Coexpression of [alpha]1,2 galactosyltransferase and UDP-galactose transporter efficiently galactosylates N- and O-glycans in Saccharomyces cerevisiae

Mami Kainuma1, Nobuhiro Ishida2, Takehiko Yoko-o1, Shigemi Yoshioka2, Makoto Takeuchi3, Masao Kawakita2 and Yoshifumi Jigami1,4

1Department of Molecular Biology, National Institute of Bioscience and Human Technology, Tsukuba, Ibaraki 305-8566 Japan, 2Department of Physiological Chemistry, The Tokyo Metropolitan Institute of Medical Science (Rinshoken), Tokyo 113-8613 Japan and 3Central Laboratories for Key Technology, KIRIN Brewery Co., Yokohama, Kanagawa 236-0004, Japan

Received on April 1, 1998; revised on June 30, 1998; accepted on July 2, 1998

We have studied in vivo neo-galactosylation in Saccharomyces cerevisiae and analyzed the critical factors involved in this system. Two heterologous genes, gma12+ encoding [alpha]1,2-galactosyltransferase ([alpha]1,2 GalT) from Schizosaccharomyces pombe and UGT2 encoding UDP-galactose (UDP-Gal) transporter from human, were functionally expressed to examine the intracellular conditions required for galactosylation. Detection by fluorescence labeled [alpha]-galactose specific lectin revealed that 50% of the cells incorporated galactose to cell surface mannoproteins only when the gma12+ and hUGT2 genes were coexpressed in galactose media. Integration of both genes in the [Delta]mnn1 background cells increased galactosylation to 80% of the cells. Correlation between cell surface galactosylation and UDP-galactose transport activity indicated that an exogenous supply of UDP-Gal transporter rather than [alpha]1,2 GalT played a key role for efficient galactosylation in S.cerevisiae. In addition, this heterologous system enabled us to study the in vivo function of S.pombe [alpha]1,2 GalT to prove that it transfers galactose to both N- and O-linked oligosaccharides. Structural analysis indicated that this enzyme transfers galactose to O-mannosyl residue attached to polypeptides and produces Gal[alpha]1,2-Man1-O-Ser/Thr structure. Thus, we have successfully generated a system for efficient galactose incorporation which is originally absent in S.cerevisiae, suggesting further possibilities for in vivo glycan remodeling toward therapeutically useful galactose containing heterologous proteins in S.cerevisiae.

Key words: [alpha]1,2 galactosyltransferase/galactosylation/in vivo glycan remodeling/S.cerevisiae/UDP-galactose transporter

Introduction

Glycosylation is one of the major posttranslational modification. This modification plays a key role in the biological function of native glycoproteins in eucaryotic cells. Two representing structures of oligosaccharides are N-glycan attached to asparagine residues and O-glycan attached to serine or threonine residues in a peptide backbone. In spite of remarkable differences in final structures between mammals and lower eucaryotes such as Saccharomyces cerevisiae, they share a common oligosaccharide core intermediate in the ER (reviewed in Lehle and Tanner, 1995; Trimble and Verostek, 1995; Orlean, 1997). Therefore, yeast has been used to study the molecular mechanisms of this common glycosylation pathway (Lehle and Tanner, 1995; Eckart and Bussineau, 1996).

Yeast has also been used as a production host of recombinant proteins. However, one of the major drawbacks for yeast as a host cell is the lack of further biosynthetic pathways for complex type oligosaccharides formation. This sometimes alters the biological function and/or immunogenicity of particular proteins (Schultz et al., 1987; Ballou, 1990; Baenziger, 1994). In order to clarify the biological function of diverse oligosaccharides and to construct a desirable yeast expression system for glycoproteins of therapeutic use, we have been manipulating the glycosylation system of S.cerevisiae by introducing mutations responsible for oligosaccharide biosynthesis. So far we have constructed [Delta]och1mnn1 double mutant cells which produce Man8GlcNAc2, high mannose-type (ER-core) oligosaccharides (Nakayama et al., 1992; Nakanishi-Shindo et al., 1993), and [Delta]och1[Delta]mnn1[Delta]mnn4 triple mutant cells (Odani et al., 1996) which produce less yeast specific mannosylphosphorylated Man8GlcNAc2. The next stage for sugar structure remodeling is to add the complex type oligosaccharides to this ER-core intermediate.

As one such example, Schwientek et al. introduced human [beta]1,4 galactosyltransferase ([beta]1,4 GalT) in an alg1 mutant strain (Schwientek et al., 1996). This strain produced Gal[beta]1,4-GlcNAc[beta]1,4-GlcNAc at the nonpermissive temperature, suggesting S.cerevisiae could import UDP-Gal into the Golgi lumen. However, it remained unsolved how UDP-Gal was transported into the Golgi lumen. Recently, we have reported in vivo galactose incorporation to mannoproteins by expressing the Schizosaccharomyces pombe [alpha]1,2 GalT gene in the S.cerevisiae with mnn1 background strain R16B (Roy et al., 1998). Furthermore, we have detected the presence of UDP-Gal transport activity in this transformed S.cerevisiae R16B strain in in vitro assay. Therefore, we reasoned that this endogenous activity allowed UDP-Gal to be transported from cytoplasm to the Golgi lumen where galactose transfer takes place. However, unlike S.pombe, only 15-20% of the total cells were galactosylated in this strain. Thus we hypothesized that not only the production and localization of GalT but also the efficient supply and its transport of UDP-Gal should be the key for the effective in vivo galactosylation in S.cerevisiae.

Recently, complementary DNAs for the human and S.pombe UDP-Gal transporters have been cloned and characterized (Ishida et al., 1996; Miura et al., 1996; Tabuchi et al., 1997; Yoshioka et al., 1997). Other nucleotide sugar transporter cDNAs including the murine CMP-sialic acid transporter have also been cloned in the past few years (Abeijon et al., 1996; Eckhardt et al., 1996; Dean et al., 1997; Ma et al., 1997). Of these, human UDP-Gal transporter and murine CMP-sialic acid transporter have been heterologously expressedin S.cerevisiae in active form to demonstrate in vitro that these cDNAs encode the transporter per se, but not regulatory proteins (Berninsone et al., 1997; Sun-Wada et al., 1998). Nonetheless, there is no in vivo study to demonstrate whether the expressed foreign sugar nucleotide transporter functions in S.cerevisiae or not.

In this report, we have first developed the in vivo neo-galactosylation system in S.cerevisiae. Using this system, we have then analyzed the limiting factor(s) for galactosylation which includes the expression level of S.pombe [alpha]1,2 GalT (Chappell and Warren, 1989; Chappell et al., 1994) and the effect of UDP-Gal transporter from human origin (Ishida et al., 1996). This is the first report to demonstrate that the human UDP-Gal transporter expressed in S.cerevisiae functions for the in vivo galactosylation and to clarify that this UDP-Gal transporter rather than [alpha]1,2 GalT is the major limiting factor for efficient galactosylation in S.cerevisiae. We have also addressed the in vivo target acceptor of S.pombe [alpha]1,2 GalT using this heterologous system and demonstrated that this enzyme transfers galactose to both N- and O-linked oligosaccharides. A further characterization indicates that this [alpha]1,2 GalT transfers galactose to O-mannosyl-Ser/Thr and generates Gal-Man-Ser/Thr structure.

Results

Coexpression of [alpha]1,2 galactosyltransferase and UDP-galactose transporter genes in S.cerevisiae

To provide galactosyltransferase activity in S.cerevisiae, the gma12+ gene encoding S.pombe [alpha]1,2-galactosyltransferase (Chappell et al., 1994) was overexpressed in strain W303-1A that was designated as MKY1. The product of gma12+ was already confirmed to retain its activity after efficient localization at the Golgi membranes in S.cerevisiae (Roy et al., 1998). To provide a high level of UDP-Gal transporter activity, human UGT2 cDNA (Ishida et al., 1996) was expressed under the control of the strong TDH3 promoter in W303-1A to construct MKY3. In S.cerevisiae exogenous galactose is converted to UDP-Gal by the endogenous enzymes, encoded by GAL1, GAL10, and GAL7 gene clusters located in the right arm of chromosome II, whose expression is induced by galactose (St. John and Davis, 1981). Therefore, the strains were grown in galactose containing media to supply UDP-Gal as a donor substrate. The galactose incorporation to the cell surface was judged by monitoring the fluorescence intensity using FITC conjugated [alpha]-galactose specific lectin, GS I-B4. The results are summarized in Table I. Neither strains MKY1 nor MKY3 were stained. When both genes were coexpressed in a multicopy plasmid ~50% of MKY5 cells were stained, indicating an increased galactose incorporation into cell surface glycoproteins compared to our previous results (15-20% of total cells; Roy et al., 1998). Microscopic views of the wild type W303-1A and strain MKY5 are shown in Figure 1A,B. The results from these constructs indicated that the presence of both [alpha]1,2 GalT and UDP-Gal transporter is essential for the efficient galactosylation in S.cerevisiae. Western blot analysis of microsomal fraction prepared from strain MKY5 demonstrated that hUGT2 expression was not affected in the presence of gma12+ expression (Figure 2, lane 3).


Figure 1. FITC conjugated GS I-B4 lectin staining of W303-1A and isogenic strains harboring [alpha]1,2 GalT and UDP-Gal transporter. W303-1A; parental strain (A), MKY5; W303-1A harboring multicopy plasmids, YEpUGAPgma12+ and pUGT2 (B), and MKY14; chromosomal integration of gma12+ and hUGT2 genes in the [Delta]mnn1 background (C). They were grown in either YP (A and C) or synthetic (B) media containing galactose and stained with FITC-GS I-B4. Light micrograph, Nomarski (left), and fluorescence (right) are shown.

Table I. S.cerevisiae strains and their evaluation of cell surface galactosylation
Strain Genotype FITC-GSI-B4b Reference
W303-1A MATa leu2-3,112 his3-11,15 ade2-1 ura3-1 trp1-1 can1-100 -c Wei et al.
MKY1 MATa leu2-3,112 his3-11,15 ade2-1 ura3-1 trp1-1 can1-100 [YEpUGAPgma12+]a - This study
MKY3 MATa leu2-3,112 his3-11,15 ade2-1 ura3-1 trp1-1 can1-100 [pUGT2] - This study
MKY5 MATa leu2-3,112 his3-11,15 ade2-1 ura3-1 trp1-1 can1-100 [YEpUGAPgma12+], [pUGT2] 50%d This study
MKY6 MATa leu2-3,112 his3-11,15 ade2-1 ura3-1 trp1-1 can1-100 URA3::gma12+ - This study
MKY8 MATa leu2-3,112 his3-11,15 ade2-1 ura3-1 trp1-1 can1-100 URA3::gma12+ [pUGT2] 50%~ This study
MKY12 MATa leu2-3,112 his3-11,15 ade2-1 ura3-1 trp1-1 can1-100 [Delta]mnn1::HIS3 URA3::gma12+ - This study
MKY13 MATa leu2-3,112 his3-11,15 ade2-1 ura3-1 trp1-1 can1-100 [Delta]mnn1::HIS3 URA3::gma12+ [pUGT2] 60-70% This study
MKY14 MATa leu2-3,112 his3-11,15 ade2-1 ura3-1 trp1-1 can1-100 [Delta]mnn1::HIS3 URA3::gma12+ LEU2::UGT2 ~80% This study
aBrackets mean plasmid-bearing forms.
bThe constructs were grown in galactose containing media to evaluate galactose attachment on cell surfaces.
cNo staining was detected.
dPercentage was calculated by FITC stained cell numbers over total cell numbers.

Overexpression of gma12+ gene impairs cell growth and morphology


Figure 2. Western immunoblot analysis of hUGT2 protein. Microsomal fractions were prepared from Had-1 cells harboring UGT2 (lane 1) and the various yeast constructs; W303-1A (lane 2), MKY5 (lane 3), MKY13 (lane 4) and MKY14 (lane 5) as described in Materials and methods. Equal amounts (40 µg) of microsomal proteins were loaded on SDS-PAGE (12%), subjected to immunoblot analysis using anti-UGT2 antibody. The bar on the right identifies the position where UGT2 migrates. Molecular mass markers are shown on the left in kilodaltons.

In the course of our experiments, we noticed that when gma12+ was overexpressed in a multicopy plasmid, yeast cells showed a slow growth and abnormal morphology with multibudded and elongated forms (Figure 3A,C). This may not be due to the overexpression of foreign membrane proteins in general because a similar phenotype was not seen in strain MKY3 which overexpressed another membrane protein, UGT2, from a multicopy plasmid (Figure 3, compare A and B). Therefore, we reduced the copy number of gma12+ by integrating this gene into the S.cerevisiae chromosome and the resultant strain was designated as MKY6. Strain MKY6 recovered to normal morphology (Figure 3D) and grew as fast as the wild type strain (data not shown). Then the extent of galactosylation was compared between strains MKY5 and MKY8, where multicopy plasmid pUGT2 was introduced into strain MKY6. A comparable or increased fluorescence intensity was observed in strain MKY8 than in strain MKY5 by the FITC-lectin staining (Table I, indicating that a single copy of gma12+ is sufficient for efficient galactose transfer to mannoproteins and overexpression of gma12+ causes impaired cell growth and abnormal morphology of S.cerevisiae.


Figure 3. The effects of gma12+ and hUGT2 dosage on cell morphology. Light micrographs of various strains grown to mid-logarithmic phase in galactose containing synthetic media are shown. MKY1; overexpression of gma12+ (A), MKY3; overexpression of hUGT2 (B), MKY5; co-overexpression of gma12+ and UGT2 (C), and MKY6; single copy expression of gma12 + (D). Magnification is the same for all strains.

[Delta]mnn1 provides a better acceptor for galactosylation in S.cerevisiae

In strain MKY8 ~50% of the cells showed a galactose incorporation into their cell surface. One reason for the partial incorporation of galactose may be due to the limitation of suitable acceptor structure for S.pombe [alpha]1,2-GalT in S.cerevisiae. When gma12+ gene was expressed in S.cerevisiae, the enzyme showed a higher activity towards mannoproteins isolated from mnn1, which lacks [alpha]1,3 mannosyltransferase, than those isolated from wild type or mnn2 strains, which lacks first [alpha]1,2 linked mannose, in vitro (Roy et al., 1998). To examine whether mannoproteins lacking [alpha]1,3 linked terminal mannose serve as better acceptors for [alpha]1,2 galactosylation in vivo, the MNN1 gene that encodes terminal [alpha]1,3 mannosyltransferase was disrupted in the MKY6 background. While the resulting strain MKY12 harboring [Delta]mnn1 and URA3::gma12+ did not show any lectin staining (Table I), strain MKY13 in which a multicopy plasmid pUGT2 was introduced into MKY12 showed approximately 60-70% of the cells staining by FITC-lectin (Table I). A direct effect of [Delta]mnn1 mutation was confirmed by comparing the fluorescence intensity between strains MKY8 and MKY13 (50% versus 65% staining; Table I). Taken together, these results indicate that mannoproteins lacking terminal [alpha]1,3 mannosyl residue indeed provide a better acceptor for Gma12p. The necessity of coexpression of [alpha]1,2 GalT and hUGT2 for galactosylation (MKY12 versus MKY13) are consistent with the previous result (MKY1 versus MKY5), independent from the supply of preferable acceptor by [Delta]mnn1 mutation.

Integration of both [alpha]1,2 GalT and hUGT2 to the yeast chromosome allowed us to grow the cells in nonselective YP media (MKY14). When strain MKY14 thus constructed was grown in YPG, 80% of the cells were uniformly stained by FITC-conjugated GS I-B4 (Table I, Figure 1C). This result indicated that a single copy of hUGT2 in addition to that of [alpha]1,2 GalT is sufficient for galactosylation. The expression level of hUGT2 in microsomal fraction among different constructs were compared by Western analysis. The amount of hUGT2 protein in yeast transformants gradually increased from strains MKY5, MKY13 to MKY14 (Figure 2, lanes 3-5) which correlated with the degree of galactose incorporation into cell surface as judged by the intensity of fluorescence staining (see Figure 1).

The fluorescence intensity of each strain was quantitatively evaluated by FACS analysis (Figure 4). Consistent with fluorescent microscopic observation (Table I, Figure 1), a gradual increase of fluorescence intensity was observed (Figure 4A-D). In addition, while no shift of intensity was observed in strain MKY14 grown in YPD (Figure 4E), ~80% of MKY14 cells grown in YPG media shifted to a higher fluorescent intensity (Figure 4F). Broad peaks in strains MKY5, MKY8, and MKY13 could be physiological difference between cells grown in synthetic media and YP media. The cells grown in synthetic media showed strong fluorescence to the bud sites and bud scars of the cell surface whereas strain MKY14, which is able to grow in YP media, showed more uniform distribution of FITC-staining on the cell surface (see Figure 1 B,C). This result also indicates that a stable segregation of the responsible genes are important for the uniform distribution of galactose on cell surface mannoproteins.


Figure 4. FACS analyses of various constructs. Cells grown in either synthetic or YP media containing galactose were stained with FITC conjugated lectin GS I-B4. Fluorescence intensity was measured by FACS as described in Materials and methods. W303-1A (A) grown in YPG and strains MKY5 (B), MKY8 (C), and MKY13 (D) grown in synthetic media containing galactose are shown. MKY14 grown in either YPD (E) or YPG (G) are also shown.

UDP-galactose transporter is the limiting factor for galactosylation

From the above results on fluorescence staining and Western blot analyses using several strains which harbor a single copy of chromosomally integrated gma12+ gene, we hypothesized that the level of UDP-galactose transporter activity should be the limiting factor for the in vivo galactosylation rather than the GalT activity. To understand the relationship between the effect of in vivo galactosylation and UDP-galactose transporter activity, we performed the in vitro UDP-galactose transport assay using strains W303-1A, MKY8, MKY12, MKY13, and MKY14 (Figure 5). The wild type strain, W303-1A, exhibited a trace amount of activity which was also reported previously (Sun-Wada et al., 1998). Consistent with the previous result, the higher activity compared to the wild type strain was obtained in strain MKY12 even without hUGT2 protein. This may be due to the presence of [alpha]1,2 GalT, which enables to generate UMP for antiport system (Roy et al., 1998), although it is not sufficient for in vivo galactosylation in W303-1A background strain. In contrast to strain MKY12, the activities of strain MKY8 and MKY13 which harbored human UGT2 in multicopy plasmid were about 3-fold higher than that of MKY12. Moreover, strain MKY14 which has stable segregation of the UGT2 gene gave ~6-fold higher activity than MKY12 has. Together with the results on fluorescence staining between MKY12 and MKY13 cells (0% versus 60-70%), these results clearly indicate that the supply of UDP-Gal transporter is the limiting factor for the in vivo galactosylation in S.cerevisiae.


Figure 5. UDP-galactose transporter activities of various constructs. P3 microsomal fractions of W303-1A, MKY8, MKY12, MKY13, and MKY14 were prepared as described in Materials and methods. Each fraction (250 µg) was incubated with 5 µM UDP-[3H]Gal (456 cpm/pmol) at 30°C in a final volume of 300 µl. After incubation the transporter activity was measured as described in Materials and methods. Values are means ± SD from quadruplicate experiments.

In vivo galactosylation of N-linked oligosaccharides in S.cerevisiae

By FITC conjugated lectin staining, we demonstrated incorporation of galactose to the cell surface of S.cerevisiae. However, it does not specify its transfer to N- or O-linked oligosaccharides of mannoproteins. Mannoproteins prepared from both wild type and MKY14 cells were subjected to Endo H digestion to release N-linked oligosaccharides. Monosaccharide contents of those oligosaccharides were analyzed as described in Materials and methods. As shown in Figure 6, no galactose peak was detected in PA-monosaccharide samples prepared from wild type cells grown in galactose containing media (panel C) or from MKY14 cells grown in glucose containing media (panel B). In contrast, when MKY14 cells were grown in galactose a new peak corresponding to galactose appeared (panel A). These results demonstrated that Gma12p transferred the galactose residues to N-linked oligosaccharides. Based on the peak area of mannose and galactose in Figure 6A, ~0.4% of total N-linked oligosaccharides contain galactose residues.


Figure 6. Monosaccharides analysis of N-linked mannoproteins from strains W303-1A and MKY14. Total cell surface mannoproteins prepared either from W303-1A grown in galactose or MKY14 grown in either galactose or glucose were subjected to Endo H digestion and mild acid treatment followed by pyridylamination. Each sample was applied on anion exchange column PALPAK Type A to separate constituent monosaccharides. Separated PA-monosaccharide samples were compared with authentic PA-monosaccharides for the identification (data not shown). MKY14 grown in galactose (A) and glucose (B); W303-1A grown in galactose (C). PA-glucose (Glc), PA-mannose (Man) and PA-galactose (Gal) peaks are assigned after comparison of their retention times with those of authentic standards. The origin of glucose is unidentified.

In vivo galactosylation of O-linked oligosaccharides in S.cerevisiae

We further examined the possibility of galactosylation of O-linked oligosaccharides. It was known that S.cerevisiae chitinase secreted into the culture medium is exclusively mannosylated with a series of short O-linked oligosaccharides (Kuranda and Robbins, 1991). Chitinase was purified from culture media by chitin binding after the wild type and MKY14 strains were grown in either YPD or YPG media. Its purity was confirmed by Coomassie staining (Figure 7A). Faster migration of the chitinase prepared from MKY14 cells was due to the [Delta]mnn1 mutation (Lussier et al., 1995). The chitinase was subjected to lectin blotting by Con A (Figure 7B) or GS I-B4 (Figure 7C). All chitinases were detected by Con A irrespective of carbon sources. On the other hand, the [alpha]-galactoside specific lectin, GS I-B4, only recognized the chitinase prepared from strain MKY14 grown in YPG and no signals were observed in other chitinase preparation.


Figure 7. Lectin blot analysis of chitinase from strains W303-1A and MKY14. Extracellular chitinase was purified as described in Materials and methods from W303-1A (lanes 1 and 2) and MKY14 (lanes 3 and 4) cells grown in either galactose (lanes 1 and 3) or glucose (lanes 2 and 4). Equal amount of protein extracts was separated on SDS-PAGE (7.5%), subjected to Commassie staining (A), or electrotransferred, then followed by ConA (B) or GS I-B4 (C) lectin detection, as described in Materials and methods. Molecular mass is indicated in kilodaltons on the left of (A).

Further, to confirm which mannose is occupied by [alpha]1,2 linked galactose(s), HPLC analysis of O-linked oligosaccharides were conducted. O-Linked oligosaccharides of purified chitinase were prepared from MKY14 cultures grown in either galactose or glucose containing YP media. The PA labeled neutral O-linked oligosaccharides were analyzed by the amine column as described in Materials and methods. When the HPLC profiles of the samples prepared from strain MKY14 grown in either glucose or galactose were compared (Figure 8A,B), an extra peak was observed in strain MKY14 grown in galactose at around 20 min which corresponded to the size of disaccharide. This peak was not digested with an [alpha]-mannosidase treatment whereas other peaks were digested to monosaccharides (Figure 8C). This 20 min peak was completely disappeared by a further digestion with [alpha]-galactosidase (data not shown). The same sample treated with [alpha]-galactosidase diminished only the 20 min peak but none of the other peaks (Figure 8D). This 20 min peak was pooled (Figure 8E) and digested with [alpha]-galactosidase. This resulted in a complete conversion from the size of disaccharide to monosaccharide position (Figure 8F). Based on peak area shown in Figure 8B, ~10% of disaccharides consist of Gal[alpha]1,2-Man1-O-Ser/Thr structure which corresponds to 5% of total O-linked oligosaccharides. Taken together, the results indicated that [alpha]1,2 GalT transferred galactose only to the core O-mannosyl residue attached to polypeptides.


Figure 8. HPLC analysis of O-linked oligosaccharides released from chitinase. O-Linked oligosaccharides were prepared from extracellular chitinase as described in Materials and methods. HPLC profiles using amine column are shown: MKY14 grown in YPD (A), MKY14 grown in YPG (B), total PA-oligosaccharides shown in panel B were digested with [alpha]-mannosidase (C), or with [alpha]-galactosidase (D), and the 20 min peak shown in (B) (asterisk) was pooled from four HPLC runs (E). The peak shown in (E) was digested with [alpha]-galactosidase resulting in a shift of the corresponding peak to 13 min (M1) (F). The peaks designated as M1 to M4 represent PA-labeled sugar chains containing one to four mannose residues after confirming their elution times with authentic PA-labeled M1-M4 oligosaccharides. Asterisk indicates the galactose containing peak.

Discussion

Generation of galactosylation system in S.cerevisiae

We have demonstrated the in vivo functional expression of a heterologous sugar nucleotide transporter, UDP-Gal transporter, in S.cerevisiae. Expression of this membrane protein directly contributed to the efficient in vivo galactosylation in S.cerevisiae. Our new strain which comprises integration of S.pombe [alpha]1,2 GalT and human UDP-Gal transporter genes in the [Delta]mnn1 background cell showed a highly efficient galactose incorporation into both N- and O-linked mannoproteins. By testing various combinations of factors involved in galactosylation, we confirmed that the following components are important for efficient in vivo glycosylation (1) expression of glycosyltransferase, (2) expression of sugar nucleotide transporter, (3) supply of donor substrates in cytosol, and (4) provision of acceptor oligosaccharides in the Golgi lumen. Our results on in vivo galactosylation and in vitro UDP-galactose transport assay strongly suggest that not the expression level of [alpha]1,2 GalT but the availability of UDP-Gal in the Golgi lumen plays a key role in de novo galactosylation in S.cerevisiae (Table I; MKY1 versus MKY5, MKY6 versus MKY8, and MKY12 versus MKY13 and 14; and Figure 5). Our data revealed that only a single copy of both [alpha]1,2 GalT and UDP-Gal transporter genes is sufficient for efficient galactosylation (Table I; MKY5 versus MKY8 and MKY13 versus MKY14, respectively).

In a previous report, the expression of S.pombe [alpha]1,2 GalT in the S.cerevisiae strain with mnn1 background displayed partial galactosylation in vivo (15-20% of total cells; Roy et al., 1998). In contrast, in this study the expression of gma12+ gene alone (MKY1) or the strain expressing gma12+ gene in the [Delta]mnn1 background (MKY12) did not evoke any in vivo galactosylation in S.cerevisiae cells, although the in vitro UDP-galactose transport activity was comparable between R16B expressing gma12+ gene and MKY12 (~10 pmol/mg protein/5 min). One possible reason is the difference in the strain backgrounds between R16B and W303-1A. Strain R16B used in the previous report (Roy et al., 1998) was constructed by various crossover with other non-isogenic strains which sometimes introduce unknown mutations. For example, different from W303-1A, aggregation of cells was observed in liquid medium when gma12+ was overexpressed in strain R16B (S. K. Roy, personal communication). In the work reported here, we performed stepwise the gene integration or gene disruption starting from well characterized haploid laboratory strain, W303-1A, without any cross. This enabled us to compare the effect of each gene function on galactosylation in parallel. The in vitro activity detected in MKY12 comparable to strain R16B may not be sufficient for in vivo galactosylation in this strain.

An aberrant cell morphology was observed when the BED1 gene was disrupted in S.cerevisiae (Mondésert and Reed, 1996). The BED1 gene was cloned based on its involvement in bud emergence and polarized growth. This gene is identical to the MNN10 gene which takes part in outer chain mannosylation in S.cerevisiae (Dean and Poster, 1996). Interestingly, the BED1/MNN10 gene has a significant sequence homology to the S.pombe gma12+ gene (Dean and Poster, 1996; Mondésert and Reed, 1996). Overexpression of the gma12+ gene under the TDH3 promoter in the multicopy plasmid also caused an abnormal cell morphology with an impaired growth phenotype. Elongated, larger and multibudded cells were observed (Figure 3A, MKY1). This alteration of S.cerevisiae phenotype was reversed by reducing the copy number of the gma12+ gene by integrating it into chromosome (Table I, MKY1 versus MKY6), while galactosylation has been comparable or even slightly increased (Table I, MKY5 versus MKY8). The results presented here and the BED1 study (Mondésert and Reed, 1996) indicate that the expression level of this glycosyltransferase gene family is somehow critical for the control of cell growth and morphology in S.cerevisiae.

The growth rate of parental strain W303-1A and strain MKY14 was measured to see how galactosylation affects cell growth in S.cerevisiae. The wild type W303-1A and MKY14 strain grown in YPG media showed a doubling time of 2.5 h and 5.0 h, respectively, while both strains grown in glucose showed no significant differences in their growth rate. The galactosylation of mannoproteins may cause some inhibition of cell growth in S.cerevisiae.

Acceptor specificity of S.pombe Gma12 produced in S.cerevisiae

The Gma12 p was originally purified based on its activity of galactose transfer from UDP-Gal to a variety of mannose-based acceptors to form an [alpha]1,2 galactosyl mannoside linkage (Chappell and Warren, 1989; Chappell et al., 1994). In in vitro studies [alpha]-methyl d-mannoside showed a fourfold increase in specific activity relative to d-mannose (Chappell and Warren, 1989). However, real in vivo acceptor substrates have not been identified and there is no direct evidence for involvement in galactosylation of N- and/or O-linked oligosaccharides. S.cerevisiae heterologously expressing GalT derived from S.pombe allowed us to study the in vivo acceptor specificity of the enzyme because no GalT has been identified in S.cerevisiae (Herscovics and Orlean, 1993). In this report, we have demonstrated that the [alpha]1,2 GalT encoded by the gma12+ gene acts on both N- and O-linked oligosaccharides in vivo (Figures 5-7). Since Gma12p favors mannoproteins isolated from mnn1 mutant strain as an acceptor (Roy et al., 1998), we initially anticipated that the galactose might be attached at either fourth or fifth position to replace [alpha]1,3 terminal mannose residues in O-linked oligosaccharides. However, our results indicated that this enzyme transferred galactose only to the first mannose residue attached to Ser/Thr residues and no further elongation of either galactose by Gma12p or mannose by endogenous mannosyltransferases was observed (Figure 7). We have no plausible answer to explain why the lack of terminal [alpha]1,3 mannose residues contribute to better [alpha]1,2 galactosylation of the first mannose residues.

We can, however, postulate two reasons for detecting only Gal-Man-Ser/Thr structure but neither Gal-Gal-Man-Ser/Thr nor Gal-Man-Man-Ser/Thr structures which are constituents in S.pombe (Ballou et al., 1994). Since it has been reported that several galactosyltransferases are present in S.pombe (T. Yoko-o, personal communication; Chappell et al., 1994), it is likely that Gma12p is responsible for the first galactose addition and the other GalTs for the subsequent galactose addition in O-linked oligosaccharides of S.pombe. This is supported by the observation that Gma12p has no activity towards mannobiose in vitro (S. K. Roy, personal communication). Alternatively, since the galactosyltransferase is expressed in a heterologous system, the [alpha]1,2 galactosyltransferase is competing for the second sugar transfer with [alpha]1,2 mannosyltransferases (Ktr1p, Ktr3p, and Kre2p/Mnt1p), all of which are known to be involved in the second and third mannose additions in O-linked oligosaccharide of S.cerevisiae (Lussier et al., 1997). The [alpha]1,2 GalT was able to compete for the second sugar transfer but not for the third sugar transfer presumably due to the improper localization or less affinity towards disaccharides such as mannobiose or mannosylgalactose. This competition between Kre2 family proteins and Gma12p for the second sugar transfer also supports the idea that [alpha]1,2 GalT may be localized in the early Golgi compartment where Kre2 protein family is localized in S.cerevisiae (Chapman and Munro,1994; Lussier et al., 1995). Previously it was speculated that Gma12p resides in a cis compartment with other mannosyltransferase activity in S.pombe (Chappell et al., 1994). Further study on the specific localization and retention mechanism of Gma12p and other galactosyltransferases in S.pombe is awaited.

Our yeast strains constructed here producing sugar chains with [alpha]1,2 linked galactose are not directly applicable to produce glycoprotein therapeutics because normal human complex-type oligosaccharides contain only [beta]-linked galactose. Also the amount of actual galactose resides incorporated into the total oligosaccharides were not in a large quantity. However, the information obtained in this study will be useful for the construction of yeast strains which produces a desired glycoform of heterologous proteins in S.cerevisiae. In addition the use of such a heterologous system will contribute to further understanding the functional relationship of the expressed foreign proteins, such as glycosyltransferases and sugar nucleotide transporters, in the Golgi apparatus.

Materials and methods

Strains and genetic manipulations

The genotypes and sources of S.cerevisiae strains used or constructed in this work are summarized in Table I. YP medium (YP) and synthetic minimal medium (S), supplemented with the relevant amino acids, bases and 2% glucose (D) or 2% galactose (G), were prepared as described previously (Rose et al., 1990). Cultures were grown at 30°C and were monitored by measuring optical density at 600 nm (1 OD600 = 5 × 10 5 cells/ml). Yeast DNA transformation was carried out as follows: aliquots of 5 × 105 cells at early stationary phase were centrifuged and resuspended in 0.1 ml of 0.2 N lithium acetate, 40% polyethylene glycol 3350-4000, 10 mM DTT. Three micrograms of salmon sperm and 1 µg of the transforming DNA were mixed into the cell suspensions and incubated for 30 min at 45°C. The cell suspension was then directly plated on selective medium and incubated at 30°C.

Plasmid and various strain constructions

YEpUGAPgma12+ constructed previously was used for [alpha]1,2 GalT expression in S.cerevisiae (Roy et al., 1998). The multicopy plasmid pUGT2 which contains human UDP-Gal transporter cDNA was constructed as follows: A 1.2 kb EcoRI fragment containing the coding region of hUGT2 from pGEM75zf(+)-UGT2 (Ishida et al., 1996) was excised and ligated into pKT10 (Tanaka et al., 1990; Roy et al., 1998) at the EcoRI site downstream of yeast TDH3 promoter. Then a 2.2 kb BamHI fragment which contained the TDH3 promoter, hUGT2, and TDH3 terminator was ligated into the BamHI site of YEp351 (Hill et al., 1986). The final plasmid was designated as pUGT2. The W303-1A strain which was transformed by YEpUGAPgma12+ was called MKY1. MKY3 was constructed by introducing pUGT2 into W303-1A. MKY1 was transformed with pUGT2 to obtain MKY5. To integrate gma12+gene in the S.cerevisiae chromosome, YEpUGAPgma12+ was linearized at the EcoRV site of the URA3 gene and introduced at the ura3 locus of W303-1A. Stable Ura+ transformants were chosen and the integration of single copy of gma12+ at the ura3 locus of W303-1A strain was confirmed by Southern blot analysis (data not shown). One of the resulting strains was named MKY6. MKY6 was transformed with pUGT2 to obtain MKY8.

To disrupt the MNN1 gene pHYH-[Delta]mnn1 was generated as follows. A 1.8 kb SphI fragment from pJJ244-[Delta]mnn1 (kindly provided by Y. Shimma) was ligated into the SphI site of pHYH (Schwelberger et al., 1993). pHYH-[Delta]mnn1 was digested at the NotI site and the linearized fragment containing the disrupted MNN1 gene was introduced into MKY6 by one-step gene disruption/deletion (Rothstein, 1983). Stable His+ transformants were selected, and the integrative disruption at the MNN1 locus was confirmed by PCR (data not shown). One of the transformants was designated as MKY12.

To integrate hUGT2 at the leu2 locus of chromosome, YIp352-UGT2 was constructed by excising the HpaI/AatII fragment to remove the 2 µm circle, followed by blunt-ending and religating the remaining fragment. The YIp352-UGT2 was linearized at the EcoRV site of the LEU2 gene and introduced into MKY12 to generate MKY14. The integration of single copy of hUGT2 at the leu2 locus was confirmed by Southern blot analysis (data not shown).

Western immunoblotting

Yeast microsomal fraction was prepared by growing the strains in either YP or synthetic media containing galactose or glucose to the mid logarithmic phase. The cells were lysed by glass beads in the buffer containing 10 mM Tris-HCl, pH 7.5, 2 mM phenylmethanesulfonyl fluoride, 10 mM DTT, and 5 µg/ml protease inhibitor (leupeptin, pepstatin A and aprotinin) and centrifuged at 1000 × g for 5 min followed by 100,000 × g for 60 min. The pellet was resuspended in the same buffer. Microsomal fraction from Had-1 cells expressing hUGT2 gene was prepared as described previously (Yoshioka et al., 1997). The samples were fractionated on a 12% SDS-PAGE and Western immunoblotting was carried out using anti-hUGT2 polyclonal antibody as described previously (Yoshioka et al., 1997).

FITC-conjugated lectin staining of various constructs

Each strain was grown in YP or synthetic media containing either glucose or galactose as a carbon source at 30°C to mid-logarithmic phase. Aliquots containing 1 × 105 cells were centrifuged and treated with fluorescein isothiocyanate (FITC)-conjugated Griffonia simplicifolia I-B4 (GS I-B4) lectin (Vector Laboratories, USA) as described previously (Roy et al., 1998). The cells were then subjected to the microscopic or FACS analysis.

Fluorescent activated cell sorting (FACS) analysis

FITC-labeled cells were analyzed by FACSCalibur (Becton Dickinson) to detect fluorescence intensity. The instrument is equipped with argon-ion laser which is operated at 488 nm with 15 mW energy. Emission of FITC was measured at 560 nm. Cells were passed through flow cytometers, and 10,000 events were counted and analyzed with CELLQuest (Becton Dickinson).

Subcellular fractionation and UDP-galactose transport assay

The microsomal fractionation was performed as described by Roy et al. (Roy et al., 1998). A P3 fraction (25 µg/µl) was used for the following UDP-galactose transport assay. The transport assay was performed as described previously (Roy et al., 1998) with some modifications. Briefly, a P3 fraction (250 µg) was incubated in a reaction mixture (20 mM Tris-HCl, pH 7.5, 0.25 M sucrose, 5 mM MgCl2, 1 mM MnCl2, 10 mM 2-mercaptoethanol) with 5 µM UDP-[3H] galactose (specific activity, 456 cpm/pmol). After incubation at 30°C for 5 min, the reaction was terminated by 10-fold dilution with an ice-cold stop buffer (20 mM Tris-HCl, pH 7.5, 150 mM KCl, 1 mM MgCl2) and placed on ice. Diluted samples were applied onto filtration apparatus containing HA filters (24 mm diameter, 0.45 µm pore size, Millipore). The filters were washed with the stop buffer, air-dried, and dissolved in 10 ml scintillation mixture (Clear-sol I, Nakarai Tesque). After 2 h at room temperature, the samples were counted in a liquid scintillation counter (Beckman LS1701). The amount of radioactivity that was bound nonspecifically to the outside of the vesicles was determined by zero time assay.

HPLC analysis of N-linked monosaccharides

Total cell surface mannoproteins were prepared as described previously (Nakanishi-Shindo et al., 1993). Endoglycosidase H (Endo H) from Streptomyces griseus (Seikagaku Corp., Japan) was used to perform N-linked oligosaccharide digestion (Tarentino and Maley, 1974) according to the manufacturer's instructions. Monosaccharides were prepared from N-linked oligosaccharides by acid hydrolysis followed by N-acetylation as described previously (Suzuki et al., 1991). Each monosaccharide was labeled with 2-aminopyridine (PA) as described previously (Nakanishi-Shindo et al., 1993). Monosaccharide analysis was performed by using anion-exchange column, PALPAK Type A (Takara, Japan), and the products were identified with the PA-sugar standards (Takara, Japan).

Isolation of extracellular chitinase by chitin binding

Extracellular chitinase was isolated as described previously with some modifications (Kuranda and Robbins, 1991). Briefly, intact chitinase (Cts1p) was isolated without any overexpression of the chitinase gene (CTS1). W303-1A and MKY14 cells were grown in 100 ml of either YPD or YPG medium until mid-logarithmic phase. The cells were pelleted and the supernatants were collected to which chitin pretreated with 1% SDS and 1% [beta]-mercaptoethanol was added to a final concentration of 1 µg/ml and then stirred at 4°C overnight. Chitinase bound to insoluble chitin was collected by filtration through Type HA membrane (0.45 mm, Millipore) and washed with Solution A (0.8% NaCl, 0.02% KCl, 0.12% Na2HPO4, 0.02% KH2PO4). Finally, the chitin-chitinase complex was suspended in 150 µl of the sample buffer (2% SDS, 5% [beta]-mercaptoethanol, 10% glycerol) and heated at 100°C for 10 min to release chitinase.

Lectin blot analysis

Chitinase samples were fractionated by 7.5% SDS-PAGE (Laemmli, 1970) and subjected to electrotransfer to PVDF membranes (Amersham, Hybond-PVDF) using Towbin buffer (Towbin et al., 1979). Lectin blot analysis was carried out as described previously with some modifications (Cho et al., 1996). The blots were blocked with 5% BSA containing TBS (20 mM Tris-HCl, 500 mM NaCl, pH 7.5) with 2 µg/ml of streptavidin, then incubated with 3.5 µg/ml of biotinylated GS I-B4 (Vector Laboratories, USA) or biotinylated Con A (Vector Laboratories, USA) in a dilution buffer (TBS, 0.1% BSA, 0.3% Tween 20), followed by incubation with alkaline phosphatase-conjugated streptavidin (GIBCO-BRL, USA). The blots were developed as described previously (Wei et al., 1995).

HPLC analysis of O-linked oligosaccharides

O-Linked oligosaccharides were prepared from chitinase bound chitin by hydrazinolysis (Hydrozinolysis Reagent C, Honen) using Hydraclub S-204 (Honen) according to the manufacturer's protocol. In brief, the sample was hydrolyzed by hydrazine at 65°C for 6 h, followed by N-acetylation by extracting the sample with 0.2 N ammonium acetate and acetic anhydride. The dried sample was pyridylaminated using pyridylamination kit (Takara Shuzo Co., Ltd.) according to the manufacturer's protocol. The PA-labeled sample was resuspended in H2O and applied to boronic acid column (Pierce Chemical Co.) and O-linked oligosaccharides were eluted by 0.2 M sorbitol. The boronic acid column binding fraction was further analyzed by Asahipak NH2P-50 (0.46 × 25 cm; Showa Denko Co., Tokyo) at the rate of 1.0 ml/min with solvent A (acetonitrile) and solvent B (200 mM acetic acid-triethylamine, pH 7.3). After the sample was injected, the proportion of B was increased linearly from 10% up to 60% for 60 min. Each peak was further collected and then subjected to either [alpha]-galactosidase (Sigma, EC 3.2.1.22) or [alpha]-mannosidase (Seikagaku Corp., EC 3.2.1.24) treatment according to manufacturers' protocols.

Acknowledgments

We are grateful to Y.Shimma and T.Odani for providing pJJ244-[Delta]mnn1; Y.Maeda and K.Nakayama for help with HPLC analysis; and S.K.Roy for suggestions on UDP-galactose transport assay, sharing unpublished data, and comments on the manuscript. M.K. is indebted to X.-D.Gao and Y.Maeda for helpful comments and discussion; and M.F.Cohen for English correction. This work was supported in part by a grant-in-aid of Research and Development Project of Basic Technologies for Future Industries from Ministry of International Trade and Industry, Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be marked 'advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. M.K. was supported by a New Energy and Industrial Technology Development Organization (NEDO) postdoctoral fellowship.

References

Abeijon ,C., Robbins,P.W., and Hirschberg,C.B. (1996) Molecular cloning of the Golgi apparatus uridine diphosphate-N-acetylglucosamine transporter from Kluyveromyces lactis. Proc. Natl. Acad. Sci. USA, 93, 5963-5968. MEDLINE Abstract

Baenziger ,J.U. (1994) Protein-specific glycosyltransferases: How and why they do it! FASEB J., 8, 1019-1025. MEDLINE Abstract

Ballou ,C.E. (1990) Isolation, characterization, and properties of Saccharomyces cerevisiae mnn mutants with nonconditional protein glycosylation defects. Methods Enzymol., 185, 440-470. MEDLINE Abstract

Ballou ,E.C., Ballou,L. and Ball,G. (1994) Schizosaccharomyces pombe glycosylation mutant with altered cell surface properties. Proc. Natl. Acad. Sci. USA, 91, 9327-9331. MEDLINE Abstract

Berninsone ,P., Eckhardt,M., Gerardy-Schahn,R. and Hirschberg,C.B. (1997) Functional expression of the murine Golgi CMP-sialic acid transporter in Saccharomyces cerevisiae. J. Biol. Chem., 272, 12616-12619. 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

Chappell ,T.G. and Warren,G. (1989) A galactosyltransferase from the fission yeast Schizosaccharomyces pombe. J. Cell Biol., 109, 2693-2702. MEDLINE Abstract

Chappell ,T.G., Hajibagheri,M. A.N., Ayscough,K., Pierce,M. and Warren,G. (1994) Localization of an [alpha]1,2 galactosyltransferase activity to the Golgi apparatus of Schizosaccharomyces pombe. Mol. Biol. Cell, 5, 519-528. MEDLINE Abstract

Cho ,K.S., Yeh,J., Cho,M. and Cummings,R.D. (1996) Transcriptional regulation of [alpha]1,3-galactosyltransferase in embryonal carcinoma cells by retinoic acid. J. Biol. Chem., 271, 3238-3246. MEDLINE Abstract

Dean ,N. and Poster,J. B. (1996) Molecular and phenotypic analysis of the S.cerevisiae MNN10 gene identifies a family of related glycosyltransferases. Glycobiology, 6, 73-81. MEDLINE Abstract

Dean ,N., Zhang,Y.B. and Poster,J.B. (1997) The VRG4 gene is required for GDP-mannose transport into the lumen of the Golgi in the yeast, Saccharomyces cerevisiae. J. Biol. Chem., 272, 31908-31914. MEDLINE Abstract

Eckart ,M.R. and Bussineau,C.M. (1996) Quality and authenticity of heterologous proteins synthesized in yeast. Curr. Opin. Biotechnol., 7, 525-530. MEDLINE Abstract

Eckhardt ,M., Mühlenhoff,M., Bethe,A. and Gerady-Schahn,R. (1996) Expression cloning of the Golgi CMP-sialic acid transporter. Proc. Natl. Acad. Sci. USA, 93, 7572-7576. MEDLINE Abstract

Herscovics ,A. and Orlean,P. (1993) Glycoprotein biosynthesis in yeast. FASEB J., 7, 540-550. MEDLINE Abstract

Hill ,J.E., Myers,A.M., Koerner,T.J. and Tzagoloff,A. (1986) Yeast/E. coli shuttle vectors with multiple unique restriction sites. Yeast, 2, 163-167. MEDLINE Abstract

Ishida ,N., Miura,N., Yoshioka,S. and Kawakita,M. (1996) Molecular cloning and characterization of a novel isoform of the human UDP-galactose transporter and of related complementary DNAs belonging to the nucleotide-sugar transporter gene family. J. Biochem. (Tokyo), 120, 1074-1078. MEDLINE Abstract

Kuranda ,M.J. and Robbins,P.W. (1991) Chitinase is required for cell separation during growth of Saccharomyces cerevisiae. J. Biol. Chem., 266, 19758-19767. MEDLINE Abstract

Laemmli ,U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, 680-685. MEDLINE Abstract

Lehle ,L. and Tanner,W. (1995) Protein glycosylation in yeast. In Montreuil,J., Schachter,H. and Vliegenthart,J.F.G. (eds.), Glycoproteins. Elsevier Science B.V., Amsterdam, pp. 475-509.

Lussier ,M., Sdicu,A.-M., Ketela,T. and Bussey,H. (1995) Localization and targeting of the Saccharomyces cerevisiae Kre2p/Mnt1p [alpha]1,2 mannosyltransferase to a medial Golgi compartment. J. Cell Biol., 131, 913-927. MEDLINE Abstract

Lussier ,M., Sdicu,A.-M., Bussersau,F., Jacquet,M. and Bussey,H. (1997) The Ktr1p, Ktr3p, and Kre2p/Mnt1p mannosyltransferases participate in the elaboration of yeast O- and N-linked carbohydrate chains. J. Biol. Chem., 272, 15527-15531. MEDLINE Abstract

Ma ,D., Russell,D.G., Beverley, S.M. and Turco,S.J. (1997) Golgi GDP-mannose uptake required Leishmania LPG2. J. Biol. Chem., 272, 3799-3805.

Miura ,N., Ishida,N., Hoshino,M., Yamauchi,M., Hara,T., Ayusawa,D. and Kawakita,M. (1996) Human UDP-galactose translocator: molecular cloning of a complementary DNA that complements the genetic defect of a mutant cell line deficient in UDP-galactose translocator. J. Biochem. (Tokyo), 120, 236-241. MEDLINE Abstract

Mondésert ,G. and Reed,S.I. (1996) BED1, a gene encoding a galactosyltransferase homologue, is required for polarized growth and efficient bud emergence in Saccharomyces cerevisiae. J. Cell Biol., 132, 137-151.

Nakanishi-Shindo ,Y., Nakayama,K., Tanaka,A., Toda,Y. and Jigami,Y. (1993) Structure of the N-linked oligosaccharides that show the complete loss of [alpha]-1,6 polymannose outer chain from och1, och1 mnn1 and och1 mnn1 alg3 mutants of Saccharomyces cerevisiae. J. Biol. Chem., 268, 26338-26345. MEDLINE Abstract

Nakayama ,K., Nagasu,T., Shimma,Y., Kuromitsu,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

Odani ,T., Shimma,Y., Tanaka,A. and Jigami,Y. (1996) Cloning and analysis of the MNN4 gene required for phosphorylation of N-linked oligosaccharides in Saccharomyces cerevisiae. Glycobiology, 6, 805-810. MEDLINE Abstract

Orlean ,P. (1997) Biogenesis of yeast wall and surface components. In Pringle,J.R., Broach,J.R. and Jones,E.W. (eds.), The Molecular and Cellular Biology of the Yeast Saccharomyces: Cell Cycle and Cell Biology. Cold Spring Harbor Laboratory Press, New York, pp. 229-362.

Rose ,M.D., Winston,F. and Hieter,P. (1990) Methods in Yeast Genetics: A Laboratory Course Manual. Cold Spring Harbor Laboratory Press, New York.

Rothstein ,R.J. (1983) One step gene disruption in yeast. Methods Enzymol., 101, 202-211. MEDLINE Abstract

Roy ,S.K., Yoko-o,T., Ikenaga,H. and Jigami,Y. (1998) Functional evidence for UDP-galactose transporter in Saccharomyces cerevisiae through the in vivo galactosylation and in vitro transport assay. J. Biol. Chem., 273, 2583-2590. MEDLINE Abstract

Schultz ,L.D., Tanner,J., Hofmann,K.J., Emini,E.A., Condra,J.H., Hones,R.E., Kieff,E. and Ellis,R.W. (1987) Expression and secretion in yeast of a 400-kDa envelope glycoprotein derived from Epstein-Barr virus. Gene, 54, 113-123. MEDLINE Abstract

Schwelberger ,H.G., Kang,H.A. and Hershey,J.W. (1993) Translation initiation factor eIF-5A expressed from either of two yeast genes or from human cDNA: functional identity under aerobic and anaerobic conditions. J. Biol. Chem., 268, 14018-14025. MEDLINE Abstract

Schwientek ,T., Narimatsu,H. and Ernst,J. (1996) Golgi localization and in vivo activity of a mammalian glycosyltransferase (human [beta]1,4-galactosyltransferase) in yeast. J. Biol. Chem., 271, 3398-3405. MEDLINE Abstract

St. John ,T.P. and Davis,R.W. (1981) The organization and transcription of the galactose gene cluster of Saccharomyces cerevisiae. J. Mol. Biol., 152, 285-315. MEDLINE Abstract

Sun-Wada ,G.-H., Yoshioka,S., Ishida,N. and Kawakita,M. (1998) Functional expression of the human UDP-galactose transporter in the yeast Saccharomyces cerevisiae. J. Biochem. (Tokyo), 123, 912-917. MEDLINE Abstract

Suzuki ,J., Kondo,A., Kato,I., Hase,S. and Ikenaka,T. (1991) Analysis of high-performance anion-exchange chromatography of component sugars as their fluorescent pyridylamino derivatives. Agric. Biol. Chem.,55, 283-284.

Tabuchi ,M., Tanaka,N., Iwahara,S. and Takegawa,K. (1997) The Schizosaccharomyces pombe gms1+ gene encodes an UDP-galactose transporter homologue required for protein galactosylation. Biochem. Biophy. Res. Commun., 232, 121-125.

Tanaka ,K., Nakafuku,M., Tamanoi,F., Matsumoto,K. and Toh-e,A. (1990) IRA2, a second gene of Saccharomyces cerevisiae that encodes a protein with a domain homologous to mammalian ras GTPase-activating protein. Mol. Cell. Biol., 10, 4303-4313. MEDLINE Abstract

Tarentino ,A.L. and Maley,F. (1974) Purification and properties of an endo-beta-N acetylglucosaminidase from Streptomyces griseus. J. Biol. Chem., 249, 811-817. MEDLINE Abstract

Towbin ,H., Staehelin,T., and Gordon,J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA, 76, 4350-4654. MEDLINE Abstract

Trimble ,R.B. and Verostek,M.F. (1995) Glycoprotein oligosaccharide synthesis and processing in yeast. Trends Glycosci. Glycotech., 7, 1-30.

Wei ,C.-L., Kainuma,M. and Hershey,J.W.B. (1995) Characterization of yeast translation initiation factor 1A and cloning of its essential gene. J. Biol. Chem., 270, 22788-22794. MEDLINE Abstract

Yoshioka ,S., Sun-Wada,G., Ishida,N. and Kawakita,M. (1997) Expression of the human UDP-galactose transporter in the Golgi membranes of murine Had-1 cells that lack endogenous transporter. J. Biochem. (Tokyo),122, 691-695.


4To whom correspondence should be addressed


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: 6 Feb 1999
Copyright©Oxford University Press, 1999.