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

Abstract The oligosaccharide profiles in glycoproteins are determined by a series of processing reactions catalyzed by Golgi glycosyltransferases and glycosidases. Recently in vivogalactose incorporation in Saccharomyces cerevisiae has been demonstrated through the expression of human β-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 pombeα-1,2-galactosyltransferase. The mnn1 mutant of S. cerevisiae provides endogenous acceptors for galactose transfer by the expressed α-1,2-galactosyltransferase. We present here three lines of evidences for the existence of UDP-galactose transporter inS. cerevisiae. (i) About 15–20% of the total transformedmnn1 cells grown in a galactose medium were stained with fluorescein isothiocyanate-conjugated α-galactose-specific lectin, indicating the presence of α-galactose residues on the cell surface. (ii) Galactomannan proteins can be precipitated with agarose-immobilized α-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. cerevisiaeis 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.