Catalyzed by specific glycosyltransferases, the lipid-linked core oligosaccharide Glc3Man9GlcNAc2 is formed by the sequential addition of sugars from their activated derivates to the lipid carrier dolichyl-pyrophosphate (Kornfeld and Kornfeld, 1985; Tanner and Lehle, 1987; Herscovics and Orlean, 1993). The "one-linkage-one glycosyltransferase" hypothesis suggests that there is one distinct glycosyltransferase for every specific glycosidic linkage (Schachter, 1995). In the yeast Saccharomyces cerevisiae alg mutant strains (defective in asparagine-linked glycosylation), which are affected in different glycosyltransferases involved in the assembly of the lipid-linked core oligosaccharide, were identified (Herscovics and Orlean, 1993; Orlean, 1997; Burda and Aebi, 1999). A common characteristic of the different alg mutant strains is the accumulation of a biosynthetic oligosaccharide intermediate specific for the defective ALG locus. Based on the assumption that the accumulating intermediate is the acceptor oligosaccharide of the reaction affected, defined glycosyltransferase activities could be assigned to the different ALG loci (Burda and Aebi, 1999). In addition, mutations in ALG loci lead to underglycosylation of secreted proteins in vivo. The reason for this underglycosylation appears to be the decreased affinity of the oligosaccharyltransferase toward incompletely assembled oligosaccharides; however, these oligosaccharides are still transferred to protein, albeit with a strongly reduced efficiency.
The synthesis of a highly branched and complex oligosaccharide requires a well organized assembly pathway and the notion that "the complex type of biosynthesis of the carbohydrate component of glycoproteins is truly amazing, and it is very difficult to see at present how this sequence of enzymatic reactions involving controlled addition and deletion of sugars to and from the glycopeptide is regulated" (Neuenberger, 1995) also applies to the lipid-linked oligosaccharide (Figure
Figure 1. Structure and assembly pathway of the dolichylpyrophosphate-linked oligosaccharide Glc3Man9GlcNAc2. The stepwise synthesis occurs at the membrane of the ER catalyzed by a series of highly specific glycosyltransferases (encoded by ALG loci). The portion of the assembly pathway occurring at the lumenal side of the ER is shown. The linkage of each individual glycosyl residue and the loci coding for the corresponding glycosyltransferases are indicated. Mild hypoglycosylation of CPY protein and altered CPY glycoforms in [Delta]alg12 strains
Previously we reported on the identification and characterization of the ALG9 locus encoding an [alpha]-1,2 mannosyltransferase. It was proposed that Alg9p adds a mannose residue to the [alpha]-1,3-linked mannose (Burda et al., 1996)(Figure
Known alg mutant strains that are unable to complete the synthesis of the lipid-linked oligosaccharide at the luminal side of the ER lead to underglycosylation of secreted proteins because of the decreased affinity of the oligosaccharyltransferase (OTase) towards truncated lipid-linked oligosaccharides. In combination with a reduced OTase activity (wbp1 mutation) (Stagljar et al., 1994; Zufferey et al., 1995) these alg mutants showed a synthetic growth defect at 30°C due to a severe glycosylation deficiency. We therefore crossed the [Delta]alg12 strain with a wbp1-2 mutant strain and a tetratype tetrad was analyzed for growth and for N-glycosylation of secreted proteins. In contrast to previously analyzed alg wbp1 mutant strains, the resulting double mutant strain [Delta]alg12 wbp1-2 (YG843) was able to grow at 30°C (data not shown). When we examined the N-glycosylation of carboxypeptidase Y (CPY) by Western blot analysis (Figure
Figure 2. The alg12 mutation alters glycosylation of CPY in vivo. Four strains derived from a tetratype tetrad of a cross [Delta]alg12×wbp1-2 were used for CPY-specific immunoblotting. The relevant genotype of the strains is indicated above the lanes. The position of mature CPY (mCPY) and the different glycoforms lacking up to four N-linked oligosaccharides (-1 to -4) are given. The position of mature CPY derived from strains containing the [Delta]alg12 mutation (mCPY*) and the different glycoforms also are indicated (-1* to -3*). Strains: YG840 ([Delta]alg12, lane 1), YG841 (wt, lane 2), YG842 (wbp1-2, lane 3), YG843 ([Delta]alg12 wbp1-2, lane 4). [Delta]alg12 mutant strains accumulate lipid-linked Man7GlcNAc2 and low levels of lipid-linked Glc3Man7GlcNAc2
We analyzed the dolichol-linked oligosaccharides that accumulate in a [Delta]alg12 strain. For that purpose, we labeled [Delta]alg12 cells in vivo with [3H]mannose and isolated the radiolabeled lipid-linked oligosaccharide. After the release of the oligosaccharide from the lipid-carrier dolichol by acidic hydrolysis we separated the oligosaccharides by HPLC. Oligosaccharides of known structure were used as standards. Indeed, an altered biosynthesis of lipid-linked oligosaccharides was observed in the [Delta]alg12 strain (Figure
Figure 3. Analysis of lipid-linked oligosaccharides of the [Delta]alg12 mutant strain FHEN005-02C(A). Cells were labeled with [3H] mannose. Lipid-linked oligosaccharides were extracted and hydrolyzed, and the liberated oligosaccharides were analyzed by HPLC. Oligosaccharides isolated from strain SS328 (wild-type) and YG248 ([Delta]alg3) served as standards. The position of mannose (M), Man5,7,8GlcNAc2 (M5,7,8N2) and Glc3Man9GlcNAc2 (G3M9N2) are indicated. The structure of the corresponding oligosaccharides is illustrated. (A) Wild-type strain SS328. (B) [Delta]alg12 mutant strain FHEN005-02C(A). (C) [Delta]alg3 mutant strain YG248.
Figure 4. HPLC analysis of exo-[alpha]-1,2 mannosidase digestion products of [Delta]alg9 and [Delta]alg12 derived oligosaccharides. The radiolabeled oligosaccharides were prepared and digested with A.saitoi [alpha]-1,2 mannosidase. The digested oligosaccharide products were analyzed by HPLC. The positions of mannose (M), Man4GlcNAc2 (M4N2) Man6GlcNAc2 (M6N2) and Man7GlcNAc2 (M7N2) and the corresponding oligosaccharide structures are shown. (A) Oligosaccharide derived from [Delta]alg9 strain YG414. (B) Oligosaccharide from [Delta]alg9 strain (A) treated with exo-[alpha]-1,2 mannosidase. (C) Oligosaccharide derived from [Delta]alg12 strain YG839. (D) Oligosaccharide from [Delta]alg12 strain (C) treated with exo-[alpha]-1,2 mannosidase.
Earlier studies in yeast cells indicated that the oligosaccharide intermediate Man5GlcNAc2-PP-Dol can be glucosylated yielding Glc3Man5GlcNAc2-PP-Dol, albeit at a low level (Verostek et al., 1993a). Thus, we speculated that in [Delta]alg12 strains the accumulating Man7GlcNAc2 might be a (suboptimal) substrate for the Alg6p glucosyltransferase (Runge et al., 1984; Reiss et al., 1996) and subsequent glucosylation by Alg8p (Stagljar, I. et al., 1994) and Alg10p (Burda and Aebi, 1998) transferases might result in the formation of Glc3Man7GlcNAc2-PP-Dol, the minor peak observed in [Delta]alg12 strains (Figure
Figure 5. Lipid-linked Man7GlcNAc2 can be glucosylated in vivo. Oligosaccharides from [Delta]alg12 (B) and [Delta]alg12 [Delta]alg5 cells (C) overexpressing the Alg6p glucosyltransferase were analyzed. Oligosaccharides isolated from wild-type strain SS328 (A) served as standard. The positions of mannose (M), Man7GlcNAc2 (M7N2) Glc3Man7GlcNAc2 (G3M7N2) and Glc3Man9GlcNAc2 (G3M9N2) are indicated. The structure of the corresponding oligosaccharides are illustrated. (A) Wild-type strain SS328. (B) [Delta]alg12 mutant strain transformed with the high copy number plasmid pALG6 (YG845, SS328 background). (C) [Delta]alg12 [Delta]alg5 mutant strain transformed with the high copy number plasmid pALG6 (YG846). Glucosylation of intermediates of the LLO assembly pathway
The observation that the [alpha]-1,2-[alpha]-1,2-di-mannose branch in [Delta]alg12 strains can be glucosylated before completion of the Man9GlcNAc2 core prompted us to test whether glucosylation of oligosaccharides lacking the two Dol-P-Man derived di-mannose arms is also possible. For that purpose the Alg6p glucosyltransferase was overexpressed in both [Delta]alg3 and [Delta]alg9 mutant strains, the LLOs accumulating in these strains were analyzed by HPLC and further characterized by exo-[alpha]-1,2 mannosidase digestion (Figure
Figure 6. Glucosylation of incompletely mannosylated lipid-linked oligosaccharides. The Alg6p glucosyltransferase was overexpressed both in [Delta]alg3 and [Delta]alg9 mutant cells. The oligosaccharides from these strains were characterized by exo-[alpha]-1,2 mannosidase digestion followed by HPLC analysis. The position of mannose (M), Man3GlcNAc2 (M3N2), Man4GlcNAc2 (M4N2) Man5GlcNAc2 (M5N2), Man6GlcNAc2 (M6N2), Glc3Man5GlcNAc2 (G3M5N2), and Glc3Man6GlcNAc2 (G3M6N2) and the corresponding oligosaccharide structures are given. (A) Oligosaccharides isolated from [Delta]alg3 strain overexpressing Alg6p glucosyltransferase (YG859). (B) Oligosaccharides isolated from [Delta]alg3 strain overexpressing Alg6p glucosyltransferase digested with exo-[alpha]-1,2 mannosidase. (C) Oligosaccharides isolated from [Delta]alg9 strain overexpressing Alg6p glucosyltransferase (YG849). (D) Oligosaccharides from [Delta]alg9 strain overexpressing Alg6p glucosyltransferase digested with exo-[alpha]-1,2 mannosidase. Glucosylated lipid-linked oligosaccharide intermediates are transferred more efficiently to protein in vivo
It has been demonstrated both in vitro (Murphy and Spiro, 1981) and in vivo (Burda and Aebi, 1998) that glucosylation of the lipid-linked oligosaccharide, in particular the presence of the terminal [alpha]-1,2-linked glucose, is necessary for efficient transfer of the core oligosaccharide to protein. To test a fully glucosylated, but partially mannosylated lipid-linked oligosaccharide as a substrate for the oligosaccharyltransferase in vivo, we analyzed the glycosylation of the model protein CPY in different [Delta]alg3 mutant strains (Figure
Figure 7. N-linked glycosylation of CPY in [Delta]alg3 strains overexpressing Alg6p glucosyltransferase. [Delta]alg3 single mutant and [Delta]alg3 wbp1-2 double mutant strain, respectively, were transformed with the high copy number plasmid pALG6. Glycosylation of CPY was analyzed by CPY-specific immunoblotting. CPY isolated from the pALG6 transformed mutant cells was compared to CPY molecules of untransformed mutant strains. The relevant genotype of the strains is indicated above the lanes. The position of mature CPY (mCPY) and the different glycoforms lacking up to three N-linked oligosaccharides (-1 to -3) are indicated. Strains: YG248 ([Delta]alg3, lane 1), YG859 ([Delta]alg3 + pALG6, lane 2), YG857 ([Delta]alg3 wbp1-2, lane 3), YG858 ([Delta]alg3 wbp1-2 + pALG6, lane 4). Acceptor specificity of Alg12p mannosyltransferase
As shown above, efficient glucosylation of the lipid-linked oligosaccharide requires the completion of the Man9GlcNAc2 structure. Nevertheless, addition of glucose residues occurred on an earlier intermediate in the biosynthesis, however with reduced efficiency. Likewise, in principle it is possible to add either [alpha]-1,3- or [alpha]-1,6 mannose to the [alpha]-1,6 mannose of the lipid-linked Man5GlcNAc2 which is (according to the currently accepted topological model of LLO biosynthesis) translocated into the lumen of the ER. However, the accumulation of Man5GlcNAc2-PP-Dol in a [Delta]alg3 strain and of Man6GlcNAc2-PP-Dol in a [Delta]alg9 strain, respectively, suggests that the order of addition is determined by the specificity of the [alpha]-1,6 mannosyltransferase Alg12p: no oligosaccharide containing the [alpha]-1,6-linked mannose was detected in an [Delta]alg3 strain (Verostek et al., 1993b). We therefore asked whether overexpression of the Alg12p activity in a [Delta]alg3 or in a alg9 mutant strain may result in oligosaccharide intermediates containing the [alpha]-1,6 mannose. For that purpose [Delta]alg3 and [Delta]alg9 cells were transformed with a high copy number vector carrying the ALG12 locus, the resulting strains were labeled with [3H]-mannose and the radiolabeled oligosaccharides isolated from these strains analyzed by HPLC. The Man5GlcNAc2 oligosaccharide was found in [Delta]alg3 cells overexpressing Alg12p (Figure
Figure 8. Analysis of lipid-linked oligosaccharides in alg mutant strains overexpressing Alg12p. Lipid-linked oligosaccharides accumulating in [Delta]alg9 and [Delta]alg3 cells overexpressing Alg12p mannosyltransferase were isolated and analyzed using HPLC. The structure of the oligosaccharides was investigated by exo-[alpha]-1,2 mannosidase digestion. The position of mannose (M), Man3GlcNAc2 (M3N2), Man4GlcNAc2 (M4N2) Man5GlcNAc2 (M5N2), Man6GlcNAc2 (M6N2), and Man7GlcNAc2 (M7N2) is shown and the corresponding oligosaccharide structures are indicated. (A) Oligosaccharides isolated from [Delta]alg3 strain transformed with pALG12 (YG855) (A) and treated with [alpha]-1,2 mannosidase (B). Oligosaccharides isolated from [Delta]alg9 strain transformed with pALG12 (YG848) (C) and treated with [alpha]-1,2 mannosidase (D).
Our results demonstrate that the Alg3p and Alg12p mannosyltransferase clearly differ in their acceptor oligosaccharide specificity, because the addition of the [alpha]-1,6-linked mannose by Alg12p requires a minimal structure including the [alpha]-1,3-linked mannose residue added by Alg3p. Moreover, these experiments provide further evidence that the ALG12 locus indeed encodes a mannosyltransferase, because overexpression of this protein in a [Delta]alg9 strain results in a novel lipid-linked oligosaccharide intermediate.
The assembly of lipid-linked Glc3Man9GlcNAc2 takes place at the membrane of the endoplasmic reticulum (ER). Current topological models suggest that the first part of lipid-linked oligosaccharide biosynthesis takes place at the cytoplasmic side of the ER membrane, whereas the synthesis continues in the lumen after flipping of the lipid-linked Man5GlcNAc2 intermediate across the ER membrane. In this discussion, we will focus on the assembly pathway of the oligosaccharide after the translocation of Man5GlcNAc2-PP-Dol to the lumen of the ER. Four mannose and three glucose residues are added by specific glycosyltransferases using as substrates dolichylphosphate-activated mannose and glucose, respectively. In recent years, different yeast loci have been identified which are supposed to encode such specific mannosyl- or glucosyltransferases (Orlean, 1997; Burda and Aebi, 1999). In this report, we describe the ALG12 locus encoding a novel [alpha]-1,6 mannosyltransferase involved in the biosynthesis of the lipid-linked oligosaccharide. ALG12 deletion strains are not able to synthesize the complete lipid-linked core oligosaccharide Glc3Man9GlcNAc2, but accumulate as a major product Man7GlcNAc2-PP-Dol. Our observation that overexpression of the ALG12 locus in a [Delta]alg9 strain results in a novel oligosaccharide species containing one additional mannose residue which is normally not present in [Delta]alg9 cells, strongly suggests that Alg12p is indeed the Dol-P-Man dependent [alpha]-1,6 mannosyltransferase. The altered mobility of glycoforms of the marker protein CPY in [Delta]alg12 cells as compared to wild-type cells shows that incompletely assembled oligosaccharide is transferred to protein. However, we detected glucosylated oligosaccharide intermediates (Glc3Man7GlcNAc2) in [Delta]alg12 mutant strains. This observation might explain the mild hypoglycosylation phenotype noticed in these cells, because the presence of the terminal [alpha]-1,2 linked glucose on the glucosylated oligosaccharide intermediate makes it a good substrate of the oligosaccharyltransferase (OTase) complex. Consistent with this observation, the [Delta]alg12 mutation does not synthetically interact with the OTase mutation wbp1-2 and was therefore not identified in a screen directed towards mutants with altered biosynthesis of lipid-linked oligosaccharides (Zufferey et al., 1995). The preference for glucosylated lipid-linked oligosaccharides also has been observed in higher eukaryotes. A CHO mutant cell line unable to synthesize Dol-P-Man accumulated Man5GlcNAc2-PP-Dol; however, a minor proportion of lipid-linked oligosaccharide was glucosylated yielding Glc3Man5GlcNAc2. Analysis of the protein-bound oligosaccharides revealed that, nevertheless, the glucosylated species were preferentially transferred to the nascent polypeptide chain (Stoll et al., 1992).
alg12 mutant strains have been found previously in a screen for mutants with altered cell wall biogenesis (Lussier et al., 1997). Due to the essential role of mannoproteins in yeast cell wall biogenesis (Klis, 1994) the incomplete oligosaccharide structure which is transferred to protein in [Delta]alg12 cells might be the cause of this cell wall phenotype.
We have identified the ALG12 locus by searching the available databases for sequences similar to the ALG9 protein. As all other ER glycosyltransferases using dolichylphosphate-activated hexose, Alg12p is a highly hydrophobic protein. In addition, Alg12p possesses a putative N-terminal signal sequence (von Heijne, 1986) directing the import of the protein into the ER membrane. The database search revealed a family of yeast proteins with a common sequence motif (Canivence-Gansel et al., 1998). The homologous proteins Gpi10p and Smp3p are essential for viability and both encode Dol-P-Man-dependent mannosyltransferases that are located in the ER and involved in the biosynthesis of GPI anchors (Benghezal et al., 1995; Canivence-Gansel et al., 1998; Sütterlin et al., 1998; Riezman, personal communication). The sequence motif shared by this mannosyltransferase family might represent a recognition sequence for the common substrate Dol-P-Man. However, we did not detect this motif in the sequence of the Dol-P-Man-dependent ALG3 mannosyltransferase.
As soon as the core oligosaccharide is transferred to protein, trimming of the protein-linked Glc3Man9GlcNAc2 occurs in the ER by the enzymes glucosidase I, glucosidase II, and mannosidase I (for a review, see Moremen et al., 1994). In recent years, it has become more and more evident that individual sugar residues of the protein-bound oligosaccharide are required for specific functions in the endoplasmic reticulum (Helenius et al., 1997; Jakob et al., 1998). In particular, the Glc1Man9GlcNAc2 structure is specifically recognized by calnexin and calreticulin, a function required in the quality control of glycoprotein folding. In vivo studies by Verostek and co-workers (Verostek et al., 1993b) showed that the complete Man9GlcNAc2 core might be important for efficient glucose trimming of protein-bound oligosaccharides. Other studies on the yeast ER processing of oligosaccharides revealed that mannosidase I activity was dependent upon the terminal [alpha]-1,2-linked mannose of the [alpha]-1,6-[alpha]-1,2 di-mannose branch of protein-linked oligosaccharide (Ziegler and Trimble, 1991). In addition, recognition and degradation of malfolded glycoproteins in the ER in yeast seems to be dependent on the correctly processed Man8GlcNAc2 oligosaccharide structure (Knop et al., 1996). Therefore, the transfer of fully assembled Glc3Man9GlcNAc2 oligosaccharide to protein has to be ensured.
The selective transfer of only complete assembled oligosaccharide is guaranteed by the substrate specificity of the oligosaccharyltransferase (Silberstein and Gilmore, 1996). However, reduced transfer rate of lipid-linked biosynthetic oligosaccharide intermediates to protein has been observed both in vitro and in vivo. Small oligosaccharides such as chitobiose (GlcNAc2; Sharma et al., 1981) and different oligosaccharide intermediates synthesized by a series of alg mutant strains (Burda and Aebi, 1999) are transferred to protein, albeit at a reduced level. For efficient transfer, the terminal [alpha]-1,2-linked glucose residue of the lipid-linked oligosaccharide is required, and from our in vivo data we estimate that oligosaccharides lacking this terminal glucose are transferred to protein with a 10-fold reduced efficiency (Burda and Aebi, 1998). Based on these data, we speculate that the OTase recognizes the oligosaccharide substrate via two structurally distant motifs, the chitobiose stem and the terminal [alpha]-1,2 glucose residue. Our observation that N-linked protein glycosylation is an efficient process in [Delta]alg12 cells (which accumulate Glc3Man7GlcNAc2) also suggests the di-mannose side branches contribute little to the substrate recognition by the OTase. It is therefore essential that the biosynthesis of the lipid-linked oligosaccharide follows a highly defined pathway which terminates in the addition of the [alpha]-1,2-linked glucose residue by the ALG10 glucosyltransferase.
This precision in the assembly of lipid-linked Glc3Man9GlcNAc2 is achieved by the high specificity of the ER glycosyltransferases towards their lipid-linked oligosaccharide substrates. Our in vivo data obtained in yeast demonstrate that the [alpha]-1,3-[alpha]-1,2 di-mannose arm is assembled prior to the [alpha]-1,6-[alpha]-1,2 di-mannose antenna resulting in lipid-linked Man9GlcNAc2 (Figure
The fact that incomplete mannosylated oligosaccharide intermediates can be glucosylated raises an interesting aspect of the substrate specificity of the Alg6p glucosyltransferase that adds the first glucose residue to lipid-linked Man9GlcNAc2 (Runge et al., 1984; Reiss et al., 1996). The significant amount of Glc3Man7GlcNAc2 observed in [Delta]alg12 mutant cells and the absence of detectable amounts of glucosylated LLO in [Delta]alg3, [Delta]alg9, and [Delta]alg9 mutant strain overexpressing Alg12p suggests that the [alpha]-1,3-[alpha]-1,2 di-mannose antenna of the oligosaccharide is an important determinant of Alg6p substrate specificity. However, the [alpha]-1,6-[alpha]-1,2 di-mannose branch must be recognized as well, because pronounced glucosylation of Man7GlcNAc2 is detected only under specific conditions. This specificity of Alg6p ensures that in wild-type cells the first glucose is predominantly attached to the fully assembled Man9GlcNAc2 core. The Alg6p acceptor specificity therefore represents a checkpoint for complete mannosylated oligosaccharide intermediates. Furthermore, Alg6p also has to recognize the outer [alpha]-1,3- [alpha]-1,2-[alpha]-1,2 tri-mannose arm in order to avoid glucose addition to the other terminal [alpha]-1,2 mannose residues. This suggests that several of the mannose residues of the Man9GlcNAc2 oligosaccharide determine the substrate specificity for the Alg6p mediated glucosylation reaction. How such a complex substrate recognition by the highly hydrophobic ALG6 protein is achieved requires further investigation. The addition of the last two glucoses catalyzed by Alg8p and Alg10p, respectively (Stagljar et al., 1994; Burda and Aebi, 1998), seems to be independent of the mannosylation state of the oligosaccharide, because we did not observe mono-or-di-glucosylated Man5-7GlcNAc2-PP-Dol. This suggests that the presence of the first [alpha]-1,2-linked glucose residue is sufficient for Alg8p acceptor specificity. Even though Alg8p shares significant sequence homology to Alg6p, the activity is highly specific for the addition of the second [alpha]-1,3 glucose (Reiss et al., 1996). Also the Alg10p glucosyltransferase adding the terminal [alpha]-1,2 glucose to the lipid-linked oligosaccharide shows a very stringent acceptor specificity towards its acceptor Glc2Man9GlcNAc2-PP-Dol (Burda and Aebi, 1998). Taken together, the highly ordered LLO assembly pathway and the specific substrate recognition by the OTase ensures that only completely assembled and correctly branched oligosaccharide is transferred to protein.
Table I.
The high conservation of the lipid-linked oligosaccharide structure required for N-linked protein glycosylation suggests that individual sugar residues fulfill specific functions in glycoprotein processing. Using the combination of alg mutant strains and overexpression of glycosyltransferases, we are now in a position to genetically tailor the structure of both lipid-linked and protein-bound oligosaccharides in yeast. This will make it possible to address the functions of the individual sugar residues in vivo. Yeast strains and media
The strains of Saccharomyces cerevisiae used in this study are listed in Table I. The pALG6 plasmid has beeb described (Reiss et al., 1996). Standard yeast media and genetic techniques were applied (Guthrie and Fink, 1991). Isolation and disruption of the ALG12 locus
The ALG12 gene (GenBank Accession No. 1302525, ORF YNR030w) was isolated using the gap repair strategy resulting in plasmid pYCG_NR030w, which includes a 2.3 kb NotI fragment (bp 10-2312) containing the complete ALG12 ORF (bp 405-2060). The disruption of the ALG12 gene in strain SS328 was performed according to the PCR-based gene disruption using the KanMX4-module (Wach et al., 1994). A 1.77-kb-long PCR fragment was amplified using pFA6a-KanMX4 as template and two primers (primer 1: 5[prime]-AAAAGAGTTGAATAAAGCCATTAAACAACGATTCAGTTGACATCGATGAATTCGAGCTC-3[prime]; primer 2: 5[prime]-GCTCGCTATATATTTTATTGGAATTGACGTTAGCTATTATCACGTACGCTGCAGGTCGAC-3[prime]) (bold sequences: homologous to pFA6a-KAnMX4; other sequences: homologous either to the region directly upstream of the startcodon (primer 1) or to the region directly downstream to the stop codon (primer 2) of the ALG12 ORF. Construction of a high copy number plasmid overexpressing Alg12p
Plasmid pYCG_NR030w (see above) was cut with NotI generating a 2.3 kb fragment that contained the complete ALG12 ORF. This fragment was cloned into the vector pRS306. The resulting plasmid was cut with KpnI and SacI (flanking the NotI sites) generating a 2.3 kb fragment which was cloned into the high copy number vector YEp352 resulting in plasmid pALG12. Immunological techniques
Western-blot analysis was performed as described previously (Burda et al., 1996) using anti-CPY specific antibodies. Extraction and analysis of lipid-linked oligosaccharides of various yeast strains
In vivo metabolic labeling with [3H]mannose (20 Ci/mmol; ICN Pharmaceuticals), extraction of lipid-linked oligosaccharides, and HPLC analysis of the radiolabeled oligosaccharides were performed as described previously (Burda and Aebi, 1998). Oligosaccharide cleavage with exo-[alpha]-1,2 mannosidase
Digestion of radiolabeled oligosaccharides with exo-[alpha]-1,2 mannosidase (from Aspergillus saitoi; Oxford GlycoSciences) was performed with 2.5 µU of enzyme in 60 µl 100 mM sodium acetate pH 5.0 for 24 h at 37°C. To inactivate the enzyme the samples were heated for 5 min at 95°C and filtered through a 0.45 µm filter (Millipore UFC3OHV00) prior to analysis of the digested oligosaccharides by HPLC.
We thank Prof. Howard Riezman for bringing the ORF YNR030w to our attention. We thank Prof. Howard Riezman, Prof. Andreas Conzelmann and Prof. Rob Trimble for sharing results prior to publication and Dr. Rob Boulianne for critically reading the manuscript. This work was supported by the Swiss National Science Foundation to M.A. (Grant 3100-040350.94).
CPY, carboxypeptidase Y; Dol, dolichol; Dol-P-Glc, dolichyl-phosphoglucose; Dol-P-Man, dolichyl-phosphomannose; ER, endoplasmic reticulum; LLO, lipid-linked oligosaccharide; -PP-Dol, dolichyl-pyrophosphate-linked; OTase, oligosaccharyltransferase.
Results
Discussion
Strain
Genotype
Reference
SS328
Mat[alpha] ade2-101 ura3-52 his3[Delta]200 lys2-801
Vijayraghavan et al., 1989
FHEN005-02C(A)
Mata ura3-52 trp1-63 [Delta]alg12::kanMX4-loxP
This study
YG839
Mata ade2-101 ura3-52 his3[Delta]200 tyr1 [Delta]alg12::kanMX4
This study
YG840
Mata ade2-101 ura3-52 his3[Delta]200 lys2-801 [Delta]alg12::kanMX4
This study
YG841
Mat[alpha] ade2-101 ade3 ura3-52 his3[Delta]200
This study
YG842
Mata ade2-101 ura3-52 his3[Delta]200 leu2 lys2-801 wbp1-2
This study
YG843
Mat[alpha] ade2-101 ade3 ura3-52 his3[Delta]200 leu2 [Delta]alg12::kanMX4 wbp1-2
This study
YG844
Mat[alpha] ade2-101 ura3-52 his3[Delta]200 tyr1 [Delta]alg12::kanMX4 pYEp352
This study
YG845
Mat[alpha] ade2-101 ura3-52 his3[Delta]200 lys2-801 [Delta]alg12::kanMX4 p[ALG6]
This study
YG846
Mat[alpha] ade2-101 ura3-52 his3[Delta]200 [Delta]alg12::kanMX4 [Delta]alg5::HIS3 p[ALG6]
This study
YG414
Mat[alpha] ade2-101 ura3-52 his3[Delta]200 lys2-801 [Delta]alg9::kanMX4
Burda et al., 1996
YG847
Mat[alpha] ade2-101 ura3-52 his3[Delta]200 lys2-801 [Delta]alg9::kanMX4 pYEp352
This study
YG848
Mat[alpha] ade2-101 ura3-52 his3[Delta]200 lys2-801 [Delta]alg9::kanMX4 p[ALG12]
This study
YG849
Mat[alpha] ade2-101 ura3-52 his3[Delta]200 lys2-801 [Delta]alg9::kanMX4 p[ALG6]
This study
YG850
Mata ade2-101 ura3-52 his3[Delta]200 lys2 [Delta]alg9::kanMX4 [Delta]alg5::HIS3 p[ALG6]
This study
YG851
Mat[alpha] ade2-101 ura3-52 his3[Delta]200 [Delta]alg9::kanMX4 [Delta]alg12::kanMX4
This study
YG852
Mat[alpha] ade2-101 ade3 ura3-52 his3[Delta]200 lys2 [Delta]alg9::kanMX4 wbp1-2 pYEp352
This study
YG853
Mat[alpha] ade2-101 ade3 ura3-52 his3[Delta]200 lys2 [Delta]alg9::kanMX4 wbp1-2 p[ALG6]
This study
YG248
Mata ade2-101 ura3-52 his3[Delta]200 lys2 [Delta]alg3::HIS3
This study
YG854
Mata ade2-101 ura3-52 his3[Delta]200 lys2 [Delta]alg3::HIS3 p[ALG8]
This study
YG855
Mata ade2-101 ura3-52 his3[Delta]200 lys2 [Delta]alg3::HIS3 p[ALG12]
This study
YG856
Mata ade2-101 ura3-52 his3[Delta]200 lys2 [Delta]alg3::HIS3 [Delta]alg5::HIS3 p[ALG6]
This study
YG857
Mat[alpha] ade2-101 ura3-52 his3[Delta]200 lys2 [Delta]alg3::HIS3 wbp1-2 pYEp352
This study
YG858
Mat[alpha] ade2-101 ura3-52 his3[Delta]200 lys2 [Delta]alg3::HIS3 wbp1-2 p[ALG6]
This study
YG859
Mata ade2-101 ura3-52 his3[Delta]200 lys2 [Delta]alg3::HIS3 p[ALG6]
This study
Materials and methods
Acknowledgments
Abbreviations
References
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