GM1 synthase depends on N-glycosylation for enzyme activity and trafficking to the Golgi complex

Glycosyltransferase cDNAs contain a variable number of potential N-glycosyl ation sites. Here we examined the occupancy and relevance for the activity and intracellular trafficking of the only potential N-glycosylation site of the mouse beta 1,3galactosyltransferase (Gal-T2 or GA1/GM1/GD1b synthase) in Gal-T2 cDNA transfected CHO-K1 cells. Transfected cells synthesize a Gol gi located active enzyme of 43 kDa whose N-glycan was metabolically labeled from [H-3]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 glucos idases by Castanospermine impaired the exit of a form of Gal-T2 having redu ced 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 c omplex. Taken together, these results indicate that Gal-T2 depends on N-gly cosylation for its activity and for proper trafficking to, but not its rete ntion in, the Golgi complex.