Golgi localization in yeast is mediated by the membrane anchor region of rat liver sialyltransferase.
To investigate the function of the membrane anchor region of a mammalian glycosyltransferase in yeast we constructed a fusion gene that encodes the 34 amino-terminal residues of rat liver beta-galactoside alpha-2,6-sialyl-transferase (EC 2.4.99.1) (ST) fused to the mature form of yeast invertase. Transformants of Saccharomyces cerevisiae expressing the fusion gene produced an intracellular heterogeneously N-glycosylated fusion protein of intermediate molecular weight between the core and fully extended N-glycosylated form of invertase, suggesting a post-endoplasmic reticulum (ER) localization. In two types of cell fractionation using sucrose density gradients the ST-invertase fusion protein cofractionated with Golgi marker proteins, whereas a minor fraction (about 30%) comigrated with a vacuolar marker; ST-invertase was not detected in other cell fractions including the ER and the plasma membrane. Consistent with Golgi localization, about 70% of the total amount of the ST-invertase fusion was immunoprecipitated with an antibody directed against alpha-1,6-mannose linkages. The results demonstrate that the membrane anchor region of a mammalian type II glycosyltransferase is able to target a protein to the secretory pathway and to a Golgi compartment of the yeast S. cerevisiae, indicating conservation of targeting mechanisms between higher and lower eukaryotes. Since typical yeast Golgi localization signals are missing in the ST-membrane anchor region the results also suggest that yeast as mammalian cells utilize diverse mechanisms to direct proteins to the Golgi.
Schwientek
T
T
Institut für Mikrobiologie, Heinrich-Heine-Universität Düsseldorf, Federal Republic of Germany.
Lorenz
C
C
Ernst
J F
JF
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002499:Centrifugation, Density Gradient; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005822:Genetic Vectors; D006026:Glycoside Hydrolases / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D006031:Glycosylation; D006056:Golgi Apparatus / Q000378:metabolism*; D015151:Immunoblotting; D008099:Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D010957:Plasmids; D011499:Protein Processing, Post-Translational; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D012799:Sialyltransferases / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D013104:Spheroplasts / Q000378:metabolism; D043324:beta-Fructofuranosidase; D000094802:beta-D-Galactoside alpha 2-6-Sialyltransferase
D011993:Recombinant Fusion Proteins; D012799:Sialyltransferases; D006026:Glycoside Hydrolases; D043324:beta-Fructofuranosidase; D000094802:beta-D-Galactoside alpha 2-6-Sialyltransferase