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.     

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).     

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.     

Studies on the N-glycosylation of the subunits of oligosaccharyl transferase in Saccharomyces cerevisiae

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 (N(535)NTWN(539)NT) 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 Man(8)GlcNAc(2) is found linked only to Asn(539). 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 N(535)NTWN(539)NT (N536Q, T537S, N540Q, and T541S), did not exhibit growth defects. Based on these studies, we conclude that N-glycosylation of Stt3p at Asn(539) is essential for its function in the OT complex.     

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.     

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.     

Studies on the function of oligosaccharyl transferase subunits. Stt3p is directly involved in the glycosylation process

In the yeast, Saccharomyces cerevisiae, oligosaccharyl transferase (OT) is composed of nine different transmembrane proteins. Using a glycosylatable peptide containing a photoprobe, we previously found that only one essential subunit, Ost1p, was specifically labeled by the photoprobe and recently have shown that it does not contain the recognition domain for the glycosylatable sequence Asn-Xaa-Thr/Ser. In this study we utilized additional glycosylatable peptides containing two photoreactive groups and found that these were linked to Stt3p and Ost3p. Stt3p is the most conserved subunit in the OT complex, and therefore 21 block mutants in the lumenal region were prepared. Of the 14 lethal mutant proteins only two, as well as one temperature-sensitive mutant protein, were incorporated into the OT complex. However, using microsomes prepared from these three strains, the labeling of Ost1p was markedly decreased upon photoactivation with the Asn-Bpa-Thr photoprobe. Based on the block mutants single amino acid mutations were prepared and analyzed. From all of these results, we conclude that the sequence from residues 516 to 520, WWDYG in Stt3p, plays a central role in glycosylatable peptide recognition and/or the catalytic glycosylation process.     

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.     

Two oligosaccharyl transferase complexes exist in yeast and associate with two different translocons

Oligosaccharyl transferase (OT) scans and selectively glycosylates -Asn-X-Thr/Ser-motifs in nascent polypeptide chains in the endoplasmic reticulum (ER). Several groups have reported different results for the composition of this enzyme complex. In this study, using a membrane protein two-hybrid approach, the split-ubiquitin system, we show that except for Ost3p and Ost6p, all of the other subunits of OT exist as dimers or oligomers in the yeast, Saccharomyces cerevisiae. Ost3p and Ost6p behave strikingly similar in a series of genetic and biochemical assays, but clearly do not exist in the same OT complex. This observation, as well as the results in an accompanying study to analyze the composition of OT complex by blue native gel electrophoresis using a series of wild-type and mutant yeast strains strongly suggests that two isoforms of the OT complex exist in the ER, differing only in the presence of Ost3p or Ost6p. Each of these two isoforms of the OT complex specifically interacts with two structurally similar, but functionally different translocon complexes: the Sec61 and the Ssh1 translocon complexes.     

Functional characterization of Ost3p. Loss of the 34-kD subunit of the Saccharomyces cerevisiae oligosaccharyltransferase results in biased underglycosylation of acceptor substrates

Within the lumen of the rough endoplasmic reticulum, oligosaccharyltransferase catalyzes the en bloc transfer of a high mannose oligosaccharide moiety from the lipid-linked oligosaccharide donor to asparagine acceptor sites in nascent polypeptides. The Saccharomyces cerevisiae oligosaccharyltransferase was purified as a heteroligomeric complex consisting of six subunits (alpha-zeta) having apparent molecular masses of 64 kD (Ost1p), 45 kD (Wbp1p), 34 kD, 30 kD (Swp1p), 16 kD, and 9 kD. Here we report a structural and functional characterization of Ost3p which corresponds to the 34-kD gamma-subunit of the oligosaccharyltransferase. Unlike Ost1p, Wbp1p, and Swp1p, expression of Ost3p is not essential for viability of yeast. Instead, ost3 null mutant yeast grow at wild-type rates on solid or in liquid media irrespective of culture temperature. Nonetheless, detergent extracts prepared from ost3 null mutant membranes are twofold less active than extracts prepared from wild-type membranes in an in vitro oligosaccharyltransferase assay. Furthermore, loss of Ost3p is accompanied by significant underglycosylation of soluble and membrane- bound glycoproteins in vivo. Compared to the previously characterized ost1-1 mutant in the oligosaccharyltransferase, and the alg5 mutant in the oligosaccharide assembly pathway, ost3 null mutant yeast appear to be selectively impaired in the glycosylation of several membrane glycoproteins. The latter observation suggests that Ost3p may enhance oligosaccharide transfer in vivo to a subset of acceptor substrates.     

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.     

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.     

A novel and simple method of production and biophysical characterization of a mini-membrane protein, Ost4p: a subunit of yeast oligosaccharyl transferase

Asparagine-linked glycosylation is an essential and highly conserved protein modification reaction. In eukaryotes, oligosaccharyl transferase (OT), a multi-subunit membrane-associated enzyme complex, catalyzes this reaction in newly synthesized proteins. In Saccharomyces cerevisiae, OT consists of nine nonidentical membrane proteins. Ost4p, the smallest subunit, bridges the catalytic subunit Stt3p with Ost3p. Mutation of transmembrane residues 18-24 in Ost4p has negative effect on OT activity, disrupts the Stt3p-Ost4p-Ost3p complex, results in temperature-sensitive phenotype, and hypoglycosylation. Heterologous expression and purification of integral membrane proteins are the bottleneck in membrane protein research. The authors report the cloning, successful overexpression and purification of recombinant Ost4p with a novel but simple method producing milligram quantities of pure protein. GB1 protein was found to be the most suitable tag for the large scale production of Ost4p. The cleavage of Ost4p conveniently leaves GB1 protein in solution eliminating further purification. The precipitated pure Ost4p is reconstituted in appropriate membrane mimetic. The recombinant protein is highly helical as indicated by the far-UV CD spectrum. The well-dispersed heteronuclear single quantum coherence spectrum indicates that this minimembrane protein is well-folded. The successful production of pure recombinant Ost4p with a novel yet simple method may have important ramification for the production of other membrane proteins.     

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.     

Heterologous expression and biophysical characterization of soluble oligosaccharyl transferase subunits

Oligosaccharyl transferase (OT) catalyzes the first committed step in N-linked protein glycosylation, a co-translational process that occurs in the lumen of the endoplasmic reticulum. The yeast Saccharomyces cerevisiae enzyme complex comprises nine integral membrane proteins, five of which are essential for catalysis. Due to the challenges with purifying the active enzyme complex for detailed biophysical studies, a systematic study to express, isolate, and characterize the soluble domains of three of the largest subunits in the complex (Nlt1p, Wbp1p, and Swp1p) is reported. The proteins are expressed using the lytic baculovirus expression system and the new constructs are well behaved, monomeric in solution, and glycosylated. Two of the proteins interact with each other as seen by gel filtration and circular dichroism. This study provides a framework to study the roles of these three essential subunits of the eukaryotic OT complex.     

Insight into functional aspects of Stt3p, a subunit of the oligosaccharyl transferase. Evidence for interaction of the N-terminal domain of Stt3p with the protein kinase C cascade

Over a decade ago, the gene STT3 was identified in a staurosporine and temperature sensitivity screen of yeast. Subsequently the product of this gene was shown to be a subunit of the endoplasmic reticulum-localized oligosaccharyl transferase (OT) complex. Although stt3 mutants are known to be staurosporine-sensitive, we found that mutants of other OT subunits (except ost4 Delta) are staurosporine-resistant, which indicates that this phenotype of stt3 mutants is not simply a consequence of their defect in glycosylation, as previously speculated. Staurosporine sensitivity was found to be an allele-specific phenotype restricted to cells harboring mutations in highly conserved residues in the N-terminal domain of the STT3 protein. Cells bearing mutations in one of the cytosolic-oriented loops (amino acids 158-168) in the N terminus of Stt3p were found to be specifically susceptible to staurosporine. Staurosporine is a specific inhibitor of Pkc1p, and a genetic link had previously been suggested between PKC1 and STT3. It is known that overexpression of PKC1 suppresses the staurosporine sensitivity of the stt3 mutants in an allele-specific manner, which is typical of mutants of Pkc1p cascade. It has been shown that the pkc1 null mutant exhibits lowered OT activity. Our results combined with these previous observations indicate that the N-terminal domain of Stt3p may interact with members of the Pkc1p cascade and consequently mutations in this domain result in staurosporine sensitivity. We further speculate that the Pkc1p regulates OT activity through the N-terminal domain of Stt3p, the C-terminal domain of which possesses the recognition and/or catalytic site of the OT complex.     

Evidence for interaction of yeast protein kinase C with several subunits of oligosaccharyl transferase

Oligosaccharyltransferase (OT) in Saccharomyces cerevisiae is an enzyme complex consisting of 8 transmembrane proteins located in the endoplasmic reticulum (ER). Studies on potential protein-protein interactions in OT using a two-hybrid library screen revealed that protein kinase C (Pkc1p) interacted with the lumenal domains of several OT subunits. Additional genetic experiments revealed that overexpression of two OT subunits rescued the growth defect caused by overexpression of a Pkc1 active site mutant, implying that there are specific genetic interactions between PKC1 and OT. These in vivo findings were complemented by in vitro studies that showed that several of the OT subunits bound to a fusion protein consisting of glutathione S-transferase linked via its C-terminus to Pkc1p. Assays of OT activity, in which glycosylation of a simple acceptor peptide was assayed in microsomes from wild-type and a pkc1 null revealed a 50% reduction in activity in the microsomes from the null strain. In contrast, strains containing null mutations of two other genes known to be downstream of Pkc1p in the PKC1-MAP kinase pathway had a level of OT activity comparable to that of wild-type cells. These in vivo and in vitro experiments suggest that in yeast cells Pkc1p may be involved in regulation of the N-glycosylation of proteins.     

Solution structure of a human minimembrane protein Ost4, a subunit of the oligosaccharyltransferase complex

Oligosaccharyltransferase (OST) is a membrane associated enzyme complex that mediates transfer of an oligosaccharide onto asparagine residue of a protein. Human Ost4 is a small membrane protein and belongs to one of the seven subunits of human OST. This study determined the solution structure of human Ost4 in solvent system using NMR spectroscopy. Ost4 was demonstrated that the residues 5–30 adopt an α-helical structure. A kink structure was observed in the transmembrane domain, which may be important for its function.     

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.