Golgi localization and in vivo activity of a mammalian glycosyltransferase (human beta1,4-galactosyltransferase) in yeast.
Gene fusions encoding the membrane anchor region of yeast alpha1, 2-mannosyltransferase (Mnt1p) fused to human beta1, 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 alpha1,6-, but not alpha1,3-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 microM). 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.
Schwientek
T
T
Institut für Mikrobiologie, Heinrich-Heine-Universität Düsseldorf, D-40225 Düsseldorf, Germany.
Narimatsu
H
H
Ernst
J F
JF
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002458:Cell Fractionation; D002499:Centrifugation, Density Gradient; D003001:Cloning, Molecular; D017931:DNA Primers; D006056:Golgi Apparatus / Q000201:enzymology*; D006801:Humans; D007700:Kinetics; D008322:Mammals; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009237:N-Acetyllactosamine Synthase / Q000096:biosynthesis* / Q000378:metabolism; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D013698:Templates, Genetic
D017931:DNA Primers; D011994:Recombinant Proteins; D009237:N-Acetyllactosamine Synthase