title: Uptake and incorporation of an epitope-tagged sialic acid donor into intact rat liver Golgi compartments. Functional localization of sialyltransferase overlaps with beta-galactosyltransferase but not with sialic acid O-acetyltransferase.
abstract: The transfer of sialic acids (Sia) from CMP-sialic acid (CMP-Sia) to N-linked sugar chains is thought to occur as a final step in their biosynthesis in the trans portion of the Golgi apparatus. In some cell types such Sia residues can have O-acetyl groups added to them. We demonstrate here that rat hepatocytes express 9-O-acetylated Sias mainly at the plasma membranes of both apical (bile canalicular) and basolateral (sinusoidal) domains. Golgi fractions also contain 9-O-acetylated Sias on similar N-linked glycoproteins, indicating that O-acetylation may take place in the Golgi. We show here that CMP-Sia-FITC (with a fluorescein group attached to the Sia) is taken up by isolated intact Golgi compartments. In these preparations, Sia-FITC is transferred to endogenous glycoprotein acceptors and can be immunochemically detected in situ. Addition of unlabeled UDP-Gal enhances Sia-FITC incorporation, indicating a substantial overlap of beta-galactosyltransferase and sialyltransferase machineries. Moreover, the same glycoproteins that incorporate Sia-FITC also accept [3H]galactose from the donor UDP-[3H]Gal. In contrast, we demonstrate with three different approaches (double-labeling, immunoelectron microscopy, and addition of a diffusible exogenous acceptor) that sialyltransferase and O-acetyltransferase machineries are much more separated from one another. Thus, 9-O-acetylation occurs after the last point of Sia addition in the trans-Golgi network. Indeed, we show that 9-O-acetylated sialoglycoproteins are preferentially segregated into a subset of vesicular carriers that concentrate membrane-bound, but not secretory, proteins.
authors.affiliation: Glycobiology Program, University of California, San Diego, Cancer Center, La Jolla 92093, USA.
authors.forename: R
authors.initials: R
authors.lastname: Chammas
authors.forename: J M
authors.initials: JM
authors.lastname: McCaffery
authors.forename: A
authors.initials: A
authors.lastname: Klein
authors.forename: Y
authors.initials: Y
authors.lastname: Ito
authors.forename: L
authors.initials: L
authors.lastname: Saucan
authors.forename: G
authors.initials: G
authors.lastname: Palade
authors.forename: M G
authors.initials: MG
authors.lastname: Farquhar
authors.forename: A
authors.initials: A
authors.lastname: Varki
mesh_terms: D000107:Acetylation; D000123:Acetyltransferases / Q000378:metabolism*; D000818:Animals; D002458:Cell Fractionation; D002462:Cell Membrane / Q000378:metabolism; D003569:Cytidine Monophosphate N-Acetylneuraminic Acid / Q000378:metabolism; D000939:Epitopes; D016650:Fluorescein-5-isothiocyanate; D005690:Galactose / Q000378:metabolism; D005700:Galactosyltransferases / Q000378:metabolism*; D006001:Glycoconjugates / Q000378:metabolism; D006023:Glycoproteins / Q000378:metabolism; D006027:Glycosides / Q000378:metabolism; D006056:Golgi Apparatus / Q000201:enzymology / Q000378:metabolism*; D006923:Hymecromone / Q000031:analogs & derivatives / Q000378:metabolism; D008099:Liver / Q000166:cytology / Q000378:metabolism; D051381:Rats; D012794:Sialic Acids / Q000378:metabolism*; D012799:Sialyltransferases / Q000378:metabolism*; D014531:Uridine Diphosphate Galactose / Q000378:metabolism
chemical_list: C102546:4-methylumbelliferyl lactoside; D000939:Epitopes; D006001:Glycoconjugates; D006023:Glycoproteins; D006027:Glycosides; D012794:Sialic Acids; D014531:Uridine Diphosphate Galactose; D003569:Cytidine Monophosphate N-Acetylneuraminic Acid; D006923:Hymecromone; D000123:Acetyltransferases; C099970:sialic acid O-acetyltransferase; D005700:Galactosyltransferases; D012799:Sialyltransferases; D016650:Fluorescein-5-isothiocyanate; D005690:Galactose