Transbilayer movement of Glc-P-dolichol and its function as a glucosyl donor: Protein-mediated transport of a water-soluble analog into sealed ER vesicles from pig brain Journal Article
| Authors: | Rush, J. S.; van Leyen, K.; Ouerfelli, O.; Wolucka, B.; Waechter, C. J. |
| Article Title: | Transbilayer movement of Glc-P-dolichol and its function as a glucosyl donor: Protein-mediated transport of a water-soluble analog into sealed ER vesicles from pig brain |
| Abstract: | The results described in the accompanying article support the model in which glucosylphosphoryldolichol (Glc-P-Dol) is synthesized on the cytoplasmic face of the ER, and functions as a glucosyl donor for three Glc-P-Dol:Glc0-2Man9-GlcNAc2P-P-Dol glucosyltransferases (GlcTases) in the lumenal compartment. In this study, the enzymatic synthesis and structural characterization by NMR and electrospray-ionization tandem mass spectrometry of a series of water-soluble β-Glc-P-Dol analogs containing 2-4 isoprene units with either the cis- or trans-stereoconfiguration in the β-position are described. The water-soluble analogs were (1) used to examine the stereospecificity of the Glc-P-Dol:Glc0-2Man9GlcNAc2-P-P-Dol glucosyltransferases (GlcTases) and (2) tested as potential substrates for a membrane protein(s) mediating the transbilayer movement of Glc-P-Dol in sealed ER vesicles from rat liver and pig brain. The Glc-P-Dol-mediated GlcTases in pig brain microsomes utilized [3H]Glc-labeled Glc-P-Dol10, Glc-P-(ω,c)Dol15, Glc-P(ω,t,t)Dol20, and Glc-P-(ω,t,c)Dol20 as glucosyl donors with [3H]Glc3Man9GlcNAc2-P-P-Dol the major product labeled in vitro. A preference was exhibited for C15-20 substrates containing an internal cis-isoprene unit in the β-position. In addition, the water-soluble analog, Glc-P-Dol10, was shown to enter the lumenal compartment of sealed microsomal vesicles from rat liver and pig brain via a protein-mediated transport system enriched in the ER. The properties of the ER transport system have been characterized. Glc-P-Dol10 was not transported into or adsorbed by synthetic PC-liposomes or bovine erythrocytes. The results of these studies indicate that (1) the internal cis-isoprene units are important for the utilization of Glc-P-Dol as a glucosyl donor and (2) the transport of the water-soluble analog may provide an experimental approach to assay the hypothetical 'flippase' proposed to mediate the transbilayer movement of Glc-P-Dol from the cytoplasmic face of the ER to the lumenal monolayer. |
| Keywords: | controlled study; unclassified drug; nonhuman; mass spectrometry; animal cell; animals; protein; animalia; endoplasmic reticulum; liver; molecular sequence data; kinetics; brain; membrane protein; rat; protein transport; nuclear magnetic resonance spectroscopy; cytoplasm; tandem mass spectrometry; glucose; cattle; erythrocyte; rats; membrane transport; stereospecificity; swine; biological transport; carbohydrate sequence; liposome; liposomes; lipid bilayers; bilayer membrane; er; enzyme synthesis; erythrocytes; microsomes, liver; solubility; cell vacuole; oligosaccharides; bovinae; bos taurus; priority journal; article; sus scrofa; glucosyltransferase; isoprene; 'flippase'; glc-p-dol synthesis; dolichol; dolichol phosphate glucose; brain microsome; glucosyltransferases; polyisoprenyl phosphate monosaccharides |
| Journal Title: | Glycobiology |
| Volume: | 8 |
| Issue: | 12 |
| ISSN: | 0959-6658 |
| Publisher: | Oxford University Press |
| Date Published: | 1998-12-01 |
| Start Page: | 1195 |
| End Page: | 1205 |
| Language: | English |
| DOI: | 10.1093/glycob/8.12.1195 |
| PUBMED: | 9858641 |
| PROVIDER: | scopus |
| DOI/URL: | |
| Notes: | Article -- Export Date: 12 December 2016 -- Source: Scopus |
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