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Uncoupling the hydrolysis of lipid-linked oligosaccharide from the oligosaccharyl transfer reaction by point mutations in yeast oligosaccharyltransferase

Open AccessPublished:September 16, 2020DOI:https://doi.org/10.1074/jbc.RA120.015013
      Oligosaccharyltransferase (OST) is responsible for the first step in the N-linked glycosylation, transferring an oligosaccharide chain onto asparagine residues to create glycoproteins. In the absence of an acceptor asparagine, OST hydrolyzes the oligosaccharide donor, releasing free N-glycans (FNGs) into the lumen of the endoplasmic reticulum (ER). Here, we established a purification method for mutated OSTs using a high-affinity epitope tag attached to the catalytic subunit Stt3, from yeast cells co-expressing the WT OST to support growth. The purified OST protein with mutations is useful for wide-ranging biochemical experiments. We assessed the effects of mutations in the Stt3 subunit on the two enzymatic activities in vitro, as well as their effects on the N-glycan attachment and FNG content levels in yeast cells. We found that mutations in the first DXD motif increased the FNG generation activity relative to the oligosaccharyl transfer activity, both in vitro and in vivo, whereas mutations in the DK motif had the opposite effect; the decoupling of the two activities may facilitate future deconvolution of the reaction mechanism. The isolation of the mutated OSTs also enabled us to identify different enzymatic properties in OST complexes containing either the Ost3 or Ost6 subunit and to find a 15-residue peptide as a better-quality substrate than shorter peptides. This toolbox of mutants, substrates, and methods will be useful for investigations of the molecular basis and physiological roles of the OST enzymes in yeast and other organisms.
      N-Glycosylation refers to the covalent attachment of an oligosaccharide chain on asparagine residues in proteins and is one of the most important post-translational protein modifications (
      • Cherepanova N.
      • Shrimal S.
      • Gilmore R.
      N-Linked glycosylation and homeostasis of the endoplasmic reticulum.
      ,
      • Aebi M.
      N-Linked protein glycosylation in the ER.
      ,
      • Breitling J.
      • Aebi M.
      N-Linked protein glycosylation in the endoplasmic reticulum.
      ). The N-glycosylation consensus, Asn-X-Ser/Thr (where X is not Pro), is the sequon, and the N-oligosaccharide chain attached to a protein is the N-glycan. The oligosaccharide donors are lipid-linked oligosaccharides (LLOs) (
      • Hartley M.D.
      • Imperiali B.
      At the membrane frontier: a prospectus on the remarkable evolutionary conservation of polyprenols and polyprenyl-phosphates.
      ). The oligosaccharide portion of LLO is transferred to the side-chain carboxamide group of asparagine residues in the sequon by the action of oligosaccharyltransferase (OST) (
      • Kohda D.
      Structural basis of protein Asn-glycosylation by oligosaccharyltransferases.
      ). In higher eukaryotes, including the budding yeast Saccharomyces cerevisiae, the transfer of the 14-residue oligosaccharide chain, Glc3Man9GlcNAc2, occurs on the luminal side of the endoplasmic reticulum (ER) membrane. Statistical studies revealed that two-thirds of the sequons are glycosylated (
      • Apweiler R.
      • Hermjakob H.
      • Sharon N.
      On the frequency of protein glycosylation, as deduced from analysis of the SWISS-PROT database.
      ). The proper selections of the sequons to be glycosylated or left unmodified are a prerequisite for the protein quality control in the ER and subsequent degradation in the cytosol (
      • Cherepanova N.
      • Shrimal S.
      • Gilmore R.
      N-Linked glycosylation and homeostasis of the endoplasmic reticulum.
      ,
      • Aebi M.
      N-Linked protein glycosylation in the ER.
      ). Interestingly, oligosaccharide chains that are not attached to proteins reportedly accumulate inside the ER and the cytosol (
      • Chantret I.
      • Moore S.E.H.
      Free oligosaccharide regulation during mammalian protein N-glycosylation.
      ). The peptide:N-glycanase (PNGase), Png1, cleaves the N-glycans from misfolded glycoproteins in the cytosol. The PNGase-derived “free” N-glycans (FNGs) account for 95% of the total FNGs in the yeast cytosol (
      • Harada Y.
      • Buser R.
      • Ngwa E.M.
      • Hirayama H.
      • Aebi M.
      • Suzuki T.
      Eukaryotic oligosaccharyltransferase generates free oligosaccharides during N-glycosylation.
      ). The yeast OST enzyme generates the remaining 5% of FNGs via LLO hydrolysis. The OST-derived FNGs are transported from the ER to the cytosol for degradation by a cytosol-vacuolar α-mannosidase, Ams1 (
      • Chantret I.
      • Kodali V.P.
      • Lahmouich C.
      • Harvey D.J.
      • Moore S.E.H.
      Endoplasmic reticulum-associated degradation (ERAD) and free oligosaccharide generation in Saccharomyces cerevisiae.
      ,
      • Hirayama H.
      • Seino J.
      • Kitajima T.
      • Jigami Y.
      • Suzuki T.
      Free oligosaccharides to monitor glycoprotein endoplasmic reticulum-associated degradation in Saccharomyces cerevisiae.
      ). The amounts of FNGs generated by OST can be measured using a png1Δams1Δ double-knockout strain, by suppressing the generation of the cytosol-originated FNGs (
      • Harada Y.
      • Buser R.
      • Ngwa E.M.
      • Hirayama H.
      • Aebi M.
      • Suzuki T.
      Eukaryotic oligosaccharyltransferase generates free oligosaccharides during N-glycosylation.
      ). In contrast, most of the FNGs in the ER and the cytosol are attributed to the hydrolytic activity of OST in the ER lumen in mammalian cells (
      • Harada Y.
      • Masahara-Negishi Y.
      • Suzuki T.
      Cytosolic-free oligosaccharides are predominantly generated by the degradation of dolichol-linked oligosaccharides in mammalian cells.
      ).
      The yeast OST enzyme is a membrane-embedded protein complex consisting of eight membrane-protein subunits in an equimolar ratio. Stt3, Wbp1, Swp1, Ost1, and Ost2 are essential gene products, whereas Ost3/Ost6, Ost4, and Ost5 are nonessential gene products (
      • Kohda D.
      Structural basis of protein Asn-glycosylation by oligosaccharyltransferases.
      ). The Stt3 subunit has the catalytic site. In general, eukaryotic genomes encode multiple paralogous stt3 genes (
      • Kelleher D.J.
      • Karaoglu D.
      • Mandon E.C.
      • Gilmore R.
      Oligosaccharyltransferase isoforms that contain different catalytic STT3 subunits have distinct enzymatic properties.
      ). The homologous Stt3 proteins expressed from these paralogous genes generate different OST complexes. Except for Caenorhabditis species, the genomes from all sequenced metazoan organisms contain two STT3 genes (STT3A and STT3B). Plant genomes also encode two copies of STT3 genes (STT3A and STT3B) (
      • Niu G.
      • Shao Z.
      • Liu C.
      • Chen T.
      • Jiao Q.
      • Hong Z.
      Comparative and evolutionary analyses of the divergence of plant oligosaccharyltransferase STT3 isoforms.
      ). In the phylum Protista, for example, the Leishmania major genome contains four stt3 genes, stt3A, stt3B, stt3C, and stt3D (
      • Nasab F.P.
      • Schulz B.L.
      • Gamarro F.
      • Parodi A.J.
      • Aebi M.
      All in one: Leishmania major STT3 proteins substitute for the whole oligosaccharyltransferase complex in Saccharomyces cerevisiae.
      ). Despite their similar names, the human, plant, and protist genes lack direct orthologous relationships (
      • Niu G.
      • Shao Z.
      • Liu C.
      • Chen T.
      • Jiao Q.
      • Hong Z.
      Comparative and evolutionary analyses of the divergence of plant oligosaccharyltransferase STT3 isoforms.
      ,
      • Kelleher D.J.
      • Gilmore R.
      An evolving view of the eukaryotic oligosaccharyltransferase.
      ). One important exception is fungi, including S. cerevisiae, with genomes encoding a single stt3 gene. The human STT3B protein sequence is more similar to the yeast Stt3 protein sequence, suggesting that the human STT3A is a newcomer in evolution and the human STT3B is orthologous to the yeast Stt3 (
      • Kelleher D.J.
      • Gilmore R.
      An evolving view of the eukaryotic oligosaccharyltransferase.
      ). In place of the single Stt3, the yeast genome encodes the two paralogous ost3 and ost6 genes, which generate two different OST forms containing either Ost3 or Ost6 (
      • Schwarz M.
      • Knauer R.
      • Lehle L.
      Yeast oligosaccharyltransferase consists of two functionally distinct sub-complexes, specified by either the Ost3p or Ost6p subunit.
      ). The ratio of the Ost3-containing to Ost6-containing OST complexes is 4:1 (
      • Harada Y.
      • Buser R.
      • Ngwa E.M.
      • Hirayama H.
      • Aebi M.
      • Suzuki T.
      Eukaryotic oligosaccharyltransferase generates free oligosaccharides during N-glycosylation.
      ). Single deletions of either Ost3 or Ost6 resulted in moderate underglycosylation of proteins, and the Ost3 and Ost6 double knockout led to severe underglycosylation in yeast cells (
      • Knauer R.
      • Lehle L.
      The oligosaccharyltransferase complex from Saccharomyces cerevisiae: isolation of the OST6 gene, its synthetic interaction with OST3, and analysis of the native complex.
      ). The Ost3 and Ost6 proteins have a thioredoxin fold in the N-terminal soluble domain, and the oxidoreductase activity was considered to suppress disulfide bond formation during the oligosaccharyl transfer reaction (
      • Poljak K.
      • Selevsek N.
      • Ngwa E.
      • Grossmann J.
      • Losfeld M.E.
      • Aebi M.
      Quantitative profiling of N-linked glycosylation machinery in yeast Saccharomyces cerevisiae.
      ,
      • Schulz B.L.
      • Stirnimann C.U.
      • Grimshaw J.P.A.
      • Brozzo M.S.
      • Fritsch F.
      • Mohorko E.
      • Capitani G.
      • Glockshuber R.
      • Grütter M.G.
      • Aebi M.
      Oxidoreductase activity of oligosaccharyltransferase subunits Ost3p and Ost6p defines site-specific glycosylation efficiency.
      ). However, the N-glycosylation status was only affected to a limited extent by the disruption of the CXXC motifs in the Ost3 and Ost6 subunits, suggesting the importance of noncovalent interactions (
      • Schulz B.L.
      • Stirnimann C.U.
      • Grimshaw J.P.A.
      • Brozzo M.S.
      • Fritsch F.
      • Mohorko E.
      • Capitani G.
      • Glockshuber R.
      • Grütter M.G.
      • Aebi M.
      Oxidoreductase activity of oligosaccharyltransferase subunits Ost3p and Ost6p defines site-specific glycosylation efficiency.
      ). The actual functions of the Ost3 and Ost6 subunits remain elusive.
      In vivo mutagenesis studies of the individual subunits of yeast OST have been performed using the spotting plate assay. Switching from the WT OST to the mutated OST in cells is accomplished by the plasmid-shuffling method (
      • Li G.
      • Yan Q.
      • Nita-Lazar A.
      • Haltiwanger R.S.
      • Lennarz W.J.
      Studies on the N-glycosylation of the subunits of oligosaccharyl transferase in Saccharomyces cerevisiae.
      ,
      • Igura M.
      • Maita N.
      • Kamishikiryo J.
      • Yamada M.
      • Obita T.
      • Maenaka K.
      • Kohda D.
      Structure-guided identification of a new catalytic motif of oligosaccharyltransferase.
      ) or the GAL1 promoter–switching method (
      • Hese K.
      • Otto C.
      • Routier F.H.
      • Lehle L.
      The yeast oligosaccharyltransferase complex can be replaced by STT3 from Leishmania major.
      ). The spotting plate assay is quite easy to execute, but its outcome is rather limited: a mutation is just classified into lethal, temperature-sensitive, or normal (i.e. nonconditional) growth phenotypes. Moreover, the different properties of the two OST complexes containing Ost3 or Ost6 in vivo could complicate the interpretation of the results obtained by the spotting plate assays. In some situations, enzymological studies using purified proteins with a defined subunit composition are preferred. However, the reconstitution of the OST complex in vitro using recombinant proteins has not been successful. Fortunately, the genetic manipulation of the yeast genome is straightforward. Previously, the 3×HA (hemagglutinin) epitope tag–encoding sequence was inserted at the 3′-ends of the stt3 and ost3 genes in the yeast genome, for immunopurification of the OST complexes and immunodetection of other subunits (
      • Li G.
      • Yan Q.
      • Nita-Lazar A.
      • Haltiwanger R.S.
      • Lennarz W.J.
      Studies on the N-glycosylation of the subunits of oligosaccharyl transferase in Saccharomyces cerevisiae.
      ,
      • Karaoglu D.
      • Kelleher D.J.
      • Gilmore R.
      The highly conserved Stt3 protein is a subunit of the yeast oligosaccharyltransferase and forms a subcomplex with Ost3p and Ost4p.
      ) The tandem IgG-binding domain–encoding sequence of Protein A was added to the 3′-end of the stt3 gene in the yeast genome for the same purpose (
      • Spirig U.
      • Glavas M.
      • Bodmer D.
      • Reiss G.
      • Burda P.
      • Lippuner V.
      • Te Heesen S.
      • Aebi M.
      The STT3 protein is a component of the yeast oligosaccharyltransferase complex.
      ). Subsequently, other types of epitope tag sequences (FLAG, 3×FLAG, and 1D4) were incorporated into the 3′-end of the ost4 gene in the yeast genome, and the tagged OST complexes were affinity-purified to homogeneity for enzymatic investigation (
      • Harada Y.
      • Buser R.
      • Ngwa E.M.
      • Hirayama H.
      • Aebi M.
      • Suzuki T.
      Eukaryotic oligosaccharyltransferase generates free oligosaccharides during N-glycosylation.
      ) and cryo-EM single-particle analyses (
      • Chavan M.
      • Chen Z.
      • Li G.
      • Schindelin H.
      • Lennarz W.J.
      • Li H.
      Dimeric organization of the yeast oligosaccharyl transferase complex.
      ,
      • Bai L.
      • Wang T.
      • Zhao G.
      • Kovach A.
      • Li H.
      The atomic structure of a eukaryotic oligosaccharyltransferase complex.
      ,
      • Wild R.
      • Kowal J.
      • Eyring J.
      • Ngwa E.M.
      • Aebi M.
      • Locher K.P.
      Structure of the yeast oligosaccharyltransferase complex gives insight into eukaryotic N-glycosylation.
      ). In the case of conditional mutations, the isolation of OST complexes containing such mutant proteins is possible, provided that the yeast strain can grow under permissive conditions. Yeast strains carrying the stt3-4 and stt3-6 (G520D), stt3-5 (G520S), and stt3-7 (S552P) alleles have temperature-sensitive phenotypes (
      • Spirig U.
      • Glavas M.
      • Bodmer D.
      • Reiss G.
      • Burda P.
      • Lippuner V.
      • Te Heesen S.
      • Aebi M.
      The STT3 protein is a component of the yeast oligosaccharyltransferase complex.
      ). These strains were grown at the permissive temperature of 23 °C, and the membrane fractions were prepared. In the membrane fractions, the OST complexes with these mutants showed very low oligosaccharyl transfer activities (
      • Spirig U.
      • Glavas M.
      • Bodmer D.
      • Reiss G.
      • Burda P.
      • Lippuner V.
      • Te Heesen S.
      • Aebi M.
      The STT3 protein is a component of the yeast oligosaccharyltransferase complex.
      ). In contrast, the introduction of lethal or very severe temperature-sensitive mutations into the Stt3 subunit is rather difficult, as the co-expression of the WT Stt3 subunit is necessary for cell culture. We must confirm that the OST preparation with the mutated Stt3 subunit is not contaminated with the native OST complex containing the WT Stt3 subunit.
      In this study, we fused a new epitope tag to the 5′-end of the mutated stt3 genes in expression plasmids. The epitope tag has a high affinity to a specific antibody (
      • Fujii Y.
      • Kaneko M.
      • Neyazaki M.
      • Nogi T.
      • Kato Y.
      • Takagi J.
      PA tag: a versatile protein tagging system using a super high affinity antibody against a dodecapeptide derived from human podoplanin.
      ) and enables the preparation of the mutated OST complex free from the native OST complex. In addition, the use of host yeast strains expressing either Ost3 or Ost6 permits the purification of the OST complexes with a defined subunit composition. We measured the oligosaccharyl transfer activity and the hydrolytic activity of LLO in vitro, using the Ost3-containing OST complexes carrying mutations in the catalytic Stt3 subunit. In parallel, we determined the N-glycosylation status of the glycoproteins and the amounts of FNGs generated by OST in yeast strains harboring the same stt3 mutations. These detailed comparisons of the two activities of a series of mutations provided new insights into the N-glycosylation reactions in vitro and in living yeast cells.

      Results

      Affinity purification of the OST complexes containing mutations in the Stt3 subunit

      The PA epitope tag sequence was inserted into the region encoding the N terminus of the Stt3 subunit via a 10-residue spacer sequence (Fig. 1A). The tagged Stt3 protein was expressed under the control of the constitutive glyceraldehyde-3-phosphate dehydrogenase (GPD) promoter from the expression plasmid, pPA-STT3. An Ost6-knockout strain (ost6Δ) was used as the host to prepare the Ost3-containing OST complex (referred to as OST[Ost3]). Deleterious effects caused by the absence of Ost6 were compensated by the extra expression of Ost3 from the pOST3 plasmid (
      • Wild R.
      • Kowal J.
      • Eyring J.
      • Ngwa E.M.
      • Aebi M.
      • Locher K.P.
      Structure of the yeast oligosaccharyltransferase complex gives insight into eukaryotic N-glycosylation.
      ,
      • Spirig U.
      • Bodmer D.
      • Wacker M.
      • Burda P.
      • Aebi M.
      The 3.4-kDa Ost4 protein is required for the assembly of two distinct oligosaccharyltransferase complexes in yeast.
      ). As mutational sites, we selected the two conserved short amino acid motifs, the first DXD motif from the two DXD motifs and the DK motif in the Stt3 subunit (Fig. 1A) (
      • Igura M.
      • Maita N.
      • Kamishikiryo J.
      • Yamada M.
      • Obita T.
      • Maenaka K.
      • Kohda D.
      Structure-guided identification of a new catalytic motif of oligosaccharyltransferase.
      ,
      • Shrimal S.
      • Gilmore R.
      Oligosaccharyltransferase structures provide novel insight into the mechanism of asparagine-linked glycosylation in prokaryotic and eukaryotic cells.
      ). These two short motifs are located close to the bound peptide substrate in the three-dimensional structure and involved in the recognition of the amino acid residues in or close to the sequon (Fig. 2). Glu45 is the first acidic residue of the DXD motif and is positioned to interact with the residue at position −1 of the sequon (
      • Wild R.
      • Kowal J.
      • Eyring J.
      • Ngwa E.M.
      • Aebi M.
      • Locher K.P.
      Structure of the yeast oligosaccharyltransferase complex gives insight into eukaryotic N-glycosylation.
      ,
      • Shrimal S.
      • Gilmore R.
      Oligosaccharyltransferase structures provide novel insight into the mechanism of asparagine-linked glycosylation in prokaryotic and eukaryotic cells.
      ), whereas Asp47 is the second acidic residue in the same motif and coordinates to the divalent metal ion in the catalytic center. Asp583, Lys586, and Met590 are the three signature amino acid residues that constitute the DK motif (
      • Igura M.
      • Maita N.
      • Kamishikiryo J.
      • Yamada M.
      • Obita T.
      • Maenaka K.
      • Kohda D.
      Structure-guided identification of a new catalytic motif of oligosaccharyltransferase.
      ). Asp583 and Lys586 are directly and Met590 is indirectly involved in the formation of the Ser/Thr-binding pocket, which recognizes the hydroxy amino acid residue at position +2 in the sequon (
      • Matsumoto S.
      • Taguchi Y.
      • Shimada A.
      • Igura M.
      • Kohda D.
      Tethering an N-glycosylation sequon-containing peptide creates a catalytically competent oligosaccharyltransferase complex.
      ).
      Figure thumbnail gr1
      Figure 1Purification of yeast OST complexes containing a point mutation in the Stt3 subunit. A, schematic representation of the gene construction for expression of the PA tag–fused Stt3 protein. The PA-tag sequence, GVAMPGAEDDVV, is inserted at the N terminus of the Stt3 protein via a 10-residue spacer sequence, (GGGGS)2. Transcription is controlled by the GPD promoter and the CYC1 terminator. The asterisks indicate the positions of the mutations. B, CBB-stained gel image of the purified OST proteins, OST[Ost3, PA-Stt3(X)], where X denotes a point mutation. The ost6Δ-pOST3 yeast strain was used as the host to prepare the OST complex containing the Ost3 subunit alone. The triangles indicate the three bands corresponding to the non-, mono-, and diglycosylated forms of the Wbp1 subunit. C, protein yields based on the Wbp1 band intensities. The yield of the OST complex containing the WT Stt3 subunit with the PA tag is set to 1. D, check WT Stt3 incorporation into the OST preparation. Immunoprecipitated samples with the anti-FLAG tag antibody (FLAG-IP) and the anti-PA tag antibody (PA-IP) were analyzed by Western blotting. The WT Stt3 expressed from the chromosome was detected with the anti-FLAG antibody (left). The amounts of the Stt3 protein in the same sample volumes were measured with anti-STT3 antibody (right).
      Figure thumbnail gr2
      Figure 2Point mutation sites in the three-dimensional structure of yeast OST. A, stereoview of the sequon-binding site of the yeast Stt3 subunit (Protein Data Bank entry 6EZN). The 7-residue peptide containing a sequon, Asn-Val-Thr, was adopted from the archaeal AglB-peptide complex (5GMY) after the superimposition of the AglB structure on the Stt3 structure and is shown in cyan. The side chains of the residues mutated in this study are highlighted in magenta. B, catalytic site around the bound divalent metal cation for the activation of the side-chain carboxamide group of the Asn residue at position 0 in the sequon. The Ser/Thr-binding pocket is formed by the residues in the WWDYG motif (Trp516-Trp517-Asp518) and the DK motif (Asp583-Lys586-Met590) for the recognition of the side-chain hydroxy group of the Ser/Thr residue at position +2 in the sequon.
      Even if a lethal mutation was introduced in the catalytic Stt3 subunit, the growth of the yeast host cells is supported by the native stt3 gene in the genome. The E45K, D47A, and D583A mutations were lethal in the spotting plate assay (Fig. S1). The OST complexes containing Ost3 and the PA-tagged Stt3 subunit with a mutation (OST[Ost3, PA-Stt3(X)], where X denotes a mutation) were affinity-purified from digitonin-solubilized microsomal fractions and subjected to SDS-PAGE and CBB staining (Fig. 1B). No bands were present in the sample from the yeast cells transformed with the empty plasmid, indicating the absence of the native OST complex without the PA tag in the purified samples. The Wbp1 subunit contains two potential N-glycosylation sites. The total amount of Wbp1 was used to estimate the relative amounts of the mutation-bearing OST complexes to the WT OST complex with the PA tag (Fig. 1B). The quantification of the other subunits is less useful, due to diffused, adjacent, or high-mobility bands. The protein yields were variable, but not lower than 30% of the WT OST with the PA tag (Fig. 1C). Finally, we checked the incorporation of the WT Stt3 protein expressed from the chromosome into the OST preparations, considering the oligomeric interactions between the OST molecules. We created a yeast strain carrying an stt3 gene with a FLAG epitope tag–encoding sequence in the chromosome. The immunoprecipitated materials with the anti-FLAG antibody (FLAG-IP) and those with the anti-PA antibody (PA-IP) were analyzed by Western blotting (Fig. 1D). As a positive control experiment, the FLAG-IP sample was analyzed with the anti-FLAG antibody, indicating the presence of a single band (Fig. 1D, lane 1). In contrast, the PA-IP sample contained no detectable bands with the anti-FLAG antibody (lane 2), even though much larger amounts of the OST complex protein were loaded to the lanes of PA-IP (lanes 2 and 4) than those of FLAG-IP (lanes 1 and 3). We concluded that the genome-derived Stt3 was virtually absent in the OST preparations purified with the PA epitope tag.

      Comparison of peptide substrates in the in vitro oligosaccharyl transfer assay

      The oligosaccharyl transfer activities of the purified OST complexes were measured with LLO prepared from yeast cells as the oligosaccharide donor and various peptides containing the sequon Asn-Val-Thr as the oligosaccharide acceptor. The glycopeptide products were separated by normal-phase UPLC and quantified with in-line fluorescence detection of the carboxytetramethylrhodamine (TAMRA) dye attached to the peptide substrates. We designed four peptide substrates for the selection of a peptide substrate suitable for the oligosaccharyl transfer assay (Table 1). Kinetic parameters were determined using the OST[Ost3, PA-Stt3(WT)] complex. There were few differences in the maximal velocity (Vm), whereas considerable variations in the Km were observed (Table 2 and Fig. S2). The 15-residue peptide substrate, tam-NVT15, had the smallest Km value, indicating the highest affinity for the OST[Ost3] enzyme.
      Table 1Substrate peptides used in this study
      Peptide nameAmino acid sequence
      The N-glycosylation sequon is underlined. The peptide concentrations were determined by the absorbance at 555 nm, with an extinction coefficient of 90,000 m−1 cm−1. The N-terminal α-amino group is either unmodified (NH2-), modified with an acetyl group (Ac-), or modified with the TAMRA group (TAMRA-). The C-terminal α-carboxyl group is either unmodified (-COOH) or modified with an amide group (-CONH2).
      Preparative method
      NVT9-tamAc-AAYNVTKRK(TAMRA) -COOHChemical synthesis
      TAMRA is attached to the side-chain ε-amino group of the C-terminal lysine residue.
      tam-NVT9(TAMRA)-GAYNVTAKR-COOHChemical synthesis
      tam-NVT15(TAMRA)-GAGGSYNVTKGAGGS-CONH2Chemical synthesis (
      • Igura M.
      • Kohda D.
      Quantitative assessment of the preferences for the amino acid residues flanking archaeal N-linked glycosylation sites.
      )
      tam-NVT25NH2-GPC(TAMRA)-GAGGSGAGGSYNVTKGAGGSGAGGS-COOHProtein expression and chemical modification
      TAMRA is attached to the side-chain sulfhydryl group of the cysteine residue via the thiol-maleimide reaction.
      a The N-glycosylation sequon is underlined. The peptide concentrations were determined by the absorbance at 555 nm, with an extinction coefficient of 90,000 m−1 cm−1. The N-terminal α-amino group is either unmodified (NH2-), modified with an acetyl group (Ac-), or modified with the TAMRA group (TAMRA-). The C-terminal α-carboxyl group is either unmodified (-COOH) or modified with an amide group (-CONH2).
      b TAMRA is attached to the side-chain ε-amino group of the C-terminal lysine residue.
      c TAMRA is attached to the side-chain sulfhydryl group of the cysteine residue via the thiol-maleimide reaction.
      Table 2Enzyme kinetic parameters of the oligosaccharyl transfer reaction of the yeast OST complex for various peptide substrates
      PeptideKmVm
      In the presence of 2 µm LLO.
      μmfmol min−1
      NVT9-tam4.9 ± 0.32.9 ± 0.04
      tam-NVT98.8 ± 1.34.1 ± 0.19
      tam-NVT151.6 ± 0.23.3 ± 0.09
      tam-NVT253.8 ± 0.33.5 ± 0.06
      a In the presence of 2 µm LLO.

      Effects of the position of the epitope tag

      In addition to the OST complexes with the PA tag added to the Stt3 subunit, we prepared the OST complexes with the PA tag in the Ost4 subunit. The tag was inserted at the region encoding the C terminus of the ost4 gene in the genome via a 15-residue spacer, (GGGGS)3. To prepare the yeast OST complexes containing the Ost3 subunit alone, the ost6Δ knockout strain was used. The resulting strain and OST complex are referred to as ost6Δ ost4PA-pOST3 and OST[Ost3, Ost4-PA], respectively. The extra expression of Ost3 from the pOST3 plasmid reduces the deleterious effects caused by the absence of Ost6. In the same manner, we constructed the strain ost3Δ ost4PA-pOST6 and purified the OST[Ost6, Ost4-PA] complex. As for the Ost3-containing OST complex, two constructs bearing the PA tag in different subunits are available. We assessed the influence of the tag position (Fig. 3). The OST[Ost3, PA-Stt3] and OST[Ost3, Ost4-PA] complexes had the same specific activities for the oligosaccharyl transfer activity with the four different peptide substrates, indicating the negligible effects of the insertion and position of the PA tag. Additionally, the growth rates of the cells expressing the PA-tagged Ost4 and the PA-tagged Stt3 were the same as that of the WT cells (data not shown). These results indicate that the added PA tag has no detectable harmful effects, and the PA-tagged Ost4 and Stt3 proteins are fully functional in vitro and in vivo.
      Figure thumbnail gr3
      Figure 3Effects of the epitope tag position on the oligosaccharyl transfer activity. The PA tag was attached to either the N terminus of the Stt3 subunit (PA-STT3) or the C terminus of the Ost4 subunit (Ost4-PA). The two OST complexes contain the Ost3 subunit alone. The four peptides in were used as acceptor peptide substrates. The relative amounts of the PA-tagged OST complexes were estimated by fluorescent Western blotting of the Wbp1 subunit and used to normalize the activities. The activities of the OST with the tag in Stt3 were set to 1. The bar heights indicate the mean values of triplicate measurements. For statistical analysis, Welch's t test was applied, but no significant differences were detected (p = 0.44, 0.14, 0.26, and 0.073).

      Effects of single-point mutations in the Stt3 subunit on the enzymatic activities in vitro

      We measured the oligosaccharyl transfer activity of the OST complexes bearing mutations in the Stt3 subunit, OST[Ost3, PA-Stt3(X)], using the tam-NVT15 peptide as the oligosaccharide acceptor (Fig. 4A). The raw chromatographic data are presented in Fig. S3. The OST complex containing the lethal Stt3(D47A) subunit had no detectable oligosaccharyl transfer activity, confirming the negligible WT OST complex contamination in the final preparations. A good correlation exists between the phenotypic level and the oligosaccharyl transfer activity (inset of Fig. 4A). In parallel, we measured the FNG generation activity (i.e. the hydrolytic activity of LLO) (Fig. 4B and Fig. S4). The mutated OST complexes were incubated with LLO in the absence of the peptide substrate. The released N-oligosaccharide chain was recovered by ethanol precipitation and fluorescently labeled by pyridylamination. Normal-phase UPLC was used to separate and quantify the 2-aminopyridine (2-AP)-labeled N-oligosaccharides. Note that the incubation time was longer (24 h) than that for the oligosaccharyl transfer assay (1 h), to compensate for the low hydrolytic activity. We checked the linearity of the reaction up to 30 h. There is no obvious correlation between the phenotypic level and the FNG generation activity (inset of Fig. 4B).
      Figure thumbnail gr4
      Figure 4Oligosaccharyl transfer and FNG generation activities of the purified OST complexes containing a mutated Stt3 subunit. A, oligosaccharyl transfer activities of the purified OST[Ost3, PA-Stt3(X)] complexes, where X denotes a mutation. The peptide substrate was tam-NVT15. The primary UPLC chromatograms are shown in . B, free N-glycan generation activities of the purified OST[Ost3, PA-Stt3(X)] complexes. The primary UPLC chromatograms are shown in . In A and B, the relative amounts of the OST complexes bearing mutations were determined by CBB staining, as shown in B, and used to normalize the two activities. The bar heights indicate the mean values of triplicate measurements, and the horizontal axes in the insets represent the growth phenotypic level scored with a three-point ordinal scale. Welch's t test was applied between the WT and each mutant. *, p < 0.05. C, correlation plot between the oligosaccharyl transfer and FNG generation activities. Error bars, S.D. Data from A and B.
      We then analyzed the correlation between the two in vitro activities (Fig. 4C). If the hydrolysis of LLO were a simple side reaction, then the two activities would show a linear relationship (the dashed line in Fig. 4C). However, the amino acid replacements of Glu45 increased, and the amino acid replacements of Lys586 and Met590 decreased, the FNG generation activity relative to the oligosaccharyl transfer activity. These results collectively indicate that the two activities can be uncoupled by single-point mutations in the catalytic Stt3 subunit.

      Effects of single-point mutations in the Stt3 subunit on the N-glycosylation and FNG levels in yeast cells

      The N-glycosylation statuses of the carboxypeptidase Y (CPY) protein and the Wbp1 subunit were measured to assess the N-glycosylation level in vivo. The CPY protein contains four potential N-glycosylation sites, and the Wbp1 subunit contains two potential sites, as mentioned above, resulting in five and three bands on the SDS-polyacrylamide gels, respectively (Fig. 5, A and B). Because the N-glycans of the two glycoproteins are dispensable for the growth of yeast cells, they are useful markers for monitoring the N-glycosylation level in yeast cells. Note that all of the in vivo experiments were conducted using yeast strains expressing both the Ost3 and Ost6 subunits from the genome (stt3Δ png1Δ ams1Δ-pSTT3(X)), and the OST complexes (OST[Ost3+Ost6, Stt3(X)]) did not contain any tags. The sequons in the two proteins were almost fully modified in yeast cells expressing OSTs containing the WT Stt3 or the Stt3(M590A) with normal growth phenotypes but were only partially modified in yeast cells expressing OSTs containing temperature-sensitive mutations, Stt3(E45A) and Stt3(K586A). A good correlation exists between the oligosaccharyl transfer activities in vitro and the N-glycosylation statuses of CPY (Fig. 5A) and Wbp1 (Fig. 5B). We also measured the quantity of the total N-glycans attached to highly glycosylated mannoproteins expressed on the cell wall of yeast cells (Fig. S5A). The mannoproteins were recovered from the soluble fractions of cells after autoclave treatment, and the N-glycans were released from the mannoproteins by PNGase F digestion. In contrast to the N-glycans on CPY and Wbp1, the total N-glycan amounts remained the same in yeast cells expressing the mutated Stt3 subunit (Fig. S5B). This result indicates that the cell-wall integrity was maintained in yeast cells expressing these Stt3 mutants.
      Figure thumbnail gr5
      Figure 5N-Glycosylation and FNG levels in mutated Stt3-expressing cells. A, N-glycosylation status of the CPY protein, determined by immunoblotting with the anti-CPY antibody. The values below the gel images are the average numbers of N-glycans attached to the CPY protein. The whole-cell lysates were obtained from the stt3Δ png1Δ ams1Δ-pSTT3(X) yeast strains, where X denotes a mutation. The right panel shows the correlation plot between the N-glycosylation levels (the number of N-glycans) of CPY in cells and the oligosaccharyl transfer activities of the purified OST[Ost3, PA-Stt3(X)], adopted from A. B, N-glycosylation status of the Wbp1 subunit of the OST complex, determined by immunoblotting with the anti-Wbp1 antibody. The values below the gel image are the average numbers of N-glycans attached to the Wbp1 protein. The whole-cell lysates were the same as those used in the CPY analyses. The right panel shows the correlation plot between the N-glycosylation levels (the number of N-glycans) of Wbp1 in cells and the oligosaccharyl transfer activities of the purified OST[Ost3, PA-Stt3(X)], adopted from A. C, contents of FNG in yeast strains, stt3Δ png1Δ ams1Δ-pSTT3(X), relative to that in the yeast strain harboring pSTT3(WT). The primary UPLC chromatograms are shown in . The right panel shows the correlation plot between the FNG contents in cells and the FNG generation activities of the purified OST[Ost3, PA-Stt3(X)], adopted from B. Welch's t test was applied for statistical analyses. In the correlation plots in A–C, the horizontal and vertical error bars represent the S.D. from three independent experiments, except for six independent experiments for WT in A and WT and E45A in C.
      We then measured the amounts of FNG generated by OST in cells (Fig. 5C and Fig. S6). As expected, a good correlation exists between the FNG generation activity of the purified mutated OST enzymes and the amounts of FNG in the stt3Δ png1Δ ams1Δ-pSTT3(X) strains (Fig. 5C). In particular, yeast cells expressing Stt3(E45A) accumulated much more OST-derived FNG. This phenomenon could be caused by the increased supply of LLO. Thus, we measured the LLO contents in whole cells (Fig. S7). LLO was extracted from yeast cells and hydrolyzed to measure the amounts of released oligosaccharides. The Stt3(E45A) cells had a similar amount of LLO relative to the WT, indicating that the excessive supply of LLO is not the reason for the elevated FNG level in the Stt3(E45A)-expressing cells.

      Different modes of action of the Ost3-containing and the Ost6-containing OST complexes in the in vitro assays

      We assessed the effects of the exchange of the homologous Ost3 and Ost6 subunits (Fig. 6). The Ost3-containing OST complex, OST[Ost3, Ost4-PA], had higher oligosaccharyl transfer activity than the Ost6-containing OST complex, OST[Ost6, Ost4-PA], whereas the two OST complexes had similar FNG generation activities. We then tested the effects of the addition of the reducing agent DTT on the two activities because Ost3 and Ost6 are redox-related subunits. DTT had no effects on the oligosaccharyl transfer activity of the Ost3-containing OST complex, OST[Ost3, Ost4-PA], but enhanced the FNG generation activity more than 2-fold. In contrast, DTT had no effects on the two activities of the Ost6-containing OST complex, OST[Ost6, Ost4-PA]. These results suggest the unique roles of the Ost3 and Ost6 subunits in the recognition of a sequon-containing polypeptide chain and the generation of FNG in cells.
      Figure thumbnail gr6
      Figure 6Effects of exchanging the Ost3 and Ost6 subunits on the enzymatic activities. A, in vitro oligosaccharyl transfer activities of the OST complexes containing either Ost3 or Ost6, in the absence and the presence of 5 mm DTT. The tam-NVT15 peptide was used in the oligosaccharyl transfer assay. B, same as A, but for FNG generation activities. In A and B, the PA tag was inserted at the C terminus of the Ost4 subunit. The relative amounts of the OST complexes containing either Ost3 or Ost6 were estimated by fluorescent Western blotting of the Wbp1 subunit and used to normalize the two activities. The activities of OST[Ost3, Ost4-PA] in the absence of DTT were set to 1. The bar heights indicate the mean values of triplicate measurements. Welch's t test was applied for statistical analyses.

      Discussion

      Cell growth spotting assays have been used for mutational analyses of the yeast OST enzyme, based on the fact that N-glycosylation is essential for the survival and growth of yeast cells. The problem with these assays is that the dysfunction of the OST enzyme results in a simple growth phenotype, either lethal or temperature-sensitive. Moreover, the OST enzyme catalyzes the hydrolysis of the oligosaccharide donor, LLO, to release FNGs into the ER lumen. Although the physiological significance of the FNGs generated in the ER remains elusive, the functional modulation of the two activities in yeast cells could have diverse influences, eventually leading to an unpredictable growth phenotype. Thus, independent assessments of the two activities are desirable, using purified enzyme samples in vitro. In this study, we inserted the recently developed PA affinity tag into the region encoding the N terminus in the mutated stt3 gene, under the control of the strong GPD promoter on the expression plasmid. The co-expression of the WT stt3 gene from the yeast genome maintains the growth of yeast cells, even if the mutation is lethal. In previous studies, affinity tags were introduced to the C terminus of the Stt3 subunit (
      • Li G.
      • Yan Q.
      • Nita-Lazar A.
      • Haltiwanger R.S.
      • Lennarz W.J.
      Studies on the N-glycosylation of the subunits of oligosaccharyl transferase in Saccharomyces cerevisiae.
      ,
      • Igura M.
      • Maita N.
      • Kamishikiryo J.
      • Yamada M.
      • Obita T.
      • Maenaka K.
      • Kohda D.
      Structure-guided identification of a new catalytic motif of oligosaccharyltransferase.
      ,
      • Karaoglu D.
      • Kelleher D.J.
      • Gilmore R.
      The highly conserved Stt3 protein is a subunit of the yeast oligosaccharyltransferase and forms a subcomplex with Ost3p and Ost4p.
      ,
      • Spirig U.
      • Glavas M.
      • Bodmer D.
      • Reiss G.
      • Burda P.
      • Lippuner V.
      • Te Heesen S.
      • Aebi M.
      The STT3 protein is a component of the yeast oligosaccharyltransferase complex.
      ). Considering the membrane topology of the OST complex, the attachment to the N terminus would be better, because the N terminus is located on the opposite side of the ER membrane and is distant from the large luminal soluble domain, to avoid the risk of affecting the enzymatic activity. The 12-residue PA tag has a high affinity for the specific mAb, NZ-1 (
      • Fujii Y.
      • Kaneko M.
      • Neyazaki M.
      • Nogi T.
      • Kato Y.
      • Takagi J.
      PA tag: a versatile protein tagging system using a super high affinity antibody against a dodecapeptide derived from human podoplanin.
      ), and the binding of the PA tag (Kd = 0.49 nm) is 10–100-fold stronger than those of other frequently used tags, including the FLAG tag (Kd = 100 nm) (
      • Einhauer A.
      • Jungbauer A.
      Affinity of the monoclonal antibody M1 directed against the FLAG peptide.
      ) and the 1D4 tag (Kd = 20 nm) (
      • Locatelli-Hoops S.C.
      • Gorshkova I.
      • Gawrisch K.
      • Yeliseev A.A.
      Expression, surface immobilization, and characterization of functional recombinant cannabinoid receptor CB2.
      ). The ultrahigh affinity withstands vigorous washing and hence ensures the minimal co-purification of the tagless OST complex containing the WT Stt3 subunit (Fig. 1). It is important to check the impacts of the tag attachment on the enzymatic activities. We compared the oligosaccharyl transfer activities between the two OST complexes containing the PA tag on the N terminus of the Stt3 subunit and that on the C terminus of the Ost4 subunit (Fig. 3). The results confirmed that the tag had no adverse effects on the enzymatic activity.
      We selected amino acid residues in the first DXD motif (Glu45–Asp47) and the DK motif (Asp583-Lys586-Met590) in the Stt3 sequence as mutational sites (
      • Igura M.
      • Maita N.
      • Kamishikiryo J.
      • Yamada M.
      • Obita T.
      • Maenaka K.
      • Kohda D.
      Structure-guided identification of a new catalytic motif of oligosaccharyltransferase.
      ). These residues are positioned close to the peptide substrates in the bound state (Fig. 2) (
      • Bai L.
      • Wang T.
      • Zhao G.
      • Kovach A.
      • Li H.
      The atomic structure of a eukaryotic oligosaccharyltransferase complex.
      ,
      • Wild R.
      • Kowal J.
      • Eyring J.
      • Ngwa E.M.
      • Aebi M.
      • Locher K.P.
      Structure of the yeast oligosaccharyltransferase complex gives insight into eukaryotic N-glycosylation.
      ). We purified the OST complexes containing the PA-tagged WT and mutated Stt3 subunits, OST[Ost3, PA-Stt3(WT or X)], and measured their oligosaccharyl transfer and FNG generation activities in the presence and absence of the peptide substrates (Fig. 4). We also determined the growth phenotypes of the mutated yeast strains (stt3Δ-pSTT3(X)) (Fig. S1). Among them, we selected the Stt3 mutants with temperature-sensitive and normal growth phenotypes for further in vivo studies. The stt3Δ png1Δ ams1Δ-pSTT3(X) strains were cultured to stationary phase in YPD medium at a permissive temperature (30 °C) until the OD600 reached 10. We measured the N-glycosylation statuses of the CPY and Wbp1 glycoproteins in vivo (Fig. 5, A and B). CPY (carboxypeptidase Y) is a hydrolytic enzyme that catalyzes the release of amino acid residues from the C terminus of peptides in the vacuole. CPY is a soluble glycoprotein containing four N-glycosylation sites. Wbp1 is one of the eight subunits of the OST enzyme, with a single-spanning transmembrane helix and a large N-terminal soluble domain on the luminal side. The soluble domain contains two N-glycosylation sites. We also measured the total amounts of N-glycans attached to the cell-wall mannoproteins (Fig. S5) and the FNG (Fig. 5C) and LLO (Fig. S7) contents in cells. Considering these results, we gained new insights as follows.
      First, we discuss the different roles of the interchangeable Ost3 and Ost6 subunits. The previous in vivo studies demonstrated that the Ost3-containing and Ost6-containing OST complexes modified distinct repertoires of N-glycosylated proteins (
      • Poljak K.
      • Selevsek N.
      • Ngwa E.
      • Grossmann J.
      • Losfeld M.E.
      • Aebi M.
      Quantitative profiling of N-linked glycosylation machinery in yeast Saccharomyces cerevisiae.
      ,
      • Schulz B.L.
      • Stirnimann C.U.
      • Grimshaw J.P.A.
      • Brozzo M.S.
      • Fritsch F.
      • Mohorko E.
      • Capitani G.
      • Glockshuber R.
      • Grütter M.G.
      • Aebi M.
      Oxidoreductase activity of oligosaccharyltransferase subunits Ost3p and Ost6p defines site-specific glycosylation efficiency.
      ). Here, we determined the different properties of the two isoforms of the OST complexes at the molecular level. The sequence identity (20%) and similarity (46%) are modest between Ost3 and Ost6 (17). The Ost3-containing OST complex had higher oligosaccharyl transfer activity than the Ost6-containing OST complex (Fig. 6A). The reducing agent DTT enhanced the FNG generation activity of the Ost3-containing OST complex more than 2-fold but had no effects on that of the Ost6-containing OST complex (Fig. 6B). Thus, the Ost3 and Ost6 subunits can affect the catalytic activity of Stt3 in unique and different manners, through a direct or indirect mechanism. Contrary to previous conceptions, a recent cell biological study has suggested that the oligosaccharyl transfer catalyzed by the yeast OST may not be a co-translational process coupled with the passage across the translocon channel (
      • Shrimal S.
      • Cherepanova N.A.
      • Mandon E.C.
      • Venev S.V.
      • Gilmore R.
      Asparagine-linked glycosylation is not directly coupled to protein translocation across the endoplasmic reticulum in Saccharomyces cerevisiae.
      ), although the Ost3 and Ost6 subunits have been considered to interact with the translocon (
      • Bai L.
      • Wang T.
      • Zhao G.
      • Kovach A.
      • Li H.
      The atomic structure of a eukaryotic oligosaccharyltransferase complex.
      ,
      • Wild R.
      • Kowal J.
      • Eyring J.
      • Ngwa E.M.
      • Aebi M.
      • Locher K.P.
      Structure of the yeast oligosaccharyltransferase complex gives insight into eukaryotic N-glycosylation.
      ). We expect that the different enzymatic properties of the Ost3-containing and Ost6-containing OST complexes in vitro will serve as a good starting point for future studies on the different roles of the Ost3/Ost6 subunits in yeast cells.
      Second, the two activities of the OST enzyme can be uncoupled by single-point mutations in the Stt3 subunit. The replacements of Glu45 by Ala, Asp, Gln, and Lys enhanced the FNG generation activity relative to the oligosaccharyl transfer activity, and conversely, the replacements of Lys586 and Met590 by Ala suppressed the FNG generation activity (Fig. 4C). Lys586 and Met590 are distant from the catalytic center formed around the divalent metal ion (Fig. 2). Interestingly, the LLO hydrolytic activity can be manipulated by modulating the peptide binding mode remotely.
      Third, under physiological conditions, the sequons are embedded in long, unfolded polypeptide chains in the co-translational mode or conformationally restricted yet flexible segments of folded polypeptides in the post-translational mode of the oligosaccharyl transfer reaction. In the oligosaccharyl transfer assays in vitro, short peptides up to 10 residues have conventionally been used. Thus, the use of peptides longer than 10 residues might be better in in vitro assays, to obtain a more realistic assessment of the OST enzyme activity inside the cells. We used the correlative relationship between the oligosaccharyl transfer activity in vitro and the N-glycosylation level of glycoproteins in vivo as an evaluating criterion. Fig. 7 shows the correlation plots for each of the four peptide substrates. Each point in the plots represents the data from a different mutation in the Stt3 subunit (Fig. 7A). The changes in the oligosaccharyl transfer activity in vitro by mutations are well-correlated with the changes in the N-glycosylation level in cells (i.e. the number of N-glycans attached to CPY) induced by mutation. With all data points considered, the 15-residue tam-NVT15 peptide shows the best correlation between the in vitro and in vivo activities (Fig. 7B). Therefore, we used this peptide in the subsequent oligosaccharyl transfer assays. A closer look at the data revealed some exceptions (the magenta and blue horizontal arrows in Fig. 7B). These exceptional preferences may be attributable to unwanted interactions of the positively charged, hydrophobic TAMRA dye with the mutation sites in the OST complex. This unexpected finding advises us to pay careful attention to the peptide substrate design.
      Figure thumbnail gr7
      Figure 7Correlation plots for the quality evaluation of peptide substrates. A, oligosaccharyl transfer activities of the OST[Ost3, PA-Stt3(X)] complexes, where X denotes a mutation, relative to those of the OST[Ost3, PA-Stt3(WT)] complex. The bar heights indicate the mean values of triplicate measurements. Welch's t test was applied between the WT and each mutant. *, p < 0.05. The four peptides listed in were used as acceptor substrates. B, correlation plots. The horizontal axis represents the oligosaccharyl transfer activity in vitro taken from A, and the vertical axis represents the N-glycosylation status of CPY taken from A. Note that the plot with tam-NVT15 is identical to the correlation plot in the inset of A. The square of the correlation coefficient, R2, is shown for each plot. The dashed lines simply connect the origin and the point corresponding to WT as a visual guide and are not the regression lines. The magenta and blue horizontal arrows indicate the outlier data points.
      Finally, the data in Figure 4, Figure 5 are replotted from the standpoint of the individual mutants, for a more detailed discussion (Fig. 8). The five Stt3 mutants in Fig. 8 have a normal or temperature-sensitive phenotype. Even if the N-glycosylation level of CPY is retained to an extent of at least 30% of that in the WT cells, yeast cells can survive and grow at the permissive temperature, 30 °C. In the Stt3(E45Q) and Stt3(E45A) cells, the contents of FNG generated by OST increased 1.5- and 4-fold, respectively, suggesting that the high FNG levels formed in the ER lumen have no adverse effects on yeast cell growth. In Stt3(M590A) cells, the content of FNG generated by OST decreased by 50%. The fact that the Stt3(M590A) strain has a normal growth phenotype indicates that a decrease in the FNG level in cells down to 50% is tolerable for yeast cell growth. These results are consistent with the previous report (
      • Harada Y.
      • Buser R.
      • Ngwa E.M.
      • Hirayama H.
      • Aebi M.
      • Suzuki T.
      Eukaryotic oligosaccharyltransferase generates free oligosaccharides during N-glycosylation.
      ). The FNG levels as low as 10% of the WT cells in the png1Δ ams1Δ ost3Δ ost6Δ and the png1Δ ams1Δ alg6Δ strains sustained yeast cell growth.
      Figure thumbnail gr8
      Figure 8Comparison of the two in vitro and two in vivo activities for each of the OST complexes containing a mutated Stt3 subunit. The in vitro data were obtained with the purified OST[Ost3, PA-Stt3(X)] proteins, whereas the in vivo data were obtained with the yeast strains stt3Δ png1Δ ams1Δ-pSTT3(X), where X denotes a mutation. These data are redisplayed from (A and B) and (A and C) for a detailed comparison. Welch's t test was performed between the WT and each mutant. *, p < 0.05. The growth phenotype and the time required to reach OD600 10 from 0.2 in the YPD medium at 30 °C are shown.
      Together, we did not find any vital functions of the FNG formed in the ER. However, it is tempting to speculate that the hydrolytic activity of LLO could be a second enzymatic activity preserved throughout eukaryotic evolution, during which the number of glycoproteins and the total number of modified sequons have expanded 10–20-fold (
      • Cherepanova N.
      • Shrimal S.
      • Gilmore R.
      N-Linked glycosylation and homeostasis of the endoplasmic reticulum.
      ). The FNGs generated in the ER might be necessary for yeast in special environments and other eukaryotic organisms even in normal growth environments and may serve as an organic osmolyte. In the case of the Gram-negative bacterium Campylobacter, the FNG generated by the bacterial OST in the periplasmic space is considered to play a role in balancing the osmotic pressure between the cytosol and extracellular environment (
      • Dwivedi R.
      • Nothaft H.
      • Reiz B.
      • Whittal R.M.
      • Szymanski C.M.
      Generation of free oligosaccharides from bacterial protein N-linked glycosylation systems.
      ,
      • Nothaft H.
      • Liu X.
      • McNally D.J.
      • Li J.
      • Szymanski C.M.
      Study of free oligosaccharides derived from the bacterial N-glycosylation pathway.
      ).
      In conclusion, detailed comparisons of the two activities of a series of mutations in the Stt3 subunit in vitro and in vivo have provided new knowledge about the yeast OST enzyme: specifically, 1) the different properties between the Ost3- and Ost6-containing OST complexes, 2) the single-point mutations that change the relative activity of the LLO hydrolytic reaction to the oligosaccharyl transfer reaction, 3) the better design of peptide substrates, and 4) the dispensable function of FNG generated by OST under standard laboratory conditions. The uncoupling of the two activities by single-point mutations can offer useful insights into the generation mechanism and the physiological roles of the FNG generated in the ER. Previous in vivo knockout analyses of HEK293 and liver cells suggested that the human STT3B-containing OST complex generated FNG in cells, but the STT3A-containing OST complex did not (
      • Lu H.
      • Fermaintt C.S.
      • Cherepanova N.A.
      • Gilmore R.
      • Yan N.
      • Lehrman M.A.
      Mammalian STT3A/B oligosaccharyltransferases segregate N-glycosylation at the translocon from lipid-linked oligosaccharide hydrolysis.
      ). The mutations in the DXD motif and DK motif in the human STT3A/STT3B subunits will be helpful to confirm and investigate the functional differences between the two catalytic STT3 subunits in the human OST enzymes.

      Experimental procedures

      Reagents

      2-AP, 5-fluoroorotic acid (5-FOA), digitonin, and borane-dimethylamine reagent were purchased from FujiFilm Wako Pure Chemical. The peptides used in the oligosaccharyl transfer assay were custom-synthesized (Hayashi Kasei, Osaka, Japan). A TAMRA group was introduced during the peptide synthesis to the N terminus and the side chain of a lysine residue. The mouse monoclonal anti-carboxypeptidase Y (CPY) antibody (10A5B5) was purchased from Abcam. The rabbit polyclonal anti-Wbp1 antibody and anti-Stt3 antibody were raised against a peptide fragment of yeast Wbp1 (residues 21–392 expressed as an N-terminally His-tagged protein) and that of yeast Stt3 (residues 485–718), respectively (
      • Igura M.
      • Maita N.
      • Kamishikiryo J.
      • Yamada M.
      • Obita T.
      • Maenaka K.
      • Kohda D.
      Structure-guided identification of a new catalytic motif of oligosaccharyltransferase.
      ). The rabbit polyclonal anti-DDDDK-tag (anti-FLAG) antibody (PM020) was purchased from MBL. The peroxidase-conjugated anti-rabbit IgG antibody (NA934VS) was purchased from Amersham Biosciences ECL. The IRDye 800CW goat anti-rabbit IgG secondary antibody was purchased from LI-COR. The anti-FLAG M2–agarose (A2220) was purchased from Millipore. The XL-Western Marker (SP-2180) was purchased from Intégrale.

      Yeast strains

      Yeast strains used in this study are listed in Table 3. Gene disruption and tag insertion to the stt3 gene were performed by the PCR-based technique (
      • Longtine M.S.
      • McKenzie A.
      • Demarini D.J.
      • Shah N.G.
      • Wach A.
      • Brachat A.
      • Philippsen P.
      • Pringle J.R.
      Additional modules for versatile and economical PCR-based gene deletion and modification in Saccharomyces cerevisiae.
      ). The N-terminal amino acid sequence fused to Stt3 is DYKDDDDK-(GGGGS)2 (the FLAG epitope tag sequence followed by spacer sequence). The genomic ost4 gene was C-terminally tagged with the PA epitope by two-step markerless gene replacement (
      • Widlund P.O.
      • Davis T.N.
      A high-efficiency method to replace essential genes with mutant alleles in yeast.
      ). The C-terminal amino acid sequence fused to Ost4 is (GGGGS)3-GVAMPGAEDDVV (spacer sequence followed by the PA epitope). Correct tag integration was confirmed by PCR and DNA sequencing. For the selection of yeast strains containing pRS313-STT3(X) alone, a fresh colony of yeast strains, stt3Δ png1Δ ams1Δ containing pRS316-pSTT3 and pRS313-pSTT3(X) together, was inoculated on synthetic dropout medium (SD) containing 5-FOA and lacking l-histidine. Plates were incubated in an inverted position at 30 °C. The series of operations was repeated three times.
      Table 3Yeast strains used in this study
      Strain nameGenotypeSourcePurpose
      BY4741MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0Laboratory strainParental strain, source of LLO
      stt3Δ-pSTT3MATa stt3Δ::KanMX BY4741- pRS316-STT3This studyPlasmid shuffling
      stt3Δ png1Δ ams1Δ- pSTT3MATa stt3Δ::KanMX png1Δ::HygMX ams1Δ::LEU2 BY4741- pRS316-STT3This studyPlasmid shuffling
      stt3Δ png1Δ ams1Δ- pSTT3/pSTT3(X)MATa stt3Δ::KanMX png1Δ::HygMX ams1Δ::LEU2 BY4741- pRS316-STT3/pRS313-STT3(X)This studyPlasmid shuffling
      stt3Δ png1Δ ams1Δ- pSTT3(X)MATa stt3Δ::KanMX png1Δ::HygMX ams1Δ::LEU2 BY4741- pRS313-STT3(X)This studyQuantification of the glycosylation status of CPY and Wbp1, and the amounts of FNG, LLO, and mannoprotein N-glycans
      ost6Δ-pOST3
      The extra expression of Ost3 from the pOST3 plasmid reduces the deleterious effects by the absence of Ost6.
      MATa ost6Δ::LEU2 BY4741- pOST3This studyProduction of OST[Ost3, PA-Stt3(X)]
      ost6Δ ost4PA-pOST3
      The extra expression of Ost3 from the pOST3 plasmid reduces the deleterious effects by the absence of Ost6.
      MATa ost6Δ::LEU2 ost4Δ::ost4-PA BY4741- pOST3This studyProduction of OST[Ost3, Ost4-PA]
      ost3Δost4PA-pOST6
      The extra expression of Ost6 from the pOST6 plasmid reduces the deleterious effects by the absence of Ost3.
      MATa ost3Δ::LEU2 ost4Δ::ost4-PA BY4741- pOST6This studyProduction of OST[Ost6, Ost4-PA]
      ost6Δ-FLAGstt3-pOST3
      The extra expression of Ost3 from the pOST3 plasmid reduces the deleterious effects by the absence of Ost6.
      MATa ost6Δ::LEU2 stt3Δ::HygMX-FLAG-stt3 BY4741- pOST3This studyCheck contamination of mutated PA-Stt3 by WT FLAG-Stt3
      a The extra expression of Ost3 from the pOST3 plasmid reduces the deleterious effects by the absence of Ost6.
      b The extra expression of Ost6 from the pOST6 plasmid reduces the deleterious effects by the absence of Ost3.

      Plasmids

      Plasmids used in this study are listed in Table 4. pRS313-STT3 was generated by the deletion of the 3×HA tag sequence (30 amino acid residues) from the pRS313-STT3-3HA plasmid constructed previously (
      • Igura M.
      • Maita N.
      • Kamishikiryo J.
      • Yamada M.
      • Obita T.
      • Maenaka K.
      • Kohda D.
      Structure-guided identification of a new catalytic motif of oligosaccharyltransferase.
      ). The STT3 region is a 2,680-bp fragment containing the entire stt3 gene (2,157 bp) plus 299 bp (native promoter) and 224 bp (native terminator) at the 5′- and 3′-ends, respectively. The pPA-STT3, pOST3, and pOST6 plasmids were constructed with a Gibson Assembly kit (New England Biolabs). The open reading frames of the stt3, ost3, and ost6 genes were amplified using genomic DNA from the yeast strain BY4741 as the PCR template. For the construction of pPA-STT3, the GPD promoter, the yeast Stt3 sequence, and the CYC1 terminator were assembled into pRS313. The GPD promoter and the CYC1 terminator were derived from pAG416-GPD-ccdB as the PCR template. pAG416GPD-ccdB was a gift from Susan Lindquist (Addgene plasmid 14148; RRID:Addgene_14148). Then the DNA sequences corresponding to the PA tag, GVAMPGAEDDVV, and the spacer sequence, (GGGGS)2, were inserted between the GPD promoter and the Stt3 sequence (Fig. 1A). For the construction of pOST3 and pOST6, the DNA sequences encoding Ost3 and Ost6 were inserted into the multiple cloning site of pAG416-GPD-ccdB. An inverse PCR-based site-directed mutagenesis kit (SMK-101, TOYOBO) was used to generate single-point mutations to generate pPA-STT3(X).
      Table 4Plasmids used in this study
      Plasmid nameDescriptionSourcePurpose
      pRS316CEN6/ARS4, URA3BYP562, National BioResource Project JapanParental plasmid
      pRS316-STT3Described previouslyRef.
      • Igura M.
      • Maita N.
      • Kamishikiryo J.
      • Yamada M.
      • Obita T.
      • Maenaka K.
      • Kohda D.
      Structure-guided identification of a new catalytic motif of oligosaccharyltransferase.
      Expression of WT Stt3 under the native promoter
      pRS313CEN6/ARS4, HIS3BYP559, National BioResource Project JapanParental plasmid, empty plasmid in Fig. 1 and Fig. S1
      pRS313-STT3-3HADescribed previouslyRef.
      • Igura M.
      • Maita N.
      • Kamishikiryo J.
      • Yamada M.
      • Obita T.
      • Maenaka K.
      • Kohda D.
      Structure-guided identification of a new catalytic motif of oligosaccharyltransferase.
      Expression of WT Stt3 under the native promoter
      pRS313-STT33×HA tag sequence was removed from pRS313-STT3-3HAThis studyExpression of WT and mutated Stt3 under the native promoter in plasmid shuffling experiments and other in vivo experiments
      pSTT3(E45A)pRS313-STT3(E45A)This study
      pSTT3(E45D)pRS313-STT3(E45D)This study
      pSTT3(E45K)pRS313-STT3(E45K)This study
      pSTT3(E45Q)pRS313-STT3(E45Q)This study
      pSTT3(D47A)pRS313-STT3(D47A)This study
      pSTT3(D583A)pRS313-STT3(D583A)This study
      pSTT3(K586A)pRS313-STT3(K586A)This study
      pSTT3(M590A)pRS313-STT3(M590A)This study
      pAG416-GPD-ccdBCEN6/ARS4, HIS3, GPD, CYC1, ccdB14148, AddgeneParental plasmid
      pOST3pAG416-GPD-OST3This studyCompensate for the lack of Ost6
      pOST6pAG416-GPD-OST6This studyCompensate for the lack of Ost3
      pPA-STT3pRS313-GPD-PA-spacer-STT3This studyExpression of WT and mutated PA-Stt3 under the GPD promoter in the strain, ost6Δ-pOST3
      pPA-STT3(E45A)pRS313-GPD-PA-spacer-STT3(E45A)This study
      pPA-STT3(E45D)pRS313-GPD-PA-spacer-STT3(E45D)This study
      pPA-STT3(E45K)pRS313-GPD-PA-spacer-STT3(E45K)This study
      pPA-STT3(E45Q)pRS313-GPD-PA-spacer-STT3(E45Q)This study
      pPA-STT3(D47A)pRS313-GPD-PA-spacer-STT3(D47A)This study
      pPA-STT3(D583A)pRS313-GPD-PA-spacer-STT3(D583A)This study
      pPA-STT3(K586A)pRS313-GPD-PA-spacer-STT3(K586A)This study
      pPA-STT3(K590A)pRS313-GPD-PA-spacer-STT3(M590A)This study

      Plasmid shuffling and spotting plate assay

      Stt3Δ cells bearing pRS316-STT3 encoding the WT stt3 were transformed with p(RS313-)STT3(X), encoding the stt3 gene containing a single-point mutation X. The transformants were grown in SD medium lacking l-histidine until the OD600 reached 1. Collected cells were diluted in a 5-fold series. Aliquots (3 μl) of each dilution were spotted onto SD plates lacking l-histidine and those lacking l-histidine supplemented with 5-FOA, at a final concentration of 1 mg ml−1. After incubations at 25, 30, and 37 °C for 2 days, cell growth was assessed.

      Extraction of LLO for in vitro assays

      The yeast cells, strain BY4741, were grown in a 3-liter flask containing 1 liter of synthetic complete medium at 30 °C until the OD600 reached 10. Cells were collected and used for the extraction of LLO, as described previously (
      • Harada Y.
      • Buser R.
      • Ngwa E.M.
      • Hirayama H.
      • Aebi M.
      • Suzuki T.
      Eukaryotic oligosaccharyltransferase generates free oligosaccharides during N-glycosylation.
      ). To determine the amount of LLO, we used the end point assay and acid-hydrolysis methods. The end point assay measures the maximum amount of the glycopeptide produced by OST in the presence of an excess of the peptide substrate. The acid hydrolysis method measures the oligosaccharide released from LLO by acid hydrolysis. The LLO was hydrolyzed using 20 mm hydrochloride in a 2-propanol/water mixture (1:1, v/v) at 100 °C for 30 min. After evaporation to dryness, the pellet was labeled with 2-AP (see below). The pyridylaminated oligosaccharides were separated by Infinity 1290 UPLC (Agilent) with an AdvanceBio Glycan Mapping column (Agilent) and quantified by an in-line fluorescence detector. Solvent A was 100 mm ammonium acetate buffer, pH 4.5, and solvent B was 100% acetonitrile. The column was equilibrated with 20% solvent A at a flow rate of 0.5 ml min−1. A linear gradient of solvent A was applied from 20 to 40% over 8.5 min. Typically, 10–20 nmol of LLO was obtained from 10 g of wet yeast cells from a 1-liter culture.

      Affinity purification of the yeast OST complex bearing the PA tag

      The OST complex was purified from yeast strains as described previously, with modifications (
      • Bai L.
      • Wang T.
      • Zhao G.
      • Kovach A.
      • Li H.
      The atomic structure of a eukaryotic oligosaccharyltransferase complex.
      ). Yeast cells transformed with pPA-STT3(X) were grown in SD medium lacking appropriate nutrients (−Ura and −His). Recovered yeast cells were lysed with glass beads, and microsome fractions were collected by ultracentrifugation at 100,000 × g. The membrane pellets were resuspended and dissolved in 20 mm Tris-HCl buffer, pH 7.5, 1.5% digitonin, 0.5 m NaCl, 1 mm MgCl2, 1 mm MnCl2, 1 mm EDTA, 1 mm phenylmethylsulfonyl fluoride, protease inhibitor mixture (Roche Applied Science), and 10% (v/v) glycerol. After a 1-h incubation, the mixture was ultracentrifuged for 30 min at 100,000 × g, and the clarified supernatant was mixed with prewashed anti-PA tag antibody beads (Fujifilm Wako Pure Chemicals) at 4 °C overnight with gentle shaking. The affinity beads were collected by centrifugation and washed five times with 20 mm Tris-HCl buffer, pH 7.5, 0.1% digitonin, 150 mm NaCl, 1 mm MgCl2, and 1 mm MnCl2. Finally, the OST complex was eluted with the same buffer containing 0.1 mg ml−1 PA-tag peptide (Fujifilm Wako Pure Chemicals). The eluted fractions were separated by SDS-PAGE using gradient gels (10–20%). Staining with CBB (Fig. 1) and fluorescence measurement after Western blotting (Figure 3, Figure 4, Figure 5, Figure 6) were used for protein quantification. In the Western blotting, proteins in gels were transferred to Immobilon-FL PVDF membranes (Millipore). Anti-Wbp1 antiserum was used as the primary antibody at a dilution of 1:5,000, and IRDye-labeled goat anti-rabbit IgG was used as the secondary antibody at a dilution of 1:25,000. The fluorescence of the IRDye was measured by an Odyssey (LI-COR) imaging system. Note that the PA-tag peptide was not removed from the final protein solutions. We confirmed that the PA-tag peptide did not influence the enzymatic activities.

      Check contamination of the yeast OST preparation by the WT Stt3

      Yeast cells (ost6Δ-FLAGstt3-pOST3) transformed with pPA-STT3(WT) were grown in SD medium lacking appropriate nutrients (−Ura and −His). The materials containing FLAG-Stt3(WT) were immunoprecipitated from yeast cells expressing the chromosomal stt3 gene using anti-FLAG affinity gel. The materials absorbed were eluted with the SDS sample buffer. Separately, the OST complex containing PA-Stt3(WT) expressed from the plasmid was immunopurified from the same yeast cells as described above. The collected materials were separated on SDS-PAGE using gradient gels (10–20%). The proteins in the gels were transferred to Immobilon-P PVDF membranes (Millipore), probed with the indicated antibodies, and visualized by chemiluminescence with the SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific). The anti-FLAG antibody and anti-STT3 antiserum were used at dilutions of 1:5000 and 1:1000, respectively. The peroxidase-conjugated anti-rabbit IgG antibody was used at a dilution of 1:12,500. The chemiluminescent images were recorded with an LAS-3000 multicolor image analyzer (Fuji Photo Film).

      Oligosaccharyl transfer assay

      The oligosaccharyl transfer assay was performed using the fluorescent peptide substrate method (
      • Igura M.
      • Kohda D.
      Quantitative assessment of the preferences for the amino acid residues flanking archaeal N-linked glycosylation sites.
      ,
      • Yamasaki T.
      • Kohda D.
      A radioisotope-free oligosaccharyltransferase assay method.
      ). The reaction mixture (total 10 μl) contained 1 μm yeast LLO, 5 μm substrate peptide, and 1–5 nm purified WT or mutated OST protein, in 20 mm Tris-HCl buffer, pH 7.5, 5 mm MnCl2, 5 mm MgCl2, and 0.1% (v/v) Triton X-100. The substrate peptides were labeled with a fluorescent dye, TAMRA, for detection (Table 1). The reaction mixture was incubated for 1 h at 30 °C. The reaction was stopped by the addition of 2 μl of 60 mm EDTA-NaOH, pH 8.0. The reaction products were separated by Infinity 1290 UPLC (Agilent) with an AdvanceBio Glycan Mapping column and quantified by an in-line fluorescence detector. Solvent A was 100 mm ammonium acetate buffer, pH 4.5, and solvent B was 100% acetonitrile. The column was equilibrated with 25% solvent A at a flow rate of 0.5 ml min−1. A linear gradient of solvent A was applied from 25 to 57% over 8.5 min. Kinetic parameters for peptide substrates were determined by nonlinear least-squares fitting of the initial rates in the presence of 2 μm LLO to the Michaelis–Menten equation, using the program Kaleidagraph version 4.5.1 (Synergy Software).

      Pyridylamination

      The reducing end of the oligosaccharides was derivatized with 2-AP as described previously, with some modifications (
      • Hirayama H.
      • Seino J.
      • Kitajima T.
      • Jigami Y.
      • Suzuki T.
      Free oligosaccharides to monitor glycoprotein endoplasmic reticulum-associated degradation in Saccharomyces cerevisiae.
      ). The dried oligosaccharides were incubated with 20 μl of 2-AP in acetic acid at 80 °C for 1 h. After the reaction, the mixture was incubated with 20 μl of dimethylamine borane reagent in acetic acid at 80 °C for 30 min. The excess 2-AP was removed using a MonoFas silica gel spin column (GL Sciences). The spin column was washed with water and then preequilibrated twice with 800 μl of 100% acetonitrile before use. The sample solution was mixed with 460 μl of 100% acetonitrile and loaded onto the spin column. The column was washed twice with 800 μl of 95% (v/v) acetonitrile. Water was added to the column to elute the fluorescently labeled oligosaccharides.

      FNG generation assay

      The purified OST complex was incubated with 1 μm LLO for 24 h at 30 °C in a 100-μl reaction solution, containing 20 mm Tris-HCl, pH 7.5, 5 mm MnCl2, 5 mm MgCl2, and 0.1% (v/v) Triton X-100. The incubation time was longer (24 h) than that for the oligosaccharyl transfer assay (1 h) to compensate for the low hydrolytic activity. The reaction was terminated by the addition of 1 μl of 0.5 m EDTA-NaOH, pH 8.0. A 300-μl aliquot of ethanol was added, and the reaction solution was incubated for 15 min at 4 °C. After centrifugation at 15,000 × g for 15 min, the supernatant was evaporated to dryness. The resultant pellet was labeled with 2-AP. The pyridylaminated oligosaccharides were separated by Infinity 1290 UPLC with an AdvanceBio Glycan Mapping column and quantified by an in-line fluorescence detector. Solvent A was 100 mm ammonium acetate buffer, pH 4.5, and solvent B was 100% acetonitrile. The column was equilibrated with 20% solvent A at a flow rate of 0.5 ml min−1. A linear gradient of solvent A was applied from 20 to 40% over 14 min.

      Quantification of the total N-glycans from mannoproteins

      The total N-glycans from mannoproteins were extracted from yeast strains as described previously, with modifications (
      • Harada Y.
      • Buser R.
      • Ngwa E.M.
      • Hirayama H.
      • Aebi M.
      • Suzuki T.
      Eukaryotic oligosaccharyltransferase generates free oligosaccharides during N-glycosylation.
      ). The yeast strains, stt3Δ png1Δ ams1Δ- pSTT3(X), were grown in YPD medium (1% yeast extract, 2% peptone, and 2% glucose) at 30 °C until the OD600 reached 10. 10 OD600 units of cells were harvested and washed twice with 4 ml of water. The washed cell pellets were resuspended in 1 ml of 10 mm sodium citrate buffer, pH 6.0, and autoclaved for 2 h at 121 °C. After centrifugation at 15,000 × g for 5 min, the supernatant was incubated with 3 ml of ethanol on ice for 15 min. The solution was centrifuged at 7,500 × g for 15 min, and the pellet was dried and dissolved in 200 μl of 0.1 m ammonium bicarbonate containing 50 units of PNGase F (New England Biolabs). The solution was incubated for 16 h at 37 °C. After inactivation by heating for 5 min at 95 °C, ethanol was added to the reaction mixture at a final volume percentage of 75%. After centrifugation at 15,000 × g for 15 min, the supernatant was dried and labeled with 2-AP. The pyridylaminated N-glycans were separated by Infinity 1290 UPLC with an AdvanceBio Glycan Mapping column and quantified by an in-line fluorescence detector. Solvent A was 100 mm ammonium acetate buffer, pH 4.5, and solvent B was 100% acetonitrile. The column was equilibrated with 28% solvent A, at a flow rate of 0.5 ml min−1. A linear gradient of solvent A was applied from 28 to 40.8% over 19 min.

      Determination of the N-glycosylation status of glycoproteins

      The stt3Δ png1Δ ams1Δ-pSTT3(X) strains were cultured in YPD medium at the permissive temperature (30 °C) until the OD600 reached 10. 10 OD600 units of cells were harvested and washed twice with 1 ml of water. The whole-cell lysate was prepared as described previously (
      • Kushnirov V.V.
      Rapid and reliable protein extraction from yeast.
      ). After centrifugation at 15,000 × g for 2 min, 6-μl aliquots of the supernatants were subjected to SDS-PAGE using gradient gels (10–20%). SDS sample buffer (50 mm Tris-HCl, pH 6.8, 1% (w/v) SDS, 10% (w/v) glycerol, 0.025% (w/v) bromphenol blue, and 100 mm DTT) was used for protein denaturation. The proteins in the gels were transferred to Immobilon-P PVDF membranes (Millipore), probed with the indicated antibodies, and visualized by chemiluminescence with the SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific). The anti-CPY antibody and anti-Wbp1 antiserum were used at dilutions of 1:10,000 and 1:5,000, respectively. The peroxidase-conjugated anti-mouse and anti-rabbit IgG antibodies were used at a dilution of 1:12,500. The chemiluminescent images were recorded with an LAS-3000 multicolor image analyzer (Fuji Photo Film). The average numbers of N-glycans attached to the CPY and the Wbp1 glycoproteins were calculated.

      Quantification of FNG in cells

      FNGs were extracted from yeast cells as described previously, with modifications (
      • Harada Y.
      • Buser R.
      • Ngwa E.M.
      • Hirayama H.
      • Aebi M.
      • Suzuki T.
      Eukaryotic oligosaccharyltransferase generates free oligosaccharides during N-glycosylation.
      ). Yeast strains, stt3Δ png1Δ ams1Δ-pSTT3(X), were cultured in YPD medium until the OD600 reached 10. 100 OD600 units of cells were harvested and washed with PBS. The washed cells were resuspended in 1 ml of lysis buffer (20 mm Tri-HCl, pH 7.5, 10 mm EDTA), and then 3 ml of ice-cold ethanol was added. The solution was vortexed for 10 s and cooled on ice for 5 min, and this procedure was repeated three times. The homogenate was centrifuged at 7,500 × g for 15 min, and the supernatant was recovered and evaporated to dryness. The dried soluble oligosaccharide fraction was desalted on a column packed with AG 501-X8 ion-exchange resin (Bio-Rad) and then on an InertSep GC column (GL Sciences). After elution, the dried pellets were labeled with 2-AP. The pyridylaminated FNGs were separated by Infinity 1290 UPLC with an AdvanceBio Glycan Mapping column and quantified by an in-line fluorescence detector. Solvent A was 100 mm ammonium acetate buffer, pH 4.5, and solvent B was 100% acetonitrile. The column was equilibrated with 20% solvent A at a flow rate of 0.5 ml min−1. A linear gradient of solvent A was applied from 20 to 40% over 25 min.

      Quantification of LLO in cells

      The yeast strains, stt3Δ png1Δ ams1Δ-pSTT3(X), were grown in YPD medium at 30 °C until the OD600 reached 10. 100 OD600 units were harvested and washed twice with 4 ml of water. The extraction and acid hydrolysis of LLO were performed as described above. After evaporation to dryness, the pellet was labeled with 2-AP. The pyridylaminated oligosaccharides were separated by Infinity 1290 UPLC with an AdvanceBio Glycan Mapping column and quantified by an in-line fluorescence detector. Solvent A was 100 mm ammonium acetate buffer, pH 4.5, and solvent B was 100% acetonitrile. The column was equilibrated with 20% solvent A at a flow rate of 0.5 ml min−1. A linear gradient of solvent A was applied from 20 to 40% over 8.5 min.

      Model building of the yeast OST-peptide complex

      The three-dimensional structure model of the yeast OST-peptide complex was constructed, based on the cryo-EM structures of the yeast OST protein (Protein Data Bank entry 6EZN) by reference to the Archaeoglobus fulgidus AglB-peptide complex (5GMY). AglB is a single-subunit OST enzyme in the archaeal domain. The figures were generated with the PyMOL program, version 2.3.2 (Schrödinger, LLC).

      Statistical analysis

      The significance of differences in values was assessed by two-sided Welch's t test.

      Data availability

      All data relevant to this work are contained within the article or available upon request.

      Acknowledgments

      We thank Prof. Takashi Ito (Kyushu University) for the gift of yeast strains. The DNA sequencing service was provided by the Laboratory for Research Support at the Medical Institute of Bioregulation, Kyushu University.

      Supplementary Material

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