutant has one band almost at the same molecular weight as wild type Wbp1p, after Endo H digestion.



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  		FIG. 4.
Determination of the occupancy of the N-linked glycosylation sites on Ost1HAp, Wbp1HAp, and Stt3HAp by Western blot analysis. A, spheroplasts were prepared from each mutant strain and wild type (WT) control. Wild type spheroplasts were lysed and subjected to PNGase F digestion. Lysates with or without PNGase F digestion were separated on a SDS-polyacrylamide gel followed by Western blot analysis using anti-HA antibody (the slower migration of the protein after PNGase F treatment is probably the result of the decreased binding of SDS, because Asn was converted to Asp after PNGase F digestion). B, the cell lysates (treated with or without Endo H) of strains carrying Wbp1p, wild type, and mutants were analyzed on 10% SDS-PAGE and then after being transferred to membrane anti-HA antibody were used for Western blot to detect the proteins. The numbers (–1 and –2) indicate the loss of the expected number of N-glycan chains. C–E, spheroplasts were lysed with harsh buffer containing 1% Triton X-100, 0.2% SDS, immunoprecipitated with monoclonal anti-HA antibodies, and recovered on recombinant protein G-agarose beads. Bound proteins were eluted from the agarose beads with 0.1 M glycine (pH 3.0). Endo H and PNGase F digestion was carried out as recommended by the manufacturer. Samples treated with (+) or without (–) EndoH-PNGase were analyzed on 8% SDS-PAGE, and polyclonal anti-HA antibody was used for Western blot analysis.

 
Based on these results, we conclude that the four N-glycosylation sites of Ost1p (N99ST, N217ET, N336FT, and N400NS) and the two N-glycosylation sites of Wbp1p (N60ST and N332DS) are occupied in mature wild type proteins (see Fig. 1), but not all the sites have the same level of N-glycosylation. To determine whether Ost6p was N-glycosylated, triple HA tag was integrated in the C terminus of the OST6 gene in the chromosome. Cell lysates treated with Endo H revealed no change in mobility on SDS-PAGE, indicating the absence of N-glycosylation (data not shown).

We could not readily detect the protein Stt3HAp using cell lysates for the Western blot analysis. Therefore, we prepared spheroplasts, lysed them in 1% Triton X-100, 0.2% SDS, and immunoprecipitated them with anti-HA monoclonal antibody. The precipitates were analyzed on SDS-PAGE, and following transfer membranes were blotted with anti-HA polyclonal antibody. Stt3HAp always displays a very broad band, but this apparently is not the result of differential N-glycosylation, because the bands are still diffuse even after the removal of the N-glycans by Endo H treatment (Fig. 4C). By comparing the molecular mass of the mutants with or without Endo H digestion, we found that the wild type and all of the single mutants were glycosylated. However, the double mutant that was blocked at both of the adjacent N-glycosylation sites of Stt3p did not exhibit any shift. These results indicate that the first N-glycosylation site (N60NS) is not utilized, and when either the N535NT or N539NT is mutated, the other site is glycosylated. This conclusion was confirmed by further block mutations (Fig. 4D) in which no N-glycosylation occurred in block mutant 537AAAA540, which blocks both of the adjacent sites; however, N-glycosylation occurred in block mutant 541AAA543, which blocks the N-glycosylation site N539NT. This result strongly supports the conclusion that when N539NT is mutated, N535NT becomes glycosylated.

Oligosaccharyl Transferase Activity of the OT Subunit Mutants—Two assay methods were used to assess the OT activity of the mutants. One method was to check the N-glycosylation pattern of carboxypeptidase Y in different mutants in vivo. We found that all of the ost1p, wbp1p mutants and the first N-glycosylation site mutant of stt3p (N60Q) failed to exhibit any N-linked glycosylation defect. However, the stt3p mutant T541A displayed a significant underglycosylation pattern in carboxypeptidase Y (Fig. 5A). The in vitro assay using a synthetic peptide as the OT substrate confirmed the in vivo result, that only mutant T541A shows significantly impaired N-glycosylation activity, by a value of 20% compared with wild type (Fig. 5B).



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  		FIG. 5.
OT activity of the N-glycosylation sites mutants of Ost1p, Wbp1p, and Stt3p. A, wild type (WT) cell lysates were subjected to Endo H or PNGase F digestion. Cell lysates of the mutants and wild type (with and without Endo H or PNGase F digestion) were analyzed on 10% SDS-PAGE followed by Western blot analysis using anti-carboxypeptidase Y antibody. The numbers (–1 to –4) indicate the loss of the expected number of N-glycan chains. B, spheroplasts of the indicated mutants were used for the in vitro N-glycosylation activity assay, as described under "Materials and Methods."

 
What Causes the Growth Phenotype of the Stt3p Glycosylation Site Mutants?—Previously, we found that some OT subunits with certain mutations failed to be incorporated into the OT complex and that these mutations were lethal (9). To determine whether the lethality of the stt3p mutants was caused by its failure to be incorporated into the OT complex or its functional importance, co-immunoprecipitation under a mild detergent condition was performed. As shown in Fig. 6, all of the mutants are incorporated into the OT complex. Based on this observation we conclude that the N-glycosylation is not required for Stt3p to be incorporated into the OT complex.



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  		FIG. 6.
Co-immunoprecipitation indicates the incorporation of Stt3p mutants into the OT complex. Microsomes from cells carrying the wild type (WT) and mutant plasmids were solubilized under mild detergent conditions and immunoprecipitated (IP) with monoclonal anti-HA antibody. The precipitates were analyzed on SDS-PAGE followed by Western blot (IB) analysis using polyclonal anti-HA, anti-Ost1p, and anti-Wbp1p antibodies.

 
All the growth defects and the defects in N-glycosylation are observed only with the stt3p mutations in the region that is conserved among eukaryotic species. Obviously, the mutations that were prepared not only blocked the N-glycosylation but changed the amino acid sequence. How can we distinguish the cause of the phenotype? Is it the lack of N-glycosylation per se or an alteration in protein conformation caused by the changes in amino acid? Because almost all of the amino acids in this region are conserved, we undertook to prepare mutations in this region that only change the amino acids but do not block the N-glycosylation. Accordingly we prepared single mutations in the two N-glycosylation sites to change the middle Asn at position +1 to Gln (N536Q, N540Q) and the Thr at position +2 to Ser (T537S, T541S). After Endo H digestion, it was shown that all the mutants were glycosylated (Fig. 4E). Surprisingly, we found that none of the mutants showed any growth defect (Fig. 3B). Based on these findings, we conclude tentatively that N-glycosylation, rather than the amino acid sequence per se of these two N-glycosylation sites, is essential for the function of Stt3p.

Mass Spectrometric Analysis Showed That Only One N-Glycosylation Site Was Utilized in Wild Type Stt3p—MS was utilized to determine whether one or both of the adjacent N-glycosylation sites were in fact glycosylated. Based on the MS result, only one glycan was found in this fragment, indicating that only one N-glycosylation site was utilized in the wild type Stt3p. Both a triply charged ion of m/z 1272.9 Da and a quadruply charged ion of m/z 954.6 Da were detected (data not shown). They correspond in molecular mass to the tryptic peptide TTLVDNNTWNNTHIAIVGK, with one high mannose-type glycan of the composition Man8GlcNAc2. This composition of an N-linked glycan is in agreement with the localization of Stt3p in the S. cerevisiae endoplasmic reticulum. The triply charged ion (1272.9) was subjected to MS/MS, and the gradual losses of 162 Da (the mass of a hexose) and 203 Da (N-acetylhexosamine) confirmed the glycan sequence (Fig. 7). No ions indicating the unglycosylated or doubly glycosylated peptide were detected. In addition, the tryptic peptide YLVNNSFYK containing the potential N-glycosylation site N60NS, which is not glycosylated, based on biochemical methods, was found only in the unglycosylated state (doubly charged ion, molecular mass of 574.68 Da) (data not shown), in agreement with the results obtained above.



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  		FIG. 7.
MS/MS of the tryptic peptide TTLVDNNTWNNTHIAIVGK containing an N-linked Man8GlcNAc2 glycan. The triply charged parent ion of m/z 1272.9 Da was fragmented. The progressive loss of mannose (Man) residues and a GlcNAc residue are indicated.

 
Because the tryptic peptide TTLVDNNTWNNTHIAIVGK contains two potential N-glycosylation sites, MS/MS/MS was used to sequence the peptide and determine the site of glycan attachment. The peptide was sequenced, and the data that were obtained indicate that the N-glycan is attached to the C-terminal-most N-glycosylation site in the peptide, that is, residue Asn539 (Fig. 8). Two doubly charged diagnostic peptides (y13 and y11) were found, along with other peptides resulting from fragmentation of this peptide.



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  		FIG. 8.
MS/MS/MS on the daughter ion of m/z 1158.9, triply charged peptide TTLVDNNTWNNTHIAIVGK containing one GlcNAc residue (obtained in MS/MS after sugar losses from the triply charged ion of m/z 1272.7). The fragment ions are indicated in boldface. The ions proving the presence of N-linked glycan are underlined and marked with boldface italics in the inset table.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
It is known that three of the essential subunits, Ost1p, Wbp1p, and Stt3p, are N-linked glycoproteins in the yeast OT complex, but no information has been available regarding whether the N-glycosylation of these three subunits is important for OT activity. In addition, the extent of occupancy of the N-glycosylation sites in these proteins is not clear (1). In this study, we first focused on these issues. We confirmed that all four potential sites on Ost1p and two potential sites on Wbp1p are occupied in the mature proteins, but none of these N-glycosylation sites is important for OT function. In Stt3p, there are three potential N-glycosylation sites, among which two (N535NT and N539NT) are highly conserved across the eukaryotic species and are separated by one amino acid. There were major growth defects and a loss of OT activity when the N-glycosylation of the conserved adjacent sites was blocked by single point mutations on either site or at both sites. However, mass spectrometric analysis showed that only the N539NT of the two adjacent sites was glycosylated in the wild type strain.

It was not surprising to find that none of the N-glycosylation sites of Ost1p and Wbp1p was essential, because alignment showed that none of them is conserved among the eukaryotic species. However, two of the three N-glycosylation sites of Stt3p were highly conserved across eukaryotic species, implying that the N-glycosylation of these two sites and/or the amino acids in this region are important for Stt3p function. Our observations indicate that mutations in the N-glycosylation among the conserved sites result in cells exhibiting significant growth defects; they are either lethal or grow extremely slowly. However, when point mutations were prepared that altered the amino acid residues in the conserved sites but did not block the possibility of N-glycosylation, there was no effect on growth. Based on these results we conclude it is likely that the N-glycosylation per se is important for the function of Stt3p. Because the mutant proteins are still incorporated into the OT, the N-glycan chains are not required for interactions with the other proteins of the OT complex.

Our current observations imply that the N-glycosylation of Stt3p is important for its function. However, what is the possible function for the glycans on Stt3p? Many studies have demonstrated the contribution of the glycan chains to protein folding, structure, and function (12). Analysis of the secondary structure around N-glycosylation sites suggests that the glycans are located at positions where changes in secondary structure occur, and some aromatic amino acids, which are distant from the N-glycosylation sites in the primary sequence, become close in the tertiary structure. These observations suggest that the N-glycans may participate directly in the folding of the protein as a consequence of glycan-protein interactions (13). In vitro experiments (14–16) based on glycan-protein hydrophobic interactions also have provided evidence to support the concept that the glycans promote folding. In Stt3p, the predicted catalytic and/or recognition region containing two aromatic amino acids, W516W, is located approximately 20 amino acids N-terminal to the adjacent N-glycosylation sites, N535NTWN539NT. Based on two secondary structure prediction programs, nnPredict and PredictProtein, these two N-glycosylation sites are located on a loop that makes it flexible and possible for interaction with the catalytic site. Based on all of these results, we propose that the glycan at N539NT may be close to the catalytic site W516WDYG via the interaction between the glycan and the aromatic amino acids W516W (Fig. 1) and that this interaction may be important for the function of Stt3p.

Finally, we asked wether the two N-glycosylation sites in Stt3p, which are separated by only one amino acid, are both glycosylated or whether only one site is glycosylated. Based on the Western blot, it was clear that when either N-glycosylation site was blocked, the other one was glycosylated, and, as expected, when both sites were blocked, there was no glycosylation. However, it is very difficult to determine whether both of the adjacent sites in Stt3p or only one site is glycosylated based on Western blot analysis. Therefore, we utilized mass spectrometry to answer this question. Interestingly, we found that only the N539NT site was glycosylated in the wild type Stt3p. However, when a similar double glycosylation site, NSTWNST, was engineered into preprolactin, the region was glycosylated on both Asn residues in 60% of the molecules during in vitro synthesis, in the presence of dog pancreas rough microsomes.3 These observations suggest that a spatial constraint might exist to prevent the glycosylation of N535NT in Stt3p in vivo. Several decades ago, similar studies were performed in chicken ovalbumin, which has two N-glycosylation sites, Asn293 and Asn312, in which only the site Asn293 is glycosylated in vivo. However, both these sites are glycosylated in vitro (17). In chicken ovalbumin, further experiments suggest that the absence of an oligosaccharide chain on Asn293 does not promote N-glycosylation of Asn312 (18). However, in Stt3p, N-glycosylation of N535NT is affected by the N-glycosylation of N539NT; and in wild type Stt3p, the N535NT is prevented from N-glycosylation. The unmodified amino acid Asn535 may be involved directly in some function of Stt3p, and this function may require N-glycosylation of Asn539. We propose that unglycosylated Asn535 serves a function that is lost when it is glycosylated because of the block of the glycosylation of N539NT. This model is consistent with the fact that all the mutants in this region are lethal or severely sick at 25 °C, and the Asn535 is glycosylated when Asn539 glycosylation site is blocked. It is also consistent with the fact that high conservation of the two adjacent sites is observed across a wide range of eukaryotic species. However, the mechanism may be different from archaeabacteria, which has been reported to have N-glycosylation activity (3). The homologue of Stt3p in Campylobacter jejuni does not have two adjacent N-glycosylation sites. It only has one N-glycosylation site, N534QS, after the predicted catalytic site, W457WDYG.

In the current study, we found that 3 of the 10 N-glycosylation sites of the four OT subunits were not occupied (N175IS on Ost6p and N60NS and N535NT on Stt3p). Previous studies (17, 19, 20) indicated that N-glycosylation sites located less than 12–14 residues from a transmembrane segment of a protein are not modified in vivo, which also suggests that the active site of the OT is located 30–40 Å from the luminal face of the ER. In our case, the site of N175IS on Ost6p is located on the N terminus of the protein and is only 13 amino acids away from the N terminus of the first transmembrane domain. The N60NS on Stt3p is located on the first loop in the ER lumen, and it is 29 amino acids distant from the C terminus of the first transmembrane segment and 17 amino acids from the N terminus of the second transmembrane segment. This should be adequate space in primary sequence for N-glycosylation, and it is not clear why this site is not utilized. Statistical analysis indicated that not all potential N-glycosylation sites are occupied and that only 65% of the sites are glycosylated (13). It is particularly interesting that N535NT is not glycosylated in the wild type cells, whereas the N539NT site, which is separated from N535NT by only one amino acid, is glycosylated. We thought initially that the N-terminal-most N-glycosylation site N535NT would be favored for N-glycosylation. In fact, N535NT can be glycosylated when N539NT is blocked. But it is clear that this does not happen with wild type Stt3p. What causes the blockage and what function the conserved N535 serves remain unknown.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grants GM33185 (to W. J. L.) and GM61126 (to R. S. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} Both authors contributed equally to this work. Back

§ Present address: Dept. of Molecular Biology, The Scripps Research Institute, MEM-L71, La Jolla, CA 92037. Back

To whom correspondence should be addressed: Dept. of Biochemistry and Cell Biology, State University of New York, Stony Brook, NY 11794-5215. Tel.: 631-632-8560; Fax: 631-632-8575; E-mail: wlennarz{at}notes.cc.sunysb.edu.

1 The abbreviations used are: OT, oligosaccharyl transferase; 5-FOA, 5-fluoroorotic acid; HA, hemagglutinin; Endo H, endo-{beta}-N-acetylglucosaminidase H; MS/MS, tandem mass spectrometry; ER, endoplasmic reticulum; PNGase F, peptide N-glycosidase F; MS/MS/MS, MS/MS spectra subjected to the next round of MS. Back

2 H. Kim, personal communication. Back

3 I. Nilsson, personal communication. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Reid Gilmore (University of Massachusetts Medical School) for anti-Ost1p and anti-Wbp1p antibodies, Dr. Randy Schekman (University of California, Berkeley) for anti-carboxypeptidase Y antibody, and Dr. Manasi Chavan for the microsomes with Ost6p tagged with 3HA. We also thank Drs. Gunnar von Heijne (University of Stockholm) and Robert Noiva (University of South Dakota) and members of Dr. Lennarz' laboratory for their comments on this work.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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