Differential effects of lobe A and lobe B of the Conserved Oligomeric Golgi complex on the stability of {beta}1,4-galactosyltransferase 1 and {alpha}2,6-sialyltransferase 1.
Initially described by Jaeken et al. in 1980, congenital disorders of glycosylation (CDG) is a rapidly expanding group of human multisystemic disorders. To date, many CDG patients have been identified with deficiencies in the conserved oligomeric Golgi (COG) complex which is a complex involved in the vesicular intra-Golgi retrograde trafficking. Composed of eight subunits that are organized in two lobes, COG subunit deficiencies have been associated with Golgi glycosylation abnormalities. Analysis of the total serum N-glycans of COG-deficient CDG patients demonstrated an overall decrease in terminal sialylation and galactosylation. According to the mutated COG subunits, differences in late Golgi glycosylation were observed and led us to address the question of an independent role and requirement for each of the two lobes of the COG complex in the stability and localization of late terminal Golgi glycosylation enzymes. For this, we used a small-interfering RNAs strategy in HeLa cells stably expressing green fluorescent protein (GFP)-tagged β1,4-galactosyltransferase 1 (B4GALT1) and α2,6-sialyltransferase 1 (ST6GAL1), two major Golgi glycosyltransferases involved in late Golgi N-glycosylation. Using fluorescent lectins and flow cytometry analysis, we clearly demonstrated that depletion of both lobes was associated with deficiencies in terminal Golgi N-glycosylation. Lobe A depletion resulted in dramatic changes in the Golgi structure, whereas lobe B depletion severely altered the stability of B4GALT1 and ST6GAL1. Only MG132 was able to rescue their steady-state levels, suggesting that B4GALT1- and ST6GAL1-induced degradation are likely the consequence of an accumulation in the endoplasmic reticulum (ER), followed by a retrotranslocation into the cytosol and proteasomal degradation. All together, our results suggest differential effects of lobe A and lobe B for the localization/stability of B4GALT1 and ST6GAL1. Lobe B would be crucial in preventing these two Golgi glycosyltransferases from inappropriate retrograde trafficking to the ER, whereas lobe A appears to be essential for maintaining the overall Golgi structure.
Unité de Glycobiologie Structurale et Fonctionnelle UMR/CNRS 8576, IFR147, Université des Sciences et Technologies de Lille, Villeneuve d'Ascq, France.
Romain
R
Peanne
Dominique
D
Legrand
Sandrine
S
Duvet
Anne-Marie
AM
Mir
Gert
G
Matthijs
Jack
J
Rohrer
François
F
Foulquier
D033942:Adaptor Proteins, Vesicular Transport / Q000235:genetics / Q000378:metabolism*; D015703:Antigens, CD / Q000235:genetics / Q000378:metabolism*; D015153:Blotting, Western; D003600:Cytosol / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D005700:Galactosyltransferases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D006031:Glycosylation; D006056:Golgi Apparatus / Q000737:chemistry / Q000502:physiology*; D006367:HeLa Cells; D006801:Humans; D007124:Immunoenzyme Techniques; D021381:Protein Transport; D012333:RNA, Messenger / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012799:Sialyltransferases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D000094802:beta-D-Galactoside alpha 2-6-Sialyltransferase
D033942:Adaptor Proteins, Vesicular Transport; D015703:Antigens, CD; D005700:Galactosyltransferases; D012333:RNA, Messenger; D012799:Sialyltransferases; C498528:COG3 protein, human; C512294:COG7 protein, human; C119701:beta-1,4-galactosyltransferase I; C093493:ST6GAL1 protein, human; D000094802:beta-D-Galactoside alpha 2-6-Sialyltransferase