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.
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