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An O‐glycosylated protein of approximately 18 kDa responsible for mating type specific agglutination has been isolated from Saccharomyces cerevisiae a cells, purified to homogeneity and via peptide sequences the gene was cloned by PCR. An open reading frame codes for a protein of 69 amino acids. A minimum of five serine and five threonine residues of the mature protein are glycosylated. alpha‐Agglutinin is a highly N‐glycosylated protein of approximately 250 kDa. Both purified agglutinins form a specific 1:1 complex in vitro. Pretreatment of alpha‐agglutinin, but not of alpha‐agglutinin, with diethylpyrocarbonate (DEPC) prevents formation of the complex; treatment of alpha‐agglutinin in the presence of alpha‐agglutinin protects the former from DEPC inactivation. By carboxy terminal shortening of the alpha‐agglutinin gene and by replacing three of its eight histidyl residues by arginine, the active region of alpha‐agglutinin for interaction with alpha‐agglutinin has been defined. Neither the N‐ nor the O‐linked saccharides of the two agglutinins seem to be essential for their interaction.

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A protease-defective strain of Succharomyces cerevisiae (BT 150) was used to express fulllength cDNA of HeLa cell P-D-N-acetylglucosarninide-P-1,4-galactosyltransferase (gal-T). To ascertain import of the recombinant gal-T into the secretory pathway of yeast cells, metabolically labeled enzyme was immunoprecipitated from extracts of yeast transformants, analysed by SDS/PAGE/ fluorography and tested for sensitivity to treatment with endoglycosidase-H. Untreated recombinant gal-T had an apparent molecular mass of 48 kDa, which was reduced to 47 kDa after treatment, indicating that the recombinant enzyme was N-glycosylated and, therefore, competent for translocation across the membranes of the endoplasmic reticulum.Using specific gal-T assays employing N-acetylglucosamine or glucose in combination with alactalbumin as exogenous acceptor substrates, recombinant gal-T enzyme activity could readily be detected in crude homogenates. Analysis of the disaccharide products by 'H-NMR spectroscopy demonstrated that only 8-1-4 linkages were formed by the recombinant gal-T. The recombinant gal-T was detergent solubilized and subsequently purified by affinity chromatography on N-acetylglucosamine-derivatized Sepharose followed by a-lactalbumin-Sepharose. The purified enzyme preparation had a specific activity comparable to that of the soluble gal-T isolated from human milk. Furthermore, kinetic parameters determined for both acceptor and donor substrates of both enzymes differed only slightly.This work shows that yeast provides an appropriate host system for the heterologous expression of mammalian glycosyltransferases. 8-D-N-Acetylglucosaminide-P-1,4-galactosyltransferase(gal-T) is a glycosyltransferase which catalyzes the transfer of galactose from UDP-galactose to GlcNAc or GlcNAc-R, forming a fi-1 + 4 linkage according to the following reaction :UDP-galactose + acceptor + galactose 8-1,4-acceptor + UDP .

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