The essential OST2 gene encodes the 16-kD subunit of the yeast oligosaccharyltransferase, a highly conserved protein expressed in diverse eukaryotic organisms

Oligosaccharyltransferase catalyzes the transfer of a preassembled high mannose oligosaccharide from a dolichol-oligosaccharide donor to consensus glycosylation acceptor sites in newly synthesized proteins in the lumen of the rough endoplasmic reticulum. The Saccharomyces cerevisiae oligosaccharyltransferase is an oligomeric complex composed of six non-identical subunits (alpha-zeta). The alpha, beta, gamma, and delta subunits of the oligosaccharyltransferase are encoded by the OST1, WBP1, OST3, and SWP1 genes, respectively. Here we describe the functional characterization of the OST2 gene that encodes the epsilon- subunit of the oligosaccharyltransferase. Genomic disruption of the OST2 locus was lethal in haploid yeast showing that expression of the Ost2 protein is essential for viability. Overexpression of the Ost2 protein suppresses the temperature-sensitive phenotype of the wbp1-2 allele and increases in vivo and in vitro oligosaccharyltransferase activity in a wbp1-2 strain. An analysis of a series of conditional ost2 mutants demonstrated that defects in the Ost2 protein cause pleiotropic underglycosylation of soluble and membrane-bound glycoproteins. Microsomal membranes isolated from ost2 mutant yeast show marked reductions in the in vitro transfer of high mannose oligosaccharide from exogenous lipid-linked oligosaccharide to a glycosylation site acceptor tripeptide. Surprisingly, the Ost2 protein was found to be 40% identical to the DAD1 protein (defender against apoptotic cell death), a highly conserved protein initially identified in vertebrate organisms. The protein sequence of ost2 mutant alleles revealed mutations at highly conserved residues in the Ost2p/DAD1 protein sequence.     

The alpha subunit of the Saccharomyces cerevisiae oligosaccharyltransferase complex is essential for vegetative growth of yeast and is homologous to mammalian ribophorin I

Oligosaccharyltransferase mediates the transfer of a preassembled high mannose oligosaccharide from a lipid-linked oligosaccharide donor to consensus glycosylation acceptor sites in newly synthesized proteins in the lumen of the rough endoplasmic reticulum. The Saccharomyces cerevisiae oligosaccharyltransferase is an oligomeric complex composed of six nonidentical subunits (alpha-zeta), two of which are glycoproteins (alpha and beta). The beta and delta subunits of the oligosaccharyltransferase are encoded by the WBP1 and SWP1 genes. Here we describe the functional characterization of the OST1 gene that encodes the alpha subunit of the oligosaccharyltransferase. Protein sequence analysis revealed a significant sequence identity between the Saccharomyces cerevisiae Ost1 protein and ribophorin I, a previously identified subunit of the mammalian oligosaccharyltransferase. A disruption of the OST1 locus was not tolerated in haploid yeast showing that expression of the Ost1 protein is essential for vegetative growth of yeast. An analysis of a series of conditional ost1 mutants demonstrated that defects in the Ost1 protein cause pleiotropic underglycosylation of soluble and membrane-bound glycoproteins at both the permissive and restrictive growth temperatures. Microsomal membranes isolated from ost1 mutant yeast showed marked reductions in the in vitro transfer of high mannose oligosaccharide from exogenous lipid-linked oligosaccharide to a glycosylation site acceptor tripeptide. Microsomal membranes isolated from the ost1 mutants contained elevated amounts of the Kar2 stress-response protein.     

The oligosaccharyltransferase complex from Saccharomyces cerevisiae. Isolation of the OST6 gene, its synthetic interaction with OST3, and analysis of the native complex.

The key step of N-glycosylation of proteins, an essential and highly conserved protein modification, is catalyzed by the hetero-oligomeric protein complex oligosaccharyltransferase (OST). So far, eight genes have been identified in Saccharomyces cerevisiae that are involved in this process. Enzymatically active OST preparations from yeast were shown to be composed of four (Ost1p, Wbp1p, Ost3p, Swp1p) or six subunits (Ost2p and Ost5p in addition to the four listed). Genetic studies have disclosed Stt3p and Ost4p as additional proteins needed for N-glycosylation. In this study we report the identification and functional characterization of a new OST gene, designated OST6, that has homology to OST3 and in particular a strikingly similar membrane topology. Neither gene is essential for growth of yeast. Disruption of OST6 or OST3 causes only a minor defect in N-glycosylation, but an Deltaost3Deltaost6 double mutant displays a synthetic phenotype, leading to a severe underglycosylation of soluble and membrane-bound glycoproteins in vivo and to a reduced OST activity in vitro. Moreover, each of the two genes has also a specific function, since agents affecting cell wall biogenesis reveal different growth phenotypes in the respective null mutants. By blue native electrophoresis and immunodetection, a approximately 240-kDa complex was identified consisting of Ost1p, Stt3p, Wbp1p, Ost3p, Ost6p, Swp1p, Ost2p, and Ost5p, indicating that probably all so far identified OST proteins are constituents of the OST complex. It is also shown that disruption of OST3 and OST6 leads to a defect in the assembly of the complex. Hence, the function of these genes seems to be essential for recruiting a fully active complex necessary for efficient N-glycosylation.     

The 3.4-kDa Ost4 protein is required for the assembly of two distinct oligosaccharyltransferase complexes in yeast

In the central reaction of N-linked glycosylation, the oligosaccharyltransferase (OTase) complex catalyzes the transfer of a lipid-linked core oligosaccharide onto asparagine residues of nascent polypeptide chains in the lumen of the endoplasmic reticulum (ER). The Saccharomyces cerevisiae OTase has been shown to consist of at least eight subunits. We analyzed this enzyme complex, applying the technique of blue native gel electrophoresis. Using available antibodies, six different subunits were detected in the wild-type (wt) complex, including Stt3p, Ost1p, Wbp1p, Swp1p, Ost3p, and Ost6p. We demonstrate that the small 3.4-kDa subunit Ost4p is required for the incorporation of either Ost3p or Ost6p into the complex, resulting in two, functionally distinct OTase complexes in vivo. Ost3p and Ost6p are not absolutely required for OTase activity, but modulate the affinity of the enzyme toward different protein substrates.     

Yeast oligosaccharyltransferase consists of two functionally distinct sub-complexes, specified by either the Ost3p or Ost6p subunit

The key step of N-glycosylation of proteins in the endoplasmic reticulum is catalyzed by the hetero-oligomeric protein complex oligosaccharyltransferase (OST). It transfers the lipid-linked core-oligosaccharide to selected Asn-X-Ser/Thr-sequences of nascent polypeptide chains. Biochemical and genetic approaches have revealed that OST from Saccharomyces cerevisiae consists of nine subunits: Wbp1p, Swp1p, Stt3p, Ost1p, Ost2p, Ost4p, Ost5p, Ostp3 and Ost6p. By blue native polyacrylamide electrophoresis we show that yeast OST consists of two isoforms with distinct functions differing only in the presence of the two related Ost3 and Ost6p proteins. The OST6-complex was found to be important for cell wall integrity and temperature stress. Ost3p and Ost6p are not essential for OST activity, and can in part displace each other in the complex when overexpressed, suggesting a dynamic regulation of the complex formation.     

An evolving view of the eukaryotic oligosaccharyltransferase

Asparagine-linked glycosylation (ALG) is one of the most common protein modification reactions in eukaryotic cells, as many proteins that are translocated across or integrated into the rough endoplasmic reticulum (RER) carry N-linked oligosaccharides. Although the primary focus of this review will be the structure and function of the eukaryotic oligosaccharyltransferase (OST), key findings provided by the analysis of the archaebacterial and eubacterial OST homologues will be reviewed, particularly those that provide insight into the recognition of donor and acceptor substrates. Selection of the fully assembled donor substrate will be considered in the context of the family of human diseases known as congenital disorders of glycosylation (CDG). The yeast and vertebrate OST are surprisingly complex hetero-oligomeric proteins consisting of seven or eight subunits (Ost1p, Ost2p, Ost3p/Ost6p, Ost4p, Ost5p, Stt3p, Wbp1p, and Swp1p in yeast; ribophorin I, DAD1, N33/IAP, OST4, STT3A/STT3B, Ost48, and ribophorin II in mammals). Recent findings from several laboratories have provided overwhelming evidence that the STT3 subunit is critical for catalytic activity. Here, we will consider the evolution and assembly of the eukaryotic OST in light of recent genomic evidence concerning the subunit composition of the enzyme in diverse eukaryotes.     

Oligosaccharyltransferase isoforms that contain different catalytic STT3 subunits have distinct enzymatic properties

Oligosaccharyltransferase (OST) is an integral membrane protein that catalyzes N-linked glycosylation of nascent proteins in the lumen of the endoplasmic reticulum. Although the yeast OST is an octamer assembled from nonhomologous subunits (Ost1p, Ost2p, Ost3p/Ost6p, Ost4p, Ost5p, Wbp1p, Swp1p, and Stt3p), the composition of the vertebrate OST was less well defined. The roles of specific OST subunits remained enigmatic. Here we show that genomes of most multicellular eukaryotes encode two homologs of Stt3p and mammals express two homologs of Ost3p. The Stt3p and Ost3p homologs are assembled together with the previously described mammalian OST subunits (ribophorins I and II, OST48, and DAD1) into complexes that differ significantly in enzymatic activity. Tissue and cell type-specific differences in expression of the Stt3p homologs suggest that the enzymatic properties of oligosaccharyltransferase are regulated in eukaryotes to respond to alterations in glycoprotein flux through the secretory pathway and may contribute to tissue-specific glycan heterogeneity.     

The STT3 protein is a component of the yeast oligosaccharyltransferase complex

N-linked protein glycosylation is an essential process in eukaryotic cells. In the central reaction, the oligosaccharyltransferase (OTase) catalyzes the transfer of the oligosaccharide Glc₃Man₉GlcNAc₂ from dolicholpyrophosphate onto asparagine residues of nascent polypeptide chains in the lumen of the endoplasmic reticulum. The product of the essential gene STT3 is required for OTase activity in vivo, but is not present in highly purified OTase preparations. Using affinity purification of a tagged Stt3 protein, we now demonstrate that other components of the OTase complex, namely Ost1p, Wbp1p and Swp1p, specifically co-purify with the Stt3 protein. In addition, different conditional stt3 alleles can be suppressed by overexpression of either OST3 and OST4, which encode small components of the OTase complex. These genetic and biochemical data show that the highly conserved Stt3p is a component of the oligosaccharyltransferase complex. Received: 3 June 1997 / Accepted: 29 July 1997     

Yeast Wbp1p and Swp1p form a protein complex essential for oligosaccharyl transferase activity.

Asparagine-linked N-glycosylation is an essential protein modification occurring in all eukaryotic cells. The central step is the co-translational transfer of the core oligosaccharide assembled on the lipid carrier dolichol phosphate to selected Asn-X-Ser/Thr residues of nascent polypeptide chains in the endoplasmic reticulum. This reaction is catalyzed by the enzyme N-oligosaccharyl transferase. In yeast, Wbp1p is an essential component of this enzyme. Using a high copy number suppression approach, the SWP1 gene was isolated as an allele specific suppressor of a wbp1 mutation. Swp1p is a 30 kDa type I transmembrane protein and essential for cell viability. Similar to Wbp1p, depletion of Swp1p results in reduced N-oligosaccharyl transferase activity in vivo and in vitro. Wbp1p and Swp1p can be chemically cross-linked, suggesting that both proteins are essential constituents of the N-oligosaccharyl transferase complex.     

The yeast oligosaccharyltransferase complex can be replaced by STT3 from Leishmania major

The key step of protein N-glycosylation is catalyzed by the multimeric oligosaccharyltransferase complex (OST). Biochemical and genetic studies have revealed that OST from Saccharomyces cerevisiae consists of nine subunits: Wbp1, Swp1, Stt3, Ost1, Ost2, Ost3, Ost4, Ost5, and Ost6. With the exception of Stt3, assumed to contain the catalytic site, little is known about the function of other OST subunits. The existence of the OST complex is suggested to allow substrate specificity and efficient transfer, a close interaction with the translocon and the prevention of protein folding to ensure the efficient co-translational modification of proteins. However, in the recently completed genome of the trypanosomatid parasite Leishmania major STT3 (of which four paralogs exist, STT3-1, STT3-2, STT3-3, and STT3-4) is the only OST subunit that can be identified. Here we report that L.m.STT3 proteins, except STT3-3, are able to complement stt3 deficiency in yeast during vegetative growth, but only poorly during sporulation. By blue native electrophoresis we demonstrate that the L.mSTT3 is active mainly as a free, monomeric enzyme. In cell-free assays and also in vivo we find that L.mSTT3, expressed in yeast, has a broad specificity for nonglucosylated lipid-linked mannose-oligosaccharides, typical for several protists. But when incorporated into the OST complex, L.mSTT3 transfers also the common eukaryotic Glc(3)Man(9)GlcNAc(2)-PP-Dol donor. Finally, three L.m.STT3 paralogs were shown to complement not only stt3 but also ost1, ost2, wbp1, or swp1 mutants. Thus, STT3 from Leishmania can substitute for the whole OST complex.     

Studies on oligosaccharyl transferase in yeast

In yeast, OT consists of nine different subunits, all of which contain one or more predicted transmembrane segments. In yeast, five of these proteins are encoded by essential genes, Swp1p, Wbp1p, Ost2p, Ost1p and Stt3p. Four others are not essential Ost3p, Ost4p, Ost5p, Ost6p. All yeast OT subunits have been cloned and sequenced (Kelleher et al., 1992; 2003; Kelleher & Gilmore, 1997; Kumar et al., 1994; 1995; Breuer & Bause, 1995) and the structure of one of them, Ost4p, has been solved by NMR (Zubkov et al., 2004). Very recently, the preliminary crystal structure of the lumenal domain of an archaeal Stt3p homolog has been reported (Mayumi et al., 2007). Homologs of all OT subunits have been identified in higher eukaryotic organisms (Kelleher et al., 1992; 2003; Kumar et al., 1994; Kelleher & Gilmore, 1997).     

The essential endoplasmic reticulum chaperone Rot1 is required for protein N- and O-glycosylation in yeast

Rot1 is an essential yeast protein originally shown to be implicated in such diverse processes such as β-1,6-glucan synthesis, actin cytoskeleton dynamics or lysis of autophagic bodies. More recently also a role as a molecular chaperone has been discovered. Here, we report that Rot1 interacts in a synthetic manner with Ost3, one of the nine subunits of the oligosaccharyltransferase (OST) complex, the key enzyme of N-glycosylation. The deletion of OST3 in the rot1-1 mutant causes a temperature sensitive phenotype as well as sensitivity toward compounds interfering with cell wall biogenesis such as Calcofluor White, caffeine, Congo Red and hygromycin B, whereas the deletion of OST6, a functional homolog of OST3, has no effect. OST activity in vitro determined in membranes from rot1-1ost3Δ cells was found to be decreased to 45% compared with wild-type membranes, and model glycoproteins of N-glycosylation, like carboxypeptidase Y, Gas1 or dipeptidyl aminopeptidase B, displayed an underglycosylation pattern. By affinity chromatography, a physical interaction between Rot1 and Ost3 was demonstrated. Moreover, Rot1 was found to be involved also in the O-mannosylation process, as the glycosylation of distinct glycoproteins of this type were affected as well. Altogether, the data extend the role of Rot1 as a chaperone required to ensure proper glycosylation.     

Yeast cell death caused by mutation of the OST2 gene encoding the epsilon-subunit of Saccharomyces cerevisiae oligosaccharyltransferase

An essential epsilon-subunit of oligosaccharyltransferase Ost2 is a yeast homolog of mammalian highly conserved DAD1 (defender against apoptotic death). In hamster cells, the Gly38Arg mutation in DAD1 causes apoptosis at restrictive temperatures due to a defect in N-linked glycosylation. To analyze the function of Ost2 in yeast cell death, we constructed Saccharomyces cerevisiae strains expressing Gly58Arg (corresponding to the Gly38Arg mutation in hamster DAD1), Gly86Arg, and Glu113Val mutant Ost2. At elevated temperatures, ost2 mutants arrested growth by decreasing cell viability. Phosphatidylserine exposure, a phenotypic marker of apoptosis in mammalian cells, was found in ost2 mutant cells at 37 degrees C, although DNA fragmentation was not clearly detected. A high concentration of sorbitol compensates for the temperature sensitivity of the ost2 mutant. These results suggest that apoptosis-like cell death in ost2 mutants is caused by the secondary effect of overall reduced protein N-linked glycosylation.     

Structure of the oligosaccharyl transferase complex at 12 A resolution

Oligosaccharyl transferase (OT) catalyzes the transfer of a lipid-linked oligosaccharide to the nascent polypeptide emerging from the translocon. Currently, there is no structural information on the membrane-embedded OT complex, which consists of eight different polypeptide chains. We report a 12 A resolution cryo-electron microscopy structure of OT from yeast. We mapped the locations of four essential OT subunits through a maltose-binding protein fusion strategy. OT was found to have a large domain in the lumenal side of endoplasmic reticulum where the catalysis occurs. The lumenal domain mainly comprises the catalytic Stt3p, the donor substrate-recognizing Wbp1p, and the acceptor substrate-recognizing Ost1p. A prominent groove was observed between these subunits, and we propose that the nascent polypeptide from the translocon threads through this groove while being scanned by the Ost1p subunit for the presence of the glycosylation sequon.     

A specific segment of the transmembrane domain of Wbp1p is essential for its incorporation into the oligosaccharyl transferase complex

Wbp1p, a type I transmembrane protein, is an essential component of oligosaccharyl transferase (OT), which consists of nine different subunits in yeast. It has been proposed that three subunits, Wbp1p, Ost2p, and Swp1p, physically interact with each other, but the mechanism of these interactions is unknown. To explore the mode of interaction, we have focused on the single-transmembrane protein, Wbp1p, and made several deletions and mutations within the short cytosolic domain and the transmembrane domain. Our results show that the deletion of the cytosolic domain has no effect on cell growth, but mutation of all 17 amino acids in the transmembrane domain to 17 Leu residues or replacement of the transmembrane and cytosolic domains with the counterparts of Ost1p results in lethality. Immunoprecipitation experiments show that Wbp1p mutated in these two ways is not incorporated into the OT complex. This finding suggests that the transmembrane domain of Wbplp may mediate its association with the other subunits. A series of mutations of the transmembrane domain have revealed that block alterations in the half of the transmembrane domain facing the lumen of the endoplasmic reticulum (ER) impaired cell viability. Seven single-Lys mutants in the same domain were temperature sensitive for growth at 37 degrees C. In contrast, block mutations in the other half of the transmembrane domain facing the cytosol did not result in lethality and indicated that this portion of the transmembrane domain was not involved in stable incorporation of Wbp1p into the OT complex.     

Yeast Cell Death Caused by Mutation of the OST2 Gene Encoding the ε-Subunit of Saccharomyces cerevisiae Oligosaccharyltransferase

An essential ε-subunit of oligosaccharyltransferase Ost2 is a yeast homolog of mammalian highly conserved DAD1 (defender against apoptotic death). In hamster cells, the Gly38Arg mutation in DAD1 causes apoptosis at restrictive temperatures due to a defect in N-linked glycosylation. To analyze the function of Ost2 in yeast cell death, we constructed Saccharomyces cerevisiae strains expressing Gly58Arg (corresponding to the Gly38Arg mutation in hamster DAD1), Gly86Arg, and Glu113Val mutant Ost2. At elevated temperatures, ost2 mutants arrested growth by decreasing cell viability. Phosphatidylserine exposure, a phenotypic marker of apoptosis in mammalian cells, was found in ost2 mutant cells at 37 °C, although DNA fragmentation was not clearly detected. A high concentration of sorbitol compensates for the temperature sensitivity of the ost2 mutant. These results suggest that apoptosis-like cell death in ost2 mutants is caused by the secondary effect of overall reduced protein N-linked glycosylation.     

Analysis of glycosylation site occupancy reveals a role for Ost3p and Ost6p in site-specific N-glycosylation efficiency

Asparagine-linked glycosylation is the most common post-translational modification of proteins catalyzed in eukaryotes by the multiprotein complex oligosaccharyltransferase. Apart from the catalytic Stt3p, the roles of the subunits are ill defined. Here we describe functional investigations of the Ost3/6p components of the yeast enzyme. We developed novel analytical tools to quantify glycosylation site occupancy by enriching glycoproteins bound to the yeast polysaccharide cell wall, tagging glycosylated asparagines using endoglycosidase H glycan release, and detecting peptides and glycopeptides with LC-ESI-MS/MS. We found that the paralogues Ost3p and Ost6p were required for efficient glycosylation of distinct defined glycosylation sites. Our results describe a novel method for relative quantification of glycosylation occupancy in the genetically tractable yeast system and show that eukaryotic oligosaccharyltransferase isoforms have different activities toward protein substrates at the level of individual glycosylation sites.     

Subunits of the translocon interact with components of the oligosaccharyl transferase complex

Following initiation of translocation across the membrane of the endoplasmic reticulum via the translocon, polypeptide chains are N-glycosylated by the oligosaccharyl transferase (OT) enzyme complex. Translocation and N-glycosylation are concurrent events and would be expected to require juxtaposition of the translocon and the OT complex. To determine whether any of the subunits of the OT complex and translocon mediate interactions between the two complexes, we initiated a systematic study in budding yeast using the split-ubiquitin approach. Interestingly, the OT subunit Stt3p was found to interact only with Sec61p, whereas another OT subunit, Ost4p, was found to interact with all three components of the translocon, Sec61p, Sbh1p, and Sss1p. The OT subunit Wbp1p was found to interact very strongly with Sec61p and Sbh1p and weakly with Sss1p. Other OT subunits, Ost1p, Ost2p, and Swp1p were found to interact with Sec61p and either Sbh1p or Sss1p. Ost3p exhibited a weak interaction with Sec61p and Sbh1p, whereas Ost5p and Ost6p interacted very weakly with Sec61p and failed to interact with Sbh1p or Sss1p. We were able to confirm these split-ubiquitin findings by a chemical cross-linking technique. Based on our findings using these two techniques, we conclude that the association of these two complexes is stabilized via multiple protein-protein contacts. Based on extrapolation of the structural parameters of the crystal structure of the prokaryotic Sec complex to the eukaryotic complex, we propose a working model to understand the organization of the translocon-OT supercomplex.     

Oligosaccharyltransferase: a complex multisubunit enzyme of the endoplasmic reticulum

The attachment of N-linked oligosaccharide chains to proteins is an important cotranslational process. These chains can, in some cases, serve to stabilize the protein, while in other cases they function as recognition elements. A key enzyme in the N-glycosylation process is oligosaccharyltransferase (OT). In yeast this enzyme, which is found in the endoplasmic reticulum, consists of nine different transmembrane protein subunits. Our general aim is to learn more about the functions of the multiple subunits of yeast OT and their mode of interaction with each other. Using a combination of biochemical and genetic techniques the subunit Ost1p has been shown to recognize Asn-X-Ser/Thr glycosylation sites. The principle tool used in the identification process was a benzophenone-based glycosylation site peptide that was shown to be crosslinked to Ost1p. Our current objective is to identify the domain in the primary structure that is involved in recognition of the glycosylation site sequence. By use of bifunctional crosslinkers, the possible interaction of Ost1p with other subunits of OT will be studied. This work and other studies on the OT subunits are concisely summarized.     

New findings on interactions among the yeast oligosaccharyl transferase subunits using a chemical cross-linker

At present, there is very limited knowledge about the structural organization of the yeast oligosaccharyl transferase (OT) complex and the function of each of its nine subunits. Because of the failure of the yeast two-hybrid system to reveal interactions between luminal domains of these subunits, we utilized a membrane permeable, thiocleavable cross-linking reagent dithiobis-succinimidyl propionate to biochemically study the interactions of various OT subunits. Four essential gene products, Ost1p, Wbp1p, Swp1p, and Stt3p were shown to be cross-linked to each other in a pairwise fashion. In addition, Ost1p was found to be cross-linked to all other eight OT subunits individually. This led us to propose that Ost1p may reside in the core of the OT complex and could play an important role in its assembly. Ost4p and Ost5p were found to only interact with specific components of the OT complex and may function as an additional anchor for optimal stability of Stt3p and Ost1p in the membrane, respectively. Interestingly, Ost3p and Ost6p subunits exhibited a surprisingly identical pattern of cross-linking to other subunits, which is consistent with their proposed redundant function. Based on these findings, we analyzed the distribution of the lysine residues that are likely to be involved in cross-linking of OT subunits and propose that the OT subunits interact with each other through either their transmembrane domains and/or a region proximal to it, rather than through their luminal or cytoplasmic domains.