The purpose of this proposal is to explore aspects of the biosyntheses of dolichyl phosphate (DolP), the lipid carrier in glycoprotein biosynthesis, including the regulation of its biosynthesis and its role in modulating glycoprotein biosynthesis rates. These areas will be addressed using as a model system the yeast Saccharomyces cerevisiae. Research will focus on a key enzyme of the DolP biosynthetic pathways, the cis-prenyltransferase (CPT). Initial studies will involve the cloning and sequencing of the yeast CPT gene. Regulation of DolP synthesis will be studied through this use of mutant strains defective in cis- prenyltransferase activity (cpt strains) and genetic deletion experiments employing the cloned CPT gene. Additional experiments will involve transforming of E. coli with a shuttle vector containing the CPT gene in order to prove the identify of the cloned yeast gene and to determine in vivo chain lengths of potential biosynthetic intermediates. These experiments take advantage of the large difference in size of the polyisoprenoid products of the yeast and E. coli enzymes. The role of DolP levels in modulating glycoprotein synthesis rates will be investigated using cell lines with abnormal level of Dolp under producers will be the mutant stains (cpt), while overproducers will employ strains bearing CPT on a multicopy plasmid. In addition, the impact of lowering the free DolP pool on protein glycosylation will be explored though the use of yeast strains transformed with multicopy plasmids bearing gene for one or more of the DolP-dependent glycosyltransferase. Such studies will test the hypothesis that Dolp levels limit the rate of protein glycosylation.
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NIH ExPorter