The co-translational N-glycosylation of many important cell surface receptors, ion channels and lysosomal enzymes is essential for their correct folding, intracellular routing and function in the CNS and other mammalian cells. The vital importance of protein N-glycosylation in humans is emphasized by the clinical consequences of a series of inherited genetic defects in this pathway classified as Congenital Disorders of Glycosylation (CDG). While the enzymology and topology of the biosynthesis of Glc3Man9GlcNAc2-P-P-Dol, the oligosaccharyl donor, in the endoplasmic reticulum (ER) have been studied extensively, many gaps remain in the understanding of how the enzymes in this pathway are organized and regulated. This proposal describes further studies to learn more about the role of ER-associated proteins in the regulation of dolichyl phosphate (Dol-P) biosynthesis and recycling, and the transbilayer movement of Man-P-Dol. ? ? Three Specific Aims are planned using biochemical, genetic and immunochemical approaches designed: 1) To utilize a recently cloned cDNA to learn more about the long-chain c/s-isoprenyltransferase (c/s-IPTase) catalyzing the elongation stage in Dol-P biosynthesis by investigating its regulation and the nature of its association with potential binding partners in the ER; 2) To utilize cloned cDNAs encoding dolichol kinase (DK) and Dol-P-P phosphatase, an ER enzyme with a lumenally-oriented active site, in in vivo and in vitro experiments aimed at elucidating their precise roles in the ote novo synthesis of Dol-P and the recycling of the glycosyl carrier lipid and 3) To purify, identify and characterize the ER protein(s) mediating the transverse diffusion of Man-P-Dol in mammalian cells (""""""""flippase""""""""). The information gained on the structure of the Man-P-Dol flippase will be relevant to related membrane proteins mediating the transbilayer movement of other dolichyl-P-(P)-saccharide intermediates in protein O-, C- and N-glycosylation, glycerophospholipids, glucosylceramide and glycosylphosphatidylinositol (GPI) anchor precursors and potential defects in patients with Congenital Disorders of Glycosylation (CDG). ? ?

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Biochemistry and Biophysics of Membranes Study Section (BBM)
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Marino, Pamela
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University of Kentucky
Schools of Medicine
United States
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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
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 (2005) Assay for the transbilayer movement of polyisoprenoid-linked saccharides based on the transport of water-soluble analogues. Methods 35:316-22
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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