This project will employ three distinct approaches to characterize the reactions in the endoplasmic reticulum of mammals which assemble 14-sugar dolichol pyrophosphate-linked oligosaccharides, the precursors of asparagine-linked oligosaccharides in glycoproteins. These approaches follow directly from the work of the previous funding period. First, a full-length cDNA clone for hamster tunicamycin-sensitive N-acetylglucosamine-l-phosphate transferase (GPT), which adds the first sugar (N-acetylglucosamine-l-phosphate) to the dolichol phosphate carrier, will be used to express and map functional domains on this enzyme. Second, a Chinese hamster ovary (CHO) cell mutant with a possible defect in mannose-P-dolichol translocation will be studied to gain information about mechanisms of dolichol """"""""flipping"""""""" involved in assembly of the dolichol-linked oligosaccharide precursor. Third, a general strategy is being developed to allow the isolation of series of mutant CHO cells, each with a deficiency at a different step in the assembly of the dolichol-linked oligosaccharide. These mutants will facilitate both biochemical and genetic analyses of the proteins involved, many of which have been extremely difficult to study. It is anticipated that the information about the assembly of the asparagine-linked oligosaccharide precursors which will result from this work will provide important clues as to the functions of these carbohydrates, which are widespread among eukaryotes. In addition, general information will be obtained about interactions between membrane-bound enzymes and lipid substrates, and eventually for sorting or """"""""retention signals"""""""" for resident endoplasmic reticulum membrane proteins.
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