Previous studies in this laboratory have established that dolichyl phosphate (Dol-P) plays a critical role as a glycosyl carrier lipid in the assembly of N-linked glycoproteins in brain, as in other mammalian tissues, and described several enzymes involved in Dol-P metabolism. This application proposes a continuation of the studies on the mechanistic details and regulation of Dol-P biosynthesis, and the factors controlling the rate of dolichol-linked oligosaccharide intermediate biosynthesis in brain. The major objectives are to: 1) continue developmental studies on the lipid intermediate pathway in cultured embryonic rat brain cells by correlating the developmental patterns for the induction of Dol-P- saccharide intermediate synthesis and for polyisoprenyl diphosphate synthase (long-chain cis-isoprenyltransferase) an activity required for the biosynthesis of Dol-P; 2) elucidate the enzymatic mechanism by which the alpha-isoprene unit of dolichol is reduced and identify the polyisoprenyl substrate and the reductant involved in the reaction; 3) select a temperature-sensitive mutant of dolichol kinase in cultured neuroblastoma cells by in situ colony autoradiography. The kinase-defective mutant will be studied to determine if the enzyme catalyzes the terminal step in the de novo biosynthesis of Dol-P or functions only in the phosphorylation of reserve pools of preformed dolichol during developmental periods when N- linked glycoproteins are actively synthesized; 4) determine if polyisoprenyl diphosphate synthase, polyisoprenyl diphosphate alpha- reductase or dolichol kinase are subject to feedback control by dolichol or Dol-P in cultured neuroblastoma cells; 5) utilize the (C10) citronellyl derivatives, Cit-P-Man and Cit-P, as water-soluble analogues of Dol-P-Man and Dol-P to investigate the potential role of membrane proteins in the transverse diffusion of the mannolipid intermediate between the cytoplasmic leaflet and the lumenal monolayer of the ER and 6) complete the purification of Dol-P phosphatase and dolichol kinase using polyisoprenylated Affi-Gel 10 as a new potential affinity support. The proposed experiments will extend the current understanding of Dol-P biosynthesis and the factors regulating dolichol-bound oligosaccharide biosynthesis in brain. The prospective studies with Cit-P-Man could provide a novel approach to characterize membrane proteins mediating the transbilayer movement of Dol-P-saccharides and Dol-P in brain and other mammalian cells.

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National Institute of General Medical Sciences (NIGMS)
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Neurology C Study Section (NEUC)
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University of Kentucky
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Rush, Jeffrey S (2015) Role of Flippases in Protein Glycosylation in the Endoplasmic Reticulum. Lipid Insights 8:45-53
Harrison, Kenneth D; Park, Eon Joo; Gao, Ningguo et al. (2011) Nogo-B receptor is necessary for cellular dolichol biosynthesis and protein N-glycosylation. EMBO J 30:2490-500
Rush, Jeffrey S; Alaimo, Cristina; Robbiani, Riccardo et al. (2010) A novel epimerase that converts GlcNAc-P-P-undecaprenol to GalNAc-P-P-undecaprenol in Escherichia coli O157. J Biol Chem 285:1671-80
Rush, Jeffrey S; Gao, Ningguo; Lehrman, Mark A et al. (2009) Suppression of Rft1 expression does not impair the transbilayer movement of Man5GlcNAc2-P-P-dolichol in sealed microsomes from yeast. J Biol Chem 284:19835-42
Hartman, Matthew C T; Jiang, Songmin; Rush, Jeffrey S et al. (2007) Glycosyltransferase mechanisms: impact of a 5-fluoro substituent in acceptor and donor substrates on catalysis. Biochemistry 46:11630-8
Rush, Jeffrey S; Waechter, Charles J (2006) Partial purification of mannosylphosphorylundecaprenol synthase from Micrococcus luteus: a useful enzyme for the biosynthesis of a variety of mannosylphosphorylpolyisoprenol products. Methods Mol Biol 347:13-30
Rush, Jeffrey S; Waechter, C J (2005) Assay for the transbilayer movement of polyisoprenoid-linked saccharides based on the transport of water-soluble analogues. Methods 35:316-22
Pakkiri, Leroy S; Waechter, Charles J (2005) Dimannosyldiacylglycerol serves as a lipid anchor precursor in the assembly of the membrane-associated lipomannan in Micrococcus luteus. Glycobiology 15:291-302
Rush, Jeffrey S; Waechter, C J (2004) Functional reconstitution into proteoliposomes and partial purification of a rat liver ER transport system for a water-soluble analogue of mannosylphosphoryldolichol. Biochemistry 43:7643-52
Fernandez, F; Rush, J S; Toke, D A et al. (2001) The CWH8 gene encodes a dolichyl pyrophosphate phosphatase with a luminally oriented active site in the endoplasmic reticulum of Saccharomyces cerevisiae. J Biol Chem 276:41455-64

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