Galactosyltransferase functions in yeast

Tilo Schwientek schwient at
Thu Dec 21 19:09:07 EST 1995

Upcoming paper in JBC Vol.271

We developed the first example of in vivo-function of a mammalian
glycosyltransferase in S.cerevisiae.

Paper summary :

Gene fusions encoding the membrane anchor region of yeast
alpha-1,2-mannosyltransferase (Mnt1p) fused to human
=DF-1,4-galactosyltransferase (Gal-Tf) were constructed and expressed in the
yeast Saccharomyces cerevisiae. Fusion proteins containing 82 or only 36
N-terminal residues of Mnt1p were produced and quantitatively
N-glycosylated; glycosyl chains were shown to contain alpha-1,6-, but not
alpha-1,3-mannose determinants, a structure typical for an early Golgi
compartment. A final Golgi localization of both fusions was confirmed by
sucrose gradient fractionations, in which Gal-Tf activity cofractionated
with Golgi Mnt1p activity, as well as by immunocytological localization
experiments using a monoclonal anti-Gal-Tf antibody. In an in vitro Gal-Tf
enzymatic assay the Mnt1/Gal-Tf fusion and soluble human Gal-Tf had
comparable Km values for UDP-Gal (about 45 =B5M). To demonstrate in vivo
activity of the Mnt1/Gal-Tf fusion the encoding plasmids were transformed in
an alg1 mutant, which at the non-permissive temperature transfers short
(GlcNAc)2 glycosyl chains to proteins. Using specific lectins the addition
of galactose to several yeast proteins in transformants could be detected.
These results demonstrate that Gal-Tf, a mammalian glycosyltransferase, is
functional in the molecular environment of the yeast Golgi, indicating
conservation between yeast and human cells. The in vivo function of human
Gal-Tf indicates that the yeast Golgi is accessible for UDP-Gal and suggests
strategies for the construction of yeast strains, in which desired
glycoforms of heterologous proteins are produced.

Inquiries welcome to schwient at

More information about the Glycosci mailing list