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

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 faceof the ER and diffuse transversely to the lumenal leaflet where thesynthesis of the lipid-bound precursor oligosaccharide is completed. Toestablish the topological sites of Glc-P-Dol synthesis and thelipid-mediated glucosyltransfer reactions involved inGlc3Man9GlcNAc2-P-P-Dol synthesis in ER vesicles from pig brain, thetrypsin-sensitivity of Glc-P-Dol synthase activity and the Glc-P-Dol:Glc0-2Man9GlcNAc2-P-P-Dol glucosyltransferases (GlcTases) was examinedin sealed microsomal vesicles. Since ER vesicles from brain do not containglucose 6-phosphate (Glc 6-P) phosphatase activity, the latency of thelumenally oriented, processing glucosidase I/II activities was used toassess the intactness of the vesicle preparations. Comparative enzymaticstudies with sealed ER vesicles from brain and kidney, a tissue thatcontains Glc 6-P phosphatase, demonstrate the reliability of using theprocessing glucosidase activities as latency markers for topologicalstudies with microsomal vesicles from non-gluconeogenic tissues lacking Glc6-P phosphatase. The results obtained from the trypsin-sensitivity assayswith sealed microsomal vesicles from brain are consistent with atopological model in which Glc-P-Dol is synthesized on the cytoplasmic faceof the ER, and subsequently utilized by the three Glc-P-Dol-mediatedGlcTases after "flip-flopping" to the lumenal monolayer.