GM1 synthase depends on N-glycosylation for enzyme activity and trafficking to the Golgi complex.
Glycosyltransferase cDNAs contain a variable number of potential N-glycosylation sites. Here we examined the occupancy and relevance for the activity and intracellular trafficking of the only potential N-glycosylation site of the mouse beta1,3galactosyltransferase (Gal-T2 or GA1/GM1/GD1b synthase) in Gal-T2 cDNA transfected CHO-K1 cells. Transfected cells synthesize a Golgi located active enzyme of 43 kDa whose N-glycan was metabolically labeled from [3H]mannose and was Endo-H sensitive. Inhibition of N-glycosylation by Tunicamycin or by point mutation of the N-glycosylation site resulted in the synthesis of a polypeptide of 40 kDa which lacked enzyme activity and was concentrated in the endoplasmic reticulum (ER). Inhibition of ER glucosidases by Castanospermine impaired the exit of a form of Gal-T2 having reduced enzyme activity from the ER. The N-terminal Gal-T2 domain (aa 1-52) was able to direct and to retain the green fluorescence protein in the Golgi complex. Taken together, these results indicate that Gal-T2 depends on N-glycosylation for its activity and for proper trafficking to, but not its retention in, the Golgi complex.
Martina
J A
JA
Centro de Investigaciones en Química Biológica de Córdoba, CIQUIBIC (UNC-CONICET), Departamento de Química Biológica Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Argentina.
Daniotti
J L
JL
Maccioni
H J
HJ
D000818:Animals; D016466:CHO Cells; D003001:Cloning, Molecular; D006224:Cricetinae; D004721:Endoplasmic Reticulum / Q000201:enzymology; D005700:Galactosyltransferases / Q000378:metabolism*; D051147:Ganglioside Galactosyltransferase; D005959:Glucosidases / Q000037:antagonists & inhibitors / Q000378:metabolism; D006031:Glycosylation; D006056:Golgi Apparatus / Q000201:enzymology*; D007212:Indolizines / Q000494:pharmacology; D007700:Kinetics; D008358:Mannose / Q000378:metabolism; D051379:Mice; D021381:Protein Transport; D011994:Recombinant Proteins / Q000378:metabolism; D014162:Transfection; D014415:Tunicamycin / Q000494:pharmacology; D054836:Indolizidines
D005700:Galactosyltransferases; D051147:Ganglioside Galactosyltransferase; D005959:Glucosidases; D007212:Indolizines; D008358:Mannose; D011994:Recombinant Proteins; D014415:Tunicamycin; C037806:castanospermine; D054836:Indolizidines