Localization of an alpha 1,2 galactosyltransferase activity to the Golgi apparatus of Schizosaccharomyces pombe.
We have cloned a gene encoding an alpha 1,2 galactosyltransferase activity from Schizosaccharomyces pombe. The open reading frame of the gene (gma12 for galactomannan, alpha 1,2), combined with the previous protein purification (Chappell and Warren, 1989), predicts an O-linked glycoprotein with type II transmembrane topology. By homologous gene disruption, we have demonstrated that the gma12 gene product (gma12p) is nonessential. The deletion strain (gma12-D10::ura4) has a significantly reduced level of galactosyltransferase activity relative to the parental strain, but both in situ lectin binding and in vitro biochemical assays demonstrate the presence of further galactosyltransferase activity in addition to gma12p. Although gma12p is not the only galactosyltransferase in S. pombe, it produces a unique carbohydrate structure on the surface of the yeast cells. We have generated a polyclonal antiserum against this carbohydrate epitope and shown that gma12p is capable of synthesizing the epitope both in vitro and in vivo. Electron microscopic localization of the gma12+ specific epitope in gma12+ cells revealed that gma12p synthesizes the carbohydrate structure in the Golgi apparatus, and subsequent intracellular transport distributes the epitope to later stages of the secretory pathway. The immunolocalization studies confirm the presence of one or more galactosyltransferase activities in the Golgi apparatus in fission yeast.
Chappell
T G
TG
Imperial Cancer Research Fund, London, United Kingdom.
Hajibagheri
M A
MA
Ayscough
K
K
Pierce
M
M
Warren
G
G
D000595:Amino Acid Sequence; D000946:Antigens, Fungal / Q000378:metabolism; D001483:Base Sequence; D050260:Carbohydrate Metabolism; D002241:Carbohydrates / Q000276:immunology; D003001:Cloning, Molecular; D004271:DNA, Fungal / Q000235:genetics; D000939:Epitopes / Q000378:metabolism; D005700:Galactosyltransferases / Q000235:genetics / Q000378:metabolism*; D005800:Genes, Fungal; D006056:Golgi Apparatus / Q000201:enzymology* / Q000648:ultrastructure; D007150:Immunohistochemistry; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D015183:Restriction Mapping; D012568:Schizosaccharomyces / Q000201:enzymology* / Q000235:genetics / Q000276:immunology
D000946:Antigens, Fungal; D002241:Carbohydrates; D004271:DNA, Fungal; D000939:Epitopes; D005700:Galactosyltransferases