title: Steady-state localization of a medial-Golgi glycosyltransferase involves transit through the trans-Golgi network.
abstract: The steady-state localization of medial-Golgi enzymes is likely to involve retrograde transport pathways; however, the trafficking of these resident enzymes through the Golgi stack is unclear. To investigate if the medial-Golgi enzyme beta-1,2-N-acetylglucosaminyltransferase I (GlcNAc-TI) is transported to the late Golgi, a modified GlcNAc-TI bearing an N-glycan site on the C-terminus was constructed. The modified GlcNAc-TI was demonstrated to be functionally active in vivo, and was localized to the Golgi stack of transfected cells. In stable Chinese-hamster ovary (CHO) cell clones, the N-glycosylated GlcNAc-TI carried sialylated complex N-glycan chains. Pulse-chase studies showed that the majority of GlcNAc-TI was sialylated within 60 min of synthesis. Treatment of transfected CHO cells with Brefeldin A resulted in the glycosylated GlcNAc-TI bearing endo-beta-N-acetylglucosaminidase H resistant chains; however, the sialylation of glycosylated GlcNAc-TI was dramatically reduced. These data imply that, in CHO cells, newly synthesized GlcNAc-TI is transported rapidly through the Golgi stack to the trans-Golgi network, suggesting that GlcNAc-TI continuously recycles from the late Golgi. Furthermore, this data suggests that retrograde transport pathways play an important role in establishing the asymmetric distribution of GlcNAc-TI within the Golgi stack.
authors.affiliation: Department of Pathology and Immunology, Monash University Medical School, Commercial Road, Prahran, Melbourne, Victoria 3181, Australia.
authors.forename: A S
authors.initials: AS
authors.lastname: Opat
authors.forename: F
authors.initials: F
authors.lastname: Houghton
authors.forename: P A
authors.initials: PA
authors.lastname: Gleeson
mesh_terms: D000581:Amidohydrolases / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D020126:Brefeldin A / Q000494:pharmacology*; D016466:CHO Cells; D002460:Cell Line; D002478:Cells, Cultured; D006224:Cricetinae; D006026:Glycoside Hydrolases / Q000494:pharmacology; D006031:Glycosylation; D016695:Glycosyltransferases / Q000378:metabolism*; D006056:Golgi Apparatus / Q000378:metabolism*; D006596:Hexosaminidases / Q000494:pharmacology; D006801:Humans; D015151:Immunoblotting; D037102:Lectins / Q000378:metabolism; D008361:Mannosidases / Q000378:metabolism; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D019158:N-Acetylneuraminic Acid / Q000494:pharmacology; D009439:Neuraminidase / Q000494:pharmacology; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D011233:Precipitin Tests; D017434:Protein Structure, Tertiary; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology*; D021381:Protein Transport; D013997:Time Factors; D014162:Transfection; D021601:trans-Golgi Network / Q000378:metabolism*
chemical_list: D037102:Lectins; D011500:Protein Synthesis Inhibitors; D020126:Brefeldin A; D016695:Glycosyltransferases; D006026:Glycoside Hydrolases; D006596:Hexosaminidases; D008361:Mannosidases; C038829:mannosyl-oligosaccharide 1,3 - 1,6-alpha-mannosidase; D009439:Neuraminidase; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D019158:N-Acetylneuraminic Acid