Abstract
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.
Highlights
N-Linked glycosylation is an essential modification that is highly conserved among all eukaryotic cells [1,2,3,4]
Yeast offers an attractive host for the production of heterologous proteins [6], and a number of recombinant glycoproteins were successfully produced in S. cerevisiae, the sugars attached to proteins were confined to high mannose type
The in vivo galactose addition was demonstrated by expressing the genes of mammalian -1,4-galactosyltransferase (GalT)1 in S. cerevisiae, which suggests the presence of unexpected UDP-Gal transporter, allowing -1,4-GalT to act inside the lumen of the Golgi to add galactose onto endogenous acceptors under nonpermissive growth conditions [11]
Summary
GalT, galactosyltransferase; GS I-B4, G. simplicifolia I lectin B4; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; FITC, fluorescein isothiocyanate; FACS, fluorescence-activated cell sorting. At present it remains unsolved without further evidence that this transport activity is from a specific UDP-Gal transporter or other sugar nucleotide transporter having a broader substrate specificity This finding is promising for the achievement of our goal for the incorporation of galactose residues into sugar chain of the glycoproteins in S. cerevisiae, when the appropriate acceptor glycans are provided
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