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. Particular emphasis will be placed on (i) a biochemical and molecular characterization of the mammalian and fungal oligosaccharyltransferases, (ii) the identification and characterization of Saccharomyces cerevisiae gene products that functionally interact with the oligosaccharyltransferase and (iii) an examination of the mechanism of transbilayer transport of lipid-linked oligosaccharides. Oligosaccharyltransferase isolated from both mammalian and yeast microsomal membranes will be characterized using a combination of biochemical, molecular and cellular biological approaches. The amino acid sequence of the subunits of the yeast oligosaccharyltransferase will be determined by the isolation and sequencing of genomic clones. The function of the yeast oligosaccharyltransferase subunits will be evaluated in vivo by the analysis of conditional mutants. The spatial and temporal relationships between the protein translocation machinery and the protein glycosylation apparatus will be evaluated in S. cerevisiae. Yeast gene products that interact with the oligosaccharyltransferase will be identified by genetic screens for high-copy suppressors of an oligosaccharyltransferase mutant. 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 transported across the membrane for subsequent elongation to Glc3Man9GlcNA2-PP-Dol. The membrane topology and transbilayer transport of lipid-linked oligosaccharides will be investigated using microsomal membranes from S. cerevisiae. Translocation of Man5GlcNAc2-PP-dolichol will be investigated in vitro using endogenous and de novo synthesized lipid-linked oligosaccharides as oligosaccharide donors and synthetic tripeptides as oligosaccharide acceptors. Experimental perturbations that interfere with lipid-linked oligosaccharide transport will be used as tools to investigate the transport process. The objective of these studies is to determine whether transport of lipid-linked oligosaccharides is a protein-mediated or spontaneous process., Carbohydrate deficient glycoprotein syndrome (CDGS) is a recently described multi-systemic human disease that appears to be caused by pleiotropic underglycosylation of newly synthesized proteins in the endoplasmic reticulum. The lesion responsible for CDGS has not been defined, but could involve a defect in the assembly of the lipid-linked oligosaccharide donor or a defect in the oligosaccharyltransferase. Thus, the research described in this proposal is of direct relevance to a human disease.
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