Ja. Martina et al., GM1 synthase depends on N-glycosylation for enzyme activity and trafficking to the Golgi complex, NEUROCHEM R, 25(5), 2000, pp. 725-731
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.