Schizosaccharomyces pombe och1(+) encodes alpha-1,6-mannosyltransferase that is involved in outer chain elongation of N-linked oligosaccharides.

The fission yeast Schizosaccharomyces pombe attaches an outer chain containing mannose and galactose to the N-linked oligosaccharides on many of its glycoproteins. We identified an S. pombe och1 mutant that did not synthesize the outer chains on acid phosphatase. The S. pombe och1(+) gene was a functional homolog of Saccharomyces cerevisiae OCH1, and its gene product (SpOch1p) incorporated alpha-1,6-linked mannose into pyridylaminated Man(9)GlcNAc(2), indicating that och1(+) encodes an alpha-1,6-mannosyltransferase. Our results indicate that SpOch1p is a key enzyme of outer chain elongation. The substrate specificity of SpOch1p was different from that of S. cerevisiae OCH1 gene product (ScOch1p), suggesting that SpOch1p may have a wider substrate specificity than that of ScOch1p.

Schizosaccharomyces pombe gmd3(+)/alg11(+) is a functional homologue of Saccharomyces cerevisiae ALG11 which is involved in N-linked oligosaccharide synthesis.

The oligosaccharide of glycoproteins in the fission yeast Schizosaccharomyces pombe is unique in containing galactose. We isolated four mutants that had reduced amounts of galactose residues on their cell surface glycoproteins by fluorescence-activated cell sorter. The isolated four recessive mutants, gmd1 to gmd4, showed a defect in glycosylation of acid phosphatase, a cell surface glycoprotein. In gmd3 mutant cells, the amounts of both mannose and galactose residues were decreased on the cell surface galactomannoproteins, suggesting an underglycosylation of galactomannoproteins. The gmd3(+) gene encodes a protein that has significant similarity with Saccharomyces cerevisiae Alg11p and is likely to be involved in N-linked core oligosaccharide synthesis. ALG11 suppressed the gmd3 mutation, indicating that gmd3(+) gene is a functional homologue of the ALG11 gene. We therefore designated gmd3(+) as alg11(+).

Coexpression of alpha1,2 galactosyltransferase and UDP-galactose transporter efficiently galactosylates N- and O-glycans in Saccharomyces cerevisiae.

We have studied in vivo neo-galactosylation in Saccharomyces cerevisiae and analyzed the critical factors involved in this system. Two heterologous genes, gma12(+) encoding alpha1, 2-galactosyltransferase (alpha1,2 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 alpha1,2 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 alpha1,2 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 Galalpha1,2-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.

Differences in in vivo acceptor specificity of two galactosyltransferases, the gmh3+ and gma12+ gene products from Schizosaccharomyces pombe.

In the fission yeast Schizosaccharomyces pombe, gmh1+, gmh2+ and gmh3+ genes encode alpha-1,2-galactosyltransferase homologues. In an in vitro galactosyltransferase assay, the gmh3+ gene product showed galactose transfer activity toward a-methyl-D-mannoside as an acceptor. The disruption of gma12+, the major galactosyltransferase gene [Chappell, T. G., Hajibagheri, M. A. N., Ayscough, K., Pierce, M. & Warren, G. (1994) Mol. Biol. Cell 5, 519-528], and of gmh3+ in S. pombe caused decreases in the total remaining galactosyltransferase activity and cell surface galactose content. Disruption of gma12+ and gmh3+ also caused an increase in electrophoretic mobility of acid phosphatase, indicating their involvement in the galactosylation of cell surface glycoproteins. The gmh3delta gma12delta double mutant cells had more severe galactose-less phenotypes than single gene mutant cells. HPLC analysis of O-linked mannoprotein oligosaccharides from wild-type and disrupted strains revealed that the gma12+ gene product is responsible for the galactosylation of O-linked oligosaccharide, whereas gmh3+ has no involvement in the process. In contrast, both the gmh3+ and gma12+ gene products are involved in the galactosylation of the N-linked core oligosaccharide Man9GlcNAc2. From these results, it is evident that there are some functional differences between the enzymes in the process of galactosylation. It appears that the gmh3+ gene product transfers galactose to N-linked oligosaccharide, while the gma12+ gene product transfers galactose to both N-linked and O-linked oligosaccharides.

Functional evidence for UDP-galactose transporter in Saccharomyces cerevisiae through the in vivo galactosylation and in vitro transport assay.

The oligosaccharide profiles in glycoproteins are determined by a series of processing reactions catalyzed by Golgi glycosyltransferases and glycosidases. Recently in vivo galactose incorporation in Saccharomyces cerevisiae has been demonstrated through the expression of human beta-1,4-galactosyltransferase in an alg1 mutant, suggesting the presence of a UDP-galactose transporter in S. cerevisiae (Schwientek, T., Narimatsu, H., and Ernst, J. F. (1996) J. Biol. Chem. 271, 3398-3405). However, this is quite unexpected, because S. cerevisiae does not have galactose residues in its glycoproteins. To address this question we have constructed S. cerevisiae mnn1 mutant strains expressing Schizosaccharomyces pombe alpha-1,2-galactosyltransferase. The mnn1 mutant of S. cerevisiae provides endogenous acceptors for galactose transfer by the expressed alpha-1,2-galactosyltransferase. We present here three lines of evidences for the existence of UDP-galactose transporter in S. cerevisiae. (i) About 15-20% of the total transformed mnn1 cells grown in a galactose medium were stained with fluorescein isothiocyanate-conjugated alpha-galactose-specific lectin, indicating the presence of alpha-galactose residues on the cell surface. (ii) Galactomannan proteins can be precipitated with agarose-immobilized alpha-galactose-specific lectin from a whole cell lysate prepared from transformed mnn1 cells grown in a galactose medium. (iii) The presence of UDP-galactose transporter was demonstrated by direct transport assay. This transport in S. cerevisiae is dependent on time, temperature, and protein concentration and is inhibited by nucleotide monophosphate and Triton X-100. The overall UDP-galactose transport in S. cerevisiae is comparable with that in S. pombe, indicating a more or less similar reaction velocity, while the rate of GDP-mannose transport is higher in S. pombe than in S. cerevisiae.

Isolation and characterization of temperature-sensitive plc1 mutants of the yeast Saccharomyces cerevisiae.

The PLC1 gene of the yeast Saccharomyces cerevisiae has been discovered to encode a homolog of mammalian phosphoinositide-specific phospholipase C (PLC). Five temperature-sensitive plc1 mutants were isolated by in vitro mutagenesis with subsequent plasmid shuffling. All of the amino acid substitutions that caused a temperature-sensitive growth phenotype were located in the X or the Y region, both of which are conserved among PLC isoenzymes. The PLC activity of all products of mutant plc1 genes was dramatically lower than that of the wild-type product, indicating that PLC activity itself is important for cell growth. At the restrictive temperature, plc1 mutant cells ceased growth at random times during the cell cycle, a result that suggests that PLC1 is required at several or all stages of the cell cycle.

The putative phosphoinositide-specific phospholipase C gene, PLC1, of the yeast Saccharomyces cerevisiae is important for cell growth.

Using the polymerase chain reaction technique, we have isolated a gene that encodes a putative phosphoinositide-specific phospholipase C (PLC) in the yeast Saccharomyces cerevisiae. The nucleotide sequence indicates that the gene encodes a polypeptide of 869 amino acid residues with a calculated molecular mass of 101 kDa. This polypeptide has both the X and Y regions conserved among mammalian PLC-beta, -gamma, and -delta, and the structure is most similar to that of mammalian PLC-delta. This putative yeast PLC gene has been designated PLC1. Disruption of PLC1 results in slow growth or lethality for cells, depending on their genetic background and the medium, indicating that PLC1 is important for cell growth. Expression of rat PLC-delta 1 cDNA suppressed the growth defect of plc1 disruptants, strongly suggesting that PLC1 encodes PLC.