title: Golgi-specific localization of transglycosylases engaged in glycoprotein biosynthesis in suspension-cultured cells of sycamore (Acer pseudoplatanus L.).
abstract: Golgi complex and endoplasmic reticulum (ER) were isolated from suspension-cultured cells of sycamore (Acer pseudoplatanus L.) by stepwise sucrose density gradient centrifugation using protoplasts as starting material. The purity of the two organelle fractions isolated was assessed by measuring marker enzyme activities. Localization of glycolipid and glycoprotein glycosyltransferase activities in the isolated Golgi and ER fractions was examined; three glycosyltransferases, i.e., galactosyltransferase, fucosyltransferase, and xylosyltransferase, proved to be almost exclusively confined to the Golgi, whereas the ER fractions contained glycolipid glycosyltransferase. The Golgi complex was further subfractionated on a discontinuous sucrose density gradient into two components, migrating at densities of 1.118 and 1.127 g/cm3. The two fractions differed in their compositional polypeptide bands discernible from Na-dodecylsulfate gel electrophoresis. Galactosyltransferase distributed nearly equally between the two protein peaks and xylosyltransferase activities using the endogenous acceptor also appeared to be localized in the two subcompartments. By contrast, fucosyltransferase, engaged in the terminal stage of glycosylation, banded in the lower density fractions. Golgi-specific alpha-mannosidase, which is presumably engaged in the sugar trimming of Asn-N-linked glycoprotein carbohydrate core, was enriched fourfold in specific activity in the fractions of the higher density. The overall experimental results indicate that the cotranslational glycosylation of Asn-N-linked glycoproteins, e.g., polyphenol oxidase (laccase), takes place in the ER, while subsequent post-translational processing of the oligosaccharide moiety proceeds successively in the two physically separable compartments of the Golgi complex.
mesh_terms: D004721:Endoplasmic Reticulum / Q000201:enzymology*; D005647:Fucosyltransferases / Q000378:metabolism; D005700:Galactosyltransferases / Q000378:metabolism; D006023:Glycoproteins / Q000096:biosynthesis*; D006056:Golgi Apparatus / Q000201:enzymology*; D008364:Mannosyltransferases / Q000378:metabolism; D010430:Pentosyltransferases / Q000378:metabolism; D010940:Plant Proteins / Q000096:biosynthesis*; D010944:Plants / Q000201:enzymology* / Q000648:ultrastructure; D014166:Transferases / Q000378:metabolism*; D000097608:UDP Xylose-Protein Xylosyltransferase
chemical_list: D005647:Fucosyltransferases; D005700:Galactosyltransferases; D006023:Glycoproteins; D008364:Mannosyltransferases; D010430:Pentosyltransferases; D010940:Plant Proteins; D014166:Transferases; D000097608:UDP Xylose-Protein Xylosyltransferase