The goal of this research is to investigate two separate but related issues pertaining to polyprenois: 1) The topological organization in endoplasmic reticulum (ER) of glucosyl- P-dollchol (Glc-P-Dol) and mannosyl-P-dollchol (Man-P-Dol) and the enzymes that catalyze their synthesis. Two steps in the overall oligosaccharide assembly process that occurs in the ER, the synthesis of Glc-P-Dol and Man-P-Dol, will be studied in yeast. These two reactions were chosen because, although they both are chemically very similar, in rat liver ER the former is believed to involve a sugar nucleotide transporter and occurs in the lumen, whereas the latter is thought to not require a transporter and occurs at the cytoplasmic face. To study their synthesis probes will be developed for the glycose moelty of each lipid intermediate in phospholipid liposomes, and then these will be applied to topology studies in yeast ER. Impermeable probes to be used will be metaperlodate, which will oxidize the sugar residues, and enzymes that will either covalently alter or release the sugar residues. Having established the topological orientation of the glycose moelties of Glc-P- Dol and Man-P-Dol, the orientation of the synthases that catalyze their formation will be studied. To accomplish this in yeast, Glc-P-Dol synthase, which has recently been identified by photoaffinity labelling will be cloned and sequenced. The deducted sequence will be used to interpret studies on the topological orientation of the protein after its 1) in vitro synthesis and insertion in the ER and 2) reconstitution in liposomes. Similar studies will be carried out with yeast Man-P-Dol synthase which recently has been cloned and sequenced. These experiments should allow one to determine if the assembly of Glc-P-Dol and Man-P-Dol involves vectorial translocation of these lipid-linked sugars. 2) The mechanism and function of polyprenol attachment to proteins. The objective of these studies will be to elucidate the mechanism and function of the protein polyprenylation process. The two systems to be utilized are cultured insect cells and developing sea urchin embryos. Preliminary experiments indicate that in vivo incorporation of labeled polyprenyl groups (such as farnesol) into proteins using labeled mevalonic acid as precursor is readily detected in insect cells, presumably because these cells lack the enzyme system that converts farnesol to cholesterol. Pulse-chase labeling experiments will be carried out to study the kinetics of synthesis and turnover of these polyprenylated proteins. In addition, the mechanism of the polyprenylation process will be studied in vitro using apoproteins or synthetic peptides as acceptors, and by varying the structure of the polyprenol donor. Efforts will be made to use exogenous acceptor peptides as substrates so as to inhibit specifically protein polyprenylation in vivo, and thereby obtain insight into the function(s) of this process. Finally, because palmitoylation of proteins has already been demonstrated in the developing sea urchin embryo, which initially undergoes rapid cell proliferation and subsequently extensive cell differentiation, protein polyprenylation will be examined in this system. These studies should define the possible relationship between the biosynthetic pathway for polyprenylated proteins, signal transduction and cell proliferation.
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