The research described here is focused upon asparagine-linked glycosylation of newly synthesized proteins in the rough endoplasmic reticulum. Particular emphasis will be placed on (i) the isolation of the oligosaccharyltransferase, (ii) biochemical and molecular characterization of the oligosaccharyltransferase and (iii) examination of the mechanism of transbilayer transport of lipid-linked oligosaccharides. The long term objective of this project is to provide insight into the structural and functional organization of the asparagine linked glycosylation apparatus of the rough endoplasmic reticulum. Oligosaccharyltransferase will be isolated from both pancreatic and yeast microsomal membranes. The purified protein will be characterized using a combination of biochemical, molecular and cellular biological approaches. The role of the glycosylation site binding protein (GSBP) in oligosaccharide attachment will be examined using purified preparations of the oligosaccharyltransferase and the glycosylation site binding protein. The membrane content and intracellular location of the enzyme will be determined by probing Western blots with oligosaccharyltransferase-specific antibodies. The amino acid sequence of the oligosaccharyltransferase will be determined by the isolation and sequencing of cDNA clones from yeast and canine pancreas cDNA libraries. Sequence analysis of the two enzymes should reveal conserved regions of functional significance, sorting sequences and transmembrane spanning segments. Reconstitution of the protein into phospholipid vesicles containing lipid-linked oligosaccharides will be used to investigate substrate recognition. Biosynthetic intermediates in the assembly of lipid-linked oligosaccharide have been shown to be asymmetrically distributed between the cytoplasmic and lumenal faces of the rough endoplasmic reticulum membrane. The largest intermediate detected upon the cytoplasmic face of mammalian microsomal membranes (Man5GlcNAc2-PP- dolichol) is apparently transported across the membrane for subsequent elongation to Glc3Man9GlcNAc2-PP-Dol. The membrane topology and transbilayer transport of lipid-linked oligosaccharides will be investigated using microsomal membranes from S. cerevisiae as a model system. Translocation of Man5GlcNAc2-PP-dolichol will be investigated in vitro using de novo synthesized lipid-linked oligosaccharide and synthetic tripeptides as oligosaccharide acceptors. Specific intermediates in assembly and transport will be trapped by perturbation of the normal assembly process. Man5GlcNAc2-PP-dolichol will be incorporated into the cytoplasmic face of microsomal membranes so that transport can be monitored under conditions where de novo synthesis of lipid-linked oligosaccharide need not be maintained. The objective of these studies is to determine whether transport of lipid-linked oligosaccharides is a protein mediated or spontaneous process.
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