Expression and characterization of rat UDP-N-acetylglucosamine: [alpha]-3-d-mannoside [beta]-1,2-N-acetylglucosaminyltransferase I in Saccharomyces cerevisiae

Satoshi Yoshida, Misa Suzuki, Shigeyuki Yamano, Makoto Takeuchi2, Hiroshi Ikenaga, Noriyuki Kioka1, Hiroshi Sakai1 and Tohru Komano1

KIRIN Brewery Co., Ltd., Central Laboratories for Key Technology, 1-13-5, Fukuura Kanazawa-ku, Yokohama-shi, Kanagawa 236-0004, Japan and 1Laboratory of Biochemistry, Department of Agricultural Chemistry, Kyoto University, Kitashirakawa-oiwake-chou, Sakyo-ku, Kyoto 606-8502, Japan

Received on March 19, 1998; revised on June 4, 1998; accepted on June 4, 1998

The yeast Saccharomyces cerevisiae is a useful host for the production of heterologous proteins through the secretory pathway. However, because of the potential antigenicity of mannan-type sugar chains in humans, yeast cannot be used as a host for the production of glycoprotein therapeutics. To overcome this problem, we are trying to breed a yeast which can produce hybrid- or complex-type carbohydrates. UDP-N-acetylglucosamine:[alpha]-3-d-mannoside [beta]-1,2-N-acetylglucosaminyltransferase I (GnT-I) is essential for the conversion of high mannose-type N-glycans to hybrid- and complex-type ones. As yeast lacks this enzyme, we have introduced the rat GnT-I cDNA into yeast cells. The transformed yeast cells expressed GnT-I activity in vitro. The expressed GnT-I was localized in all organella, including the endoplasmic reticulum (ER), Golgi apparatus, and vacuole, suggesting that the mammalian Golgi retention signal of GnT-I did not function in yeast cells. Analysis of the GnT-I gene product with a c-Myc epitope tag at the C-terminus elucidates that the N-terminal region of GnT-I, including the mammalian Golgi retention signal, should be removed in the yeast ER.

Key words: GlcNAc transferase/glycoprotein synthesis/N-glycans/Saccharomyces cerevisiae/yeast

Introduction

The yeast S.cerevisiae is a useful host for the production of high molecular weight therapeutics such as growth factors, cytokines etc. These secretory proteins undergo posttranslational modifications including limited proteolysis, folding, disulfide bond formation, phosphorylation and glycosylation. High level expression of numerous heterologous proteins has been achieved in prokaryotes such as Escherichia coli. However, the products may have aberrant disulfide bonds and no protein-glycosylation. Therefore, yeast is a preferable host for the production of glycoproteins such as human erythropoietin (Elliott et al., 1989) and alpha-1-antitrypsin (Moir and Dumais, 1987).

Glycosylation has essential functions in all eukaryotic cells. In both yeast and mammals, the transfer of precursor oligosaccharides from lipid intermediates to asparagine (Asn) residues in the Asn-X-Thr/Ser signal initiates the biosynthesis of the sugar chains. Subsequently, a series of enzymes in the ER, Golgi apparatus and vacuole process these glycans to the mature forms (Kornfeld and Kornfeld, 1985; Kukuruzinska et al., 1987; Moremen et al., 1994). Glycosidases and glycosyltransferases convert typical Glc3Man9GlcNAc2 glycans to Man8GlcNAc2 glycans and then process them to mannan-type glycans in yeasts, or hybrid- and complex-type glycans in mammals. The first step towards the formation of complex-type glycans is catalyzed by the Golgi enzyme GnT-I. The enzyme takes Man5GlcNAc2 N-glycan as a substrate, and catalyzes the transfer of a GlcNAc from UDP-GlcNAc to the terminal [alpha]1-3 mannose residue which is linked to the [beta]-mannose of the N-glycan core. After the GnT-I and [alpha]-mannosidase II steps, the biosynthesis pathways for mammalian complex-glycans diversify from bi- to tetra-antennary oligosaccharides.

The GnT-I genes have been cloned from rabbit (Sarkar et al., 1990), human (Kumar et al., 1990), mouse (Kumar et al., 1992), rat (Fukada et al., 1994), chicken (Warren et al., 1992), and Xenopus laevis (Mucha et al., 1995). GnT-I is a type II membrane protein which is localized in the medial-Golgi (Burke et al., 1994). GnT-I shares typical membrane orientation with glycosyltransferases that have been cloned. It has a short cytoplasmic tail, a transmembrane signal/anchor domain, a stem region at the N-terminal and a lumenal catalytic domain at the C-terminal. The transmembrane region is essential for the localization of GnT-I in the Golgi apparatus (Tang et al., 1992). The GnT-I gene has been successfully expressed in E.coli (Kumar et al., 1992) and Aspergillus nidulans (Kalsner et al., 1995). Here, we describe the expression and characterization of GnT-I in S.cerevisiae. The GnT-I expressed in yeast is also active in vitro. The C-terminal catalytic region faces the lumenal side, and it is distributed in the ER, Golgi apparatus, and vacuole, probably because the transmembrane region of GnT-I has been removed in the yeast ER. We are now working on the biosynthesis of Man5GlcNAc2 sugar chains in S.cerevisiae to serve an acceptor substrate for the GnT-I expressed in the same yeast cells.

Results

Expression of a rat GnT-I cDNA in S.cerevisiae

We constructed a rat GnT-I expression plasmid (pGNI-8) based on the YEp13K harboring the GPD promoter, PGK terminator, and the LEU2 marker as described in Materials and methods (Figure 1). The yeast cells with pGNI-8 expressed in vitro GnT-I activity, indicating that this GPD/GnT-I/PGK expression cassette is functional in yeast cells. For integration of the GPD/GnT-I/PGK cassette into the genome, the SalI fragment (Figure 1) was inserted into the integration vector pRS305 with the LEU2 selective marker (pGNI-9). The resulting plasmid, pGNI-9, was linearized by digestion with the AflI or BstEII restriction enzyme at the LEU2 locus, and this fragment was used to transform the yeast cells (GN-1, GN-2, and GN-3; Figure 2). The ratio of DNA content derived from the GPD/GnT-I/PGK cassette was determined by Southern hybridization (Materials and methods, Figure 2A): GN-1/GN-2/GN-3/YCp/YEp is 4.4/7.0/1.0/1.7/6.3.


Figure 1. Construction of the expression vector for rat GnT-I cDNA. The multicopy plasmid pSY114 was used for the insertion of the entire open reading frame encoding rat GnT-I. The 5[prime]-end of the gene was modified by polymerase chain reaction (PCR) amplification in order to introduce a HindIII restriction site at the ATG translation start signal (Materials and methods). Each number in the figure shows the position of base pairs relative to the initiation codon. The TAG at 1350 base pairs terminates translation. Abbreviations for restriction enzymes are as follows; H, HindIII; Sm, SmaI; Sl, SalI.

Figure 2. Subcellular localization of GnT-I in yeast cells expressing the rat GnT-I cDNA at different levels. (A) Southern hybridization and Western blot analyses of yeast cells expressing the rat GnT-I cDNA. GN-1, GN-2, and GN-3 are yeast cells transformed with the integration plasmid pGNI-9 digested with AflII (GN-1 and GN-2) or BstEII (GN-3). YCp and YEp indicate yeast cells transformed with pRS315/GnT-I and pYO325/GnT-I, respectively; 1.0 × 107 cells were homogenated with glass beads and subjected to DNA electrophoresis for Southern hybridization. For Western analysis, the samples were suspended and boiled in a sample buffer for SDS-PAGE and subjected to SDS-PAGE on 10% (w/w) gel. The probe for the Southern hybridization was a 1.5 kb HindIII/SmaI fragment of the rat GnT-I cDNA. GnT-I was detected by Western blotting using an anti-GnT-I polyclonal antibody. (B) Subcellular distribution of GnT-I activity in yeast cells expressing the rat GnT-I cDNA. GnT-I and GDPase activities were assayed as described in Materials and methods. The control indicates yeast cells transformed with pRS305 digested with BstEII. CL, Crude lysate; LSP, low speed pellet; HSP, high speed pellet; HSS, high speed supernatant.

Subcellular localization of the GnT-I enzyme

We performed subcellular fractionation experiments upon the three yeast cell lines with different copy numbers of GnT-I gene as described in Materials and methods to determine whether overproduction causes mislocalization of the products. Figure 2 clearly shows that the amount of the gene product and the activity of GnT-I are proportional to the copy number of the gene in the genome and that copy number does not affect the localization of GnT-I. Therefore, we used the GN-2 cells, which expressed GnT-I at the highest level among the strains tested for further investigation.

GN-2 cells were grown in SD medium, converted to spheroplasts, homogenized and subjected to differential centrifugation. Figure three shows the distribution of marker enzymes for the ER and Golgi apparatus in the three fractions LSP, HSP, and HSS. Overproduction of GnT-I had minimal effect on the distribution of these marker enzymes (data not shown). Although the LSP and HSP fractions were crude mixtures of membranes, it should be noted here that the ER marker Sec12p was preferentially distributed in the LSP, and GDPase for the Golgi marker was not. The Sec12p was not detected in the crude lysate fraction probably by proteolysis. The GnT-I product and activity were recovered in the LSP and HSP fractions. Split bands of GnT-I were observed in the LSP fraction. These bands will be mentioned below. The LSP fraction was further subjected to sucrose bilayer centrifugation (1.2/1.5 M) to fractionate the ER from the vacuole, as described in Materials and methods (Figure 4). These results indicate that GnT-I is localized universally in the ER, Golgi apparatus, and vacuole in yeast.

Orientation of GnT-I in the yeast membranes

GnT-I is a type II membrane protein localized in the Golgi apparatus in mammals. We checked whether the C-terminus of GnT-I is located in the lumen or cytoplasm in yeast. If the catalytic domain of GnT-I is lumenal in yeast organella, as in mammals, its C-terminal domain should be non-susceptible to trypsin. We also expressed GnT-I cDNA with a c-Myc tag at the C-terminus of GnT-I in yeast (pGNI-8-Myc). The GnT-I/c-Myc product was detected with anti-c-Myc or anti-GnT-I antibody. In the absence of the detergent Triton X-100, GnT-I was not degraded during 30 min incubation at 0°C, while in the presence of the detergent, GnT-I was degraded to a 45 kDa fragment (Figure 5; detected by the anti-c-Myc antibody). No shift of the GnT-I band was detected after incubation with the detergent but no trypsin. Cytosolic proteins were easily digested with trypsin without detergent. These results indicate that the C-terminus of GnT-I is located in the lumen in yeast.


Figure 3. Subcellular localization of GnT-I in GN-2 cells. Lysed spheroplasts from 1 × 107 cells of GN-2 were subjected to differential centrifugation as described in Materials and methods and analyzed by Western blot analysis using the anti-GnT-I and Sec12p antibodies, respectively. Sec12p (Nakano et al., 1988) and GDPase (Abeijon et al., 1988) were used as markers for the ER and Golgi apparatus, respectively. The enzyme activities of GnT-I and GDPase were assayed as described in Materials and methods. The enzyme activity is shown as nmol/min/1010 cells. Abbreviations for the fractions are as described in Figure 2.

According to the SDS-PAGE results shown in Figures 3 and 4, there are split bands (51 and 48 kDa) of GnT-I only in the ER fraction. This limited proteolysis was examined. If the N-terminal region of GnT-I had been removed, GnT-I should be detected as a 51 kDa protein with both anti-c-Myc and anti-GnT-I antibodies. If the C-terminal region had been removed, GnT-I should be detected as a 48 kDa protein with the anti-GnT-I antibody, but not detected with anti-c-Myc antibody. The Western blotting analyses showed that only the 51 kDa protein was detected with both antibodies (data not shown), indicating that the N-terminal region of GnT-I (~3 kDa) was removed in the ER fraction of yeast.


Figure 4. Sucrose bilayer fractionation of the LSP fraction in the GN-2 cells. Eighty percent of the LSP fraction was resuspended in 50 µl of lysis buffer and layered on the top of a 1.2/1.5 M sucrose gradient. After an 85,000 × g spin for 1 h, 10 equivalent fractions were removed and subjected to Western blot analysis and a GDPase assay. Sec12p (Nakano et al., 1988), Vma1p (Hirata et al., 1990), and GDPase (Abeijon et al., 1988) were used as markers for the ER, vacuole, and Golgi apparatus, respectively. For Western blot analysis (GnT-I, Vma1p, and Sec12p), samples derived from 1 × 107 of cells were used. GDPase was assayed as described in Materials and methods. The enzyme activity is shown as nmol/min/1010 cells.

Figure 5. Susceptibility of GnT-I in the membrane fraction to tryptic digestion. The homogenate from yeast cells (YPH500P/pGNI-8-Myc) was treated with or without trypsin in the absence or presence of Triton X-100 (TX-100) on ice. After the indicated times (0-30 min), the tryptic action was stopped by the addition of trypsin inhibitor and the samples were analyzed by SDS-PAGE followed by immunoblotting with the anti-c-Myc (c-Myc) and anti-GnT-I (GnT-I) antibodies, respectively. Arrowheads indicate the mature 51 kDa form and 45 kDa fragment of GnT-I/c-Myc.

Discussion

We have described here the expression and localization of the rat GnT-I cDNA in the yeast S.cerevisiae. The GnT-I enzyme expressed in the yeast is active in vitro, but its location differs from that of mammals. The GnT-I enzyme is localized throughout the membranes in the secretory pathway, including the ER, Golgi apparatus, and vacuole. This is probably because signal peptidases in the yeast ER cleave off the membrane anchor region of the GnT-I protein. To retain GnT-I in the Golgi apparatus, we have constructed the OCH1/GnT-I chimeric gene which is under the control of the GAL1 promoter, and characterized the gene product. It is localized mainly in the LSP fraction containing the ER and vacuole (unpublished observations). This result is consistent with results that the overexpressed OCH1 gene product is mainly localized in the vacuole (Chapman and Munro, 1994). We are currently constructing a vector that expresses a low level of the OCH1/GnT-I product to locate it in the Golgi apparatus.

Several mammalian glycosyltransferases have been expressed and characterized in the yeast S.cerevisiae. The human [beta]-1,4-galactosyltransferase (GalT) has been expressed in yeast, but it was localized in the ER (Krezdorn et al., 1994). Schwientek and Ernst (1994) showed that the MNT1/GalT chimeric gene products can be expressed effectively and localized in the yeast Golgi apparatus.

The [alpha]-2,6-sialyltransferase for Asn-linked sugar chains is localized in the ER, but not in the Golgi apparatus of yeast (Krezdorn et al. 1994). However, the chimeric gene product, a transmembrane anchor region of [alpha]-2,6-sialyltransferase fused to yeast invertase Suc2p, is localized in the yeast Golgi apparatus, suggesting that the transmembrane anchor region of [alpha]-2,6-sialyltransferase also functions as a Golgi retention signal in yeast (Schwientek et al., 1995). The discrepancy between these results is probably due to the quality control in the yeast ER, including folding and glycosylation in the case of expression of the full-length [alpha]-2,6-sialyltransferase. Alternatively, it is possible that the chimera with a yeast gene supports the efficient expression and folding of foreign gene products in yeast.

It has been reported that the GnT-I cDNA is expressed in E.coli (Kumar et al., 1992), Asperugillus nidulans (Kalsner et al., 1995) and Arabidopsis thaliana (Gomez and Chrispeels, 1994). The GnT-I products thus expressed are active in vitro. The GnT-I expressed in A.thaliana is shows in vivo activity, because it complements a mutation of A.thaliana lacking Asn-linked complex type glycans. In A.nidulans, GnT-I is active in vitro, but does not function in vivo, because there were no Endo H-resistant glycoproteins nor GalT-dependent incorporation of galactose into glycoproteins (Kalsner et al., 1995). A.nidulans may have no precursor sugar chains to act as a substrate for GnT-I and/or no UDP-GlcNAc transporter (Abeijon et al., 1996) to import UDP-GlcNAc into the Golgi apparatus lumen. As S.cerevisiae has no acceptor substrate for GnT-I and no UDP-GlcNAc transporter, S.cerevisiae resembles A.nidulans. Genetic engineering and gene disruption are easier to perform in S.cerevisiae than in A.nidulans, thus we believe that yeast is a preferable host for the production of mammalian-type glycoproteins. Now we are planning to introduce [alpha]1,2-mannosidase, UDP-GlcNAc:[alpha]-6-d-mannoside [beta]-1,2-N-acetylglucosaminyltransferase II, sialyltransferases, and other glycosyltransferases, sugar-nucleotide transporters and sugar-nucleotide synthesis enzymes into yeast if they are necessary for the production of glycoproteins with human compatible sugar chains.

Materials and methods

Organisms and growth conditions

The protease-deficient S.cerevisiae strains used in this study were EHA-1C (Mat[alpha] leu2-3, 112 pep4-3) (Nakayama et al., 1992) obtained from Dr. Y. Jigami (National Institute of Bioscience and Human Technology) and YPH500P (Mat[alpha] ura3-52 lys2-801 ade2-101 trp1-[Delta]63 his3-[Delta]200 leu2-[Delta]1 [Delta]pep4::ADE2; Yoshida et al., 1994). Yeast cells were grown at 30°C in SD medium (Rose et al., 1990). Transformation of yeast cells was carried out as described previously (Rose et al., 1990).

Plasmid construction

GnT-I cDNA was isolated from rat liver by the screening of a cDNA library (Fukada et al., 1994). For heterologous expression in S.cerevisiae, we used the GPD promoter and the PGK terminater of pSY114 (~10 kb), which is based on YEp13K (Sone et al., 1988; Yamano et al., 1994). This plasmid also has a LEU2 yeast selective marker. The 5[prime]-end of the gene was modified by amplification by the polymerase chain reaction (PCR) to introduce a HindIII restriction site at the ATG translation start signal. The GnT-I cDNA was inserted into the GPD/PGK cassette between the HindIII and SmaI sites (Figure 1). In order to obtain centromeric- and multicopy plasmids, the GPD/GnT-I/PGK cassette was inserted in the centromeric-plasmid pRS315 (Sikorski and Hieter, 1989) and the multicopy plasmid pYO325 (Qadota et al., 1992), both of which have the LEU2 marker. For the integration of the GPD/GnT-I/PGK cassette into the chromosome of yeast, the SalI fragment (Figure 1) was inserted into the integration vector pRS305 (Sikorski and Hieter, 1989), which possesses the LEU2 selective marker. The expression plasmid pGNI-8-Myc, for GnT-I with c-Myc tag (EQKLISEEDL) at the C-terminus, was based on the pSY114 vector constructed by PCR as described previously (Evan et al., 1985; Yoko-o et al., 1995).

Southern blot analysis

DNA was prepared from the transformants as described by Rose et al. (1990) and Southern blot analysis was carried out. These DNA samples were digested with SalI and blotted onto nylon transfer membrane using 0.4 N NaOH. The 1.0 kb probe DNA, which is derived from the GPD promoter region of pSY114, was labeled with 32P using Megaprime DNA labeling systems (Amersham). The membrane was subjected to x-ray film autoradiography. The intensity of each band derived from the GPD/GnT-I/PGK cassette was measured using the Bioimaging analyzer (BAS2000, Fuji Film). The chromosomal GPD gene was used as a standard for calculating the intensity.

GnT-I assays

GnT-I activity was measured as described previously (Schachter et al., 1989), except for the use of a pyridylaminated (PA) oligosaccharide as a substrate. The assay mixture contained 100 mM MES (pH 6.1), 100 mM GlcNAc, 5 mM AMP, prewarmed 0.5% BSA, 20 mM MnCl2, 10 mM UDP-GlcNAc, 2 µM PA oligosaccharide, and yeast extract in a total volume of 50 µl. The substrate oligosaccharide, Man[alpha]1-6(Man[alpha]1-3)Man[alpha]1-6(Man[alpha]1-3)Man[beta]1-4GlcNAc[beta]1-4GlcNAc-PA (PA017), was purchased from Takara Shuzo. The assay mixture was incubated at 37°C for 60 min. The reaction was stopped by boiling for 2 min, filtered through a Ultrafree C3GV microtube (Millipore), and subjected to HPLC analysis.

The Tsk-gel Amide-80 column ([phis]4.6 × 250 mm, Toso) was equilibrated with a solvent containing acetonitril and 3% acetic acid, 10 mM triethanolamine (pH 7.3) at a ratio of 70/30 (v/v). The assay products were eluted isocratically at a rate of 1 ml/min using the same solvent and were detected by the fluorescence of the PA tag; [lambda]ex 320 nm to [lambda]m 400 nm. The structure of the reaction product was identified by proton-NMR.

Determination of protein concentration

Protein concentration was determined using the bicinchoninic acid reagent (Pierce) with bovine serum albumin as a standard.

Polyacrylamide gel electrophoresis and Western blot analysis

Proteins were separated on 10% sodium dodecyl sulfate (SDS) polyacrylamide gels. The gels were electrophoretically blotted onto PVDF membranes (Millipore). The membranes were probed with a rabbit polyclonal antibody raised against the recombinant rat GnT-I fusion protein or with a mouse monoclonal c-Myc antibody (9E10) purchased from Santa Cruz Biotechnology Inc. The rabbit and mouse antibodies were detected using an ECL detection system (Amersham) with a goat anti-rabbit and anti-mouse IgG coupled to horseradish peroxidase (HRP), respectively.

Subcellular fractionation of yeast cell lysate

Yeast cells were grown to a density of 1 × 107 cells/ml in 500 ml of SD medium. The cells were converted to spheroplasts as described previously (Nishikawa and Nakano, 1991). The spheroplasts were resuspended in 10 ml of ice-cold lysis buffer (10 mM triethanolamine acetate (pH 7.5), 250 mM sorbitol, 2 µg/ml antipain, 2 µg/ml chymostatin, 3 µg/ml pepstatin A, 2 µg/ml leupeptin, 1 mM benzamidine-HCl, 1 mM EDTA, 1 mM EGTA, and 1 mM phenylmethylsulfonylfluoride (PMSF)) and were immediately homogenized six times for 1 min period with 1 min intervals on ice using a motor-driven 30 ml Potter-Elvehjem homogenizer (Wheaton). The lysate was centrifuged three times at 500 × g for 5 min to remove unbroken spheroplasts. The supernatant was subjected to centrifugation at 10,000 × g for 15 min at 4°C in an RT100.3 rotor (Beckman) to yield a low-speed pellet (LSP) and a low-speed supernatant (LSS) fraction. The LSS fraction was further centrifuged at 100,000 × g for 60 min at 4°C in an RT100.3 rotor to yield a high-speed pellet (HSP) and a high-speed supernatant (HSS) fraction.

The LSP fraction was subjected to differential centrifugation as described by Gaynor et al. (1994). Eighty percent of the LSP fraction was resuspended in 50 µl of lysis buffer and layered on a sucrose bilayer consisting of 900 µl of 1.5 M sucrose and 900 µl of 1.2 M sucrose in lysis buffer. After an 85,000 × g spin for 1 hr, 10 equivalent fractions were removed and subjected to Western blot analysis and were assayed for GDPase. Anti-Sec12p and anti-Vma1p antibodies were used in Western blotting to detect marker proteins in the ER and vacuole, respectively. These antibodies were kindly provided by Drs. Nakano and Anraku, respectively. GDPase activity was assayed as a marker enzyme for the Golgi apparatus as described previously (Abeijon et al., 1989).

Orientation of GnT-I in the yeast membrane

The susceptibility of GnT-I in the membrane fractions to tryptic digestion was examined as described previously (Nakano et al., 1988). The yeast cell homogenate, which was prepared as described above, was treated with or without 0.5 mg/ml trypsin in the absence or presence of 0.1% Triton X-100 on ice. After the indicated time (0-30 min), the tryptic digestion was stopped by the addition of 1.1 mg/ml soybean trypsin inhibitor (Sigma). Each sample equivalent to 1.0 OD600 of cells was analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), followed by immunoblotting with anti-c-Myc or anti-GnT-I antibody.

Acknowledgments

We thank Drs. Nakano, Ohya, and Anraku (University of Tokyo) for providing antibodies against Sec12p and Vma1p; Dr. Jigami (National Institute of Bioscience and Human Technology) for providing EHA-1C strain; and Dr. Miura for technical assistance for Southern blot analysis. This work was performed as a part of the R&D Project of Industrial Science and Technology Frontier Program supported by NEDO (New Energy and Industrial Technology Development Organization).

Abbreviations

Asn, asparagine; ER, endoplasmic reticulum; GalT, UDP-galactose:N-acetylglucosamine [beta]-1,4-galactosyltransferase (EC 2.4.1.38/90); GnT-I, UDP-N-acetylglucosamine:[alpha]-3-d-mannoside b-1,2-N-acetylglucosaminyltransferase I (EC 2.4.1.101); PA, pyridylaminated; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; PMSF, phenylmethylsulfonylfluoride; SDS, sodium dodecylsulfate; SD, synthetic minimum dextrose.

References

Abeijon ,C., Orlean,P., Robbins,P.W. and Hirschberg,C.B. (1989) Topography of glycosylation in yeast: Characterization of GDP mannose transport and lumenal guanosine diphosphatase activities in Golgi-like vesicles. Proc. Natl. Acad. Sci. USA, 86, 6935-6939. MEDLINE Abstract

Abeijon ,C., Mandon,E.C., Robbins,P.W. and Hirschberg,C.B. (1996) A mutant yeast deficient in Golgi transport of uridine diphosphate N-acetylglucosamine. J. Biol. Chem., 271, 8851-8854. MEDLINE Abstract

Burke ,J., Pettitt,J.M., Humphris,D. and Gleeson,P.A. (1994) Medial-Golgi retention of N-acetylglucosaminyltransferase I. J. Biol. Chem., 269, 12049-12059. MEDLINE Abstract

Chapman ,R.E. and Munro,S. (1994) The functioning of the yeast Golgi apparatus requires an ER protein encoded by ANP1, a member of a new family of genes affecting the secretory pathway. EMBO J., 13, 4896-4907. MEDLINE Abstract

Elliott ,S., Giffin,J., Suggs,S., Lau,E.P. and Banks,A.R. (1989) Secretion of glycosylated human erythropoietin from yeast directed by the [alpha]-factor leader region. Gene, 79, 167-180. MEDLINE Abstract

Evan ,G.I., Lewis,G.K., Ramsay,G. and Bishop,J.M. (1985) Isolation of monoclonal antibodies specific for human c-myc proto-oncogene product. Mol. Cell. Biol., 5, 3610-3616. MEDLINE Abstract

Fukada ,T., Iida,K., Kioka,N., Sakai,H. and Komano,T. (1994) Cloning of a cDNA encoding N-acetylglucosaminyltransferase I from rat liver and analysis of its expression in rat tissue. Biosci. Biotech. Biochem., 58, 200-201.

Gaynor ,E.C., te Heesen,S., Graham,T.R., Aebi,M. and Emr,S.D. (1994) Signal-mediated retrieval of a membrane protein from the Golgi to the ER in yeast. J. Cell Biol., 127, 653-665. MEDLINE Abstract

Gomez ,L. and Chrispeels,M.J. (1994) Complementation of an Arabidopsis thaliana mutant that lacks complex asparagine-linked glycans with the human cDNA encoding N-acetylglucosaminyltransferase I. Proc. Natl. Acad. Sci. USA, 91, 1829-1833. MEDLINE Abstract

Hirata ,R., Ohsumi,Y., Nakano,A., Kawasaki,H., Suzuki,K. and Anraku,Y. (1990) Molecular structure of a gene, VMA1, encoding the catalytic subunit of H+-translocating adenosine trisphosphatase from vacuolar membranes of Saccharomyces cerevisiae. J. Biol. Chem., 265, 6726-2733. MEDLINE Abstract

Kalsner ,I., Hintz,W., Reid,L.S. and Schachter,H. (1995) Insertion into Aspergillus nidulans of functional UDP-GlcNAc:[alpha]3-d-mannoside [beta]-1,2-N-acetylglucosaminyltransferase I, the enzyme catalyzing the first step committed step from oligomannose to hybrid and complex N-glycan. Glycoconjugate J., 12, 360-370.

Kornfeld ,R. and Kornfeld,S. (1985) Assembly of asparagine-linked oligosaccharides. Annu. Rev. Biochem., 54, 631-664. MEDLINE Abstract

Krezdorn ,C.H., Kleene,R.B., Watzele,M., Ivanov,S.X., Hokke,C.H., Kamerling,J.P. and Berger,E.G. (1994) Human [beta]1,4 galactosyltransferase and [beta]2,6 sialyltransferase expressed in Saccharomyces cerevisiae are retained as active enzymes in the endoplasmic reticulum. Eur. J. Biochem., 220, 809-817. MEDLINE Abstract

Kukuruzinska ,M.A., Bergh,M.L.E. and Jackson,B.J. (1987) Protein glycosylation in yeast. Annu. Rev. Biochem., 56, 915-944 MEDLINE Abstract

Kumar ,R., Yang,J., Larsen.,R.D. and Stanley,P. (1990) Cloning and expression of N-acetylglucosaminyltransferase I, the medial Golgi transferase that initiate complex N-linked carbohydrate formation. Proc. Natl. Acad. Sci. USA, 87, 9948-9952. MEDLINE Abstract

Kumar ,R., Yang,J., Eddy,R.L., Byers,M.G., Shows,T.B. and Stanley,P. (1992) Cloning and expression of the murine gene and chromosomal location of the human gene encoding N-acetylglucosaminyltransferase I. Glycobiology, 2, 383-393. MEDLINE Abstract

Moir ,D.T. and Dumais,D.R. (1987) Glycosylation and secretion of human alpha-1-antitrypsin. Gene, 56, 209-217. MEDLINE Abstract

Moremen ,K.W., Trimble,R.B. and Herscovics,A. (1994) Glycosidases of the asparagine-linked oligosaccharide processing pathway. Glycobiology, 4, 113-125. MEDLINE Abstract

Mucha ,J., Kappel,S., Schachter,H., Hane,W. and Glossl,J. (1995) Molecular cloning and characterization of cDNAs coding for N-acetylglucosaminyltransferase I and II from Xenopus laevis ovary. Glycoconjugate J., 12, 473.

Nakano ,A., Brada,D. and Schekman,R. (1988) A membrane glycoprotein, Sec12p, required for protein transport from the endoplasmic reticulum to the Golgi apparatus in yeast. J. Cell Biol., 107, 851-863. MEDLINE Abstract

Nakayama ,K., Nagasu,T., Shimma,Y., Kuromatsu,J. and Jigami,Y. (1992) OCH1 encodes a novel membrane bound mannosyltransferase: outer chain elongation of asparagine-linked oligosaccharides. EMBO J., 11, 2511-2519. MEDLINE Abstract

Nishikawa ,S. and Nakano,A. (1991) The GTP-binding Sar1 protein is localized to the early compartment of the yeast secretory pathway. Biochim. Biophys. Acta, 1093, 135-143. MEDLINE Abstract

Qadota ,H., Ishii,I., Fujiyama,A., Ohya,Y. and Anraku,Y. (1992) RHO gene products, putative small GTP-binding proteins, are important for activation of the CAL1/CDC43 gene product, a protein geranylgeranyltransferase in Saccharomyces cerevisiae. Yeast, 8, 735-741. MEDLINE Abstract

Rose ,M., Winston,F. and Hieter,P. (1990) Methods in Yeast Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

Sarkar ,M., Hull,E., Nishikawa,Y., Simpson,R.J., Moritz,R.L., Dunn,R. and Schachter,H. (1991) Molecular cloning and expression of cDNA encoding the enzyme that controls conversion of high-mannose to hybrid and complex N-glycans: UDP-N-acetylglucosamine:[alpha]-3-d-mannoside [beta]-1,2-N-acetylglucosaminyltransferase I. Proc. Natl. Acad. Sci. USA, 88, 234-238. MEDLINE Abstract

Schachter ,H., Brockhasusen,I. and Hull,E. (1989) High-performance liquid chromatography assays for N-acetylglucosaminyltransferase involved in N- and O- glycan synthesis. Methods Enzymol., 179, 351-397. MEDLINE Abstract

Schwientek ,T. and Ernst,J.F. (1994) Efficient intra- and extracellullar production of human [beta]-1,4-galactosyltransferase in Saccharomyces cerevisiae is mediated by yeast secretion leaders. Gene, 145, 299-303. MEDLINE Abstract

Schwientek ,T., Lorenz,C. and Ernst,J.F. (1995) Golgi localization in yeast is mediated by the membrane anchor region of rat lever sialylrransferase. J. Biol. Chem., 270, 5483-5489. MEDLINE Abstract

Sikorski ,R.S. and Hieter,H. (1989) A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics, 122, 19-27. MEDLINE Abstract

Sone ,H., Fujii,T., Kondo,K., Shimizu,F., Tanaka,J. and Inoue,T. (1988) Nucleotide sequence and expression of the Enterobacter aerogenes [alpha]-acetolactate decarboxylase gene in baker's yeast. Appl. Environ. Microbiol., 54, 38-42. MEDLINE Abstract

Tang ,B.L., Wong,S.H., Low,S.H. and Hong,W. (1992) The transmembrane domain of N-glucosaminyltransferase I contains a Golgi retention signal. J. Biol. Chem., 267, 10122-10126. MEDLINE Abstract

Warren ,C.E., Cummings,L., Narasimhan,S., Schachter,H. and Dennis,J.W. (1992) Expression cloning of chicken [beta]-N-acetylglucosaminyltransferase I (GnT-I) using lectin selection. Glycobiology, 2, 488.

Yamano ,S., Tanaka,J. and Inoue,T. (1994) Cloning and expression of the gene encoding a-acetolactate decarboxylase from Acetobacter aceti ssp. xylinum in baker's yeast. J. Biotechnol., 32, 165-171. MEDLINE Abstract

Yoko-o ,T., Kato,H., Matsui,Y., Takenawa,T. and Toh-e,A. (1995) Isolation and characterization of temperature-sensitive plc1 mutants of the yeast Saccharomyces cerevisiae. Mol. Gen. Genet., 247, 148-156. MEDLINE Abstract

Yoshida ,S., Ohya,Y., Hirose,R., Nakano,A. and Anraku,Y. (1994) STT10, a novel class-D VPS yeast gene required for osmotic integrity related to the PKC1/STT1 protein kinase pathway. Gene, 160, 117-122.


2To whom correspondence should be addressed: KIRIN Brewery Co., Ltd., Central Laboratories for Key Technology, 1-13-5, Fukuura Kanazawa-ku, Yokohama-shi, Kanagawa 236-0004, Japan


This page is run by Oxford University Press, Great Clarendon Street, Oxford OX2 6DP, as part of the OUP Journals
Comments and feedback: www-admin{at}oup.co.uk
Last modification: 12 Jan 1999
Copyright©Oxford University Press, 1999.