Protein N-glycosylation is a critical stage in the assembly of membrane glycoproteins mediating important neurobiological functions in the CNS. N-linked oligosaccharide chains are initially synthesized as the lipid-linked precursor oligosaccharide, Glc3Man9GlcNAc2-P-P-dolichol (Oligo-P-P-Dol) by a multi-stage assembly process that begins with the formation of Man5GlcNAc2- P-P-Dol, mannosylphosphoryidolichol (Man-P-Dol) and glucosylphosphoryldolichol (Glc-P-Dol) on the cytoplasmic face of the endoplasmic reticulum (ER) with sugar nucleotides serving as the glycosyl donors. Biosynthesis is completed after the polar phosphosaccharide head groups of the three lipid intermediates diffuse transversely to the lumenal monolayer. Although many details of the biosynthesis of the glycosyl carrier lipid, dolichyl phosphate (Dol-P), and Oligo-P-P-Dol have been elucidated, there are still many crucial gaps in the understanding of the number and regulation of the enzymes involved in the biosynthesis of Dol-P de novo and the transbilayer movement of three lipid intermediates. Since it has been established that increased levels of the long chain cis-isoprenyltransferase (cis-lPTase) system, catalyzing the chain elongation stage in Dol-P biosynthesis, is a key regulatory event in the induction of the N-glycosylation apparatus in developing brain cells, this application proposes extensive new enzymological and molecular studies on the cis-lPTase system. Another major aspect of the application addresses basic questions about the membrane proteins postulated to mediate the transbilayer movement of Man-P-Dol, Glc-P-Dol and Man5GlcNAc2-P-P-Dol in the ER. This part of the project extends the finding that Man-P-citronellol (Man-P-Cit), a water-soluble analogue of Man-P-Dol, is transported into sealed ER vesicles by a protein-mediated system. A key objective is to establish that the protein(s) mediating Man-P-Cit transport is related to Man-P-Dol """"""""flippase"""""""", and to explore the use of other pertinent water-soluble analogues for similar studies. The proposed experiments will provide important new information about the structure and regulation of the cis-lPTase system in brain, and contribute to the understanding of the mechanisms by which polar lipids diffuse transversely in the ER, a fundamentally important problem in membrane biology.
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