GOLGI LOCALIZATION AND IN-VIVO ACTIVITY OF A MAMMALIAN GLYCOSYLTRANSFERASE (HUMAN BETA-1,4-GALACTOSYLTRANSFERASE) IN YEAST

Gene fusions encoding the membrane anchor region of yeast alpha 1,2-ma nnosyltransferase (Mnt1p) fused to human beta 1,4-galactosyltransferas e (Gal-Tf) were constructed and expressed in the yeast Saccharomyces c erevisiae, Fusion proteins containing 82 or only 36 N-terminal residue s of Mnt1p were produced and quantitatively N-glycosylated; glycosyl c hains were shown to contain alpha 1,6-, but not alpha 1,3-mannose dete rminants, a structure typical for an early Golgi compartment, A final Golgi localization of both fusions was confirmed by sucrose gradient f ractionations, in which Gal-Tf activity cofractionated with Golgi Mnt1 p activity, as well as by immunocytological localization experiments u sing a monoclonal anti-Gal-Tf antibody, In an in vitro Gal-Tf enzymati c assay the Mnt1/Gal-Tf fusion and soluble human Gal-Tf had comparable K-m values for UDP-Gal (about 45 mu M). To demonstrate in vivo activi ty of the Mnt1/Gal-Tf fusion the encoding plasmids were transformed in an alg1 mutant, which at the non-permissive temperature transfers sho rt (GlcNAc)(2) glycosyl chains to proteins. Using specific lectins the addition of galactose to several yeast proteins in transformants coul d be detected, These results demonstrate that Gal-Tf, a mammalian glyc osyltransferase, is functional in the molecular environment of the yea st Golgi, indicating conservation between yeast and human cells, The i n vivo function of human Gal-Tf indicates that the yeast Golgi is acce ssible for UDP-Gal and suggests strategies for the construction of yea st strains, in which desired glycoforms of heterologous proteins are p roduced.