Glycobiology, Vol 8, 1207-1213, Copyright © 1998 by Society for Glycobiology
JS Rush and CJ Waechter
In the current model for Glc3Man9GlcNAc2-P-P-Dol assembly, Man5GlcNAc2-
P-P-Dol, Man-P-Dol, and Glc-P-Dol are synthesized on the cytoplasmic face
of the ER and diffuse transversely to the lumenal leaflet where the
synthesis of the lipid-bound precursor oligosaccharide is completed. To
establish the topological sites of Glc-P-Dol synthesis and the
lipid-mediated glucosyltransfer reactions involved in
Glc3Man9GlcNAc2-P-P-Dol synthesis in ER vesicles from pig brain, the
trypsin-sensitivity of Glc-P-Dol synthase activity and the Glc-P-
Dol:Glc0-2Man9GlcNAc2-P-P-Dol glucosyltransferases (GlcTases) was examined
in sealed microsomal vesicles. Since ER vesicles from brain do not contain
glucose 6-phosphate (Glc 6-P) phosphatase activity, the latency of the
lumenally oriented, processing glucosidase I/II activities was used to
assess the intactness of the vesicle preparations. Comparative enzymatic
studies with sealed ER vesicles from brain and kidney, a tissue that
contains Glc 6-P phosphatase, demonstrate the reliability of using the
processing glucosidase activities as latency markers for topological
studies with microsomal vesicles from non-gluconeogenic tissues lacking Glc
6-P phosphatase. The results obtained from the trypsin-sensitivity assays
with sealed microsomal vesicles from brain are consistent with a
topological model in which Glc-P-Dol is synthesized on the cytoplasmic face
of the ER, and subsequently utilized by the three Glc-P-Dol-mediated
GlcTases after "flip-flopping" to the lumenal monolayer.
ORIGINAL ARTICLES
Topological studies on the enzymes catalyzing the biosynthesis of Glc-P- dolichol and the triglucosyl cap of Glc3Man9GlcNAc2-P-P-dolichol in microsomal vesicles from pig brain: use of the processing glucosidases I/II as latency markers
Department of Biochemistry, University of Kentucky College of Medicine, Lexington, KY 40536, USA.
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