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.
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-GlcNAc2-P-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 beta-Glc-P-Dol analogs containing 2-4 isoprene units with either the cis - or trans -stereoconfiguration in the beta-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-(omega, c )Dol15, Glc-P(omega, t,t )Dol20, and Glc-P-(omega, t,c )Dol20as 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 beta-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-Dol10was 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.
Rush
J S
JS
Department of Biochemistry, University of Kentucky College of Medicine, Lexington, KY 40536, USA.
van Leyen
K
K
Ouerfelli
O
O
Wolucka
B
B
Waechter
C J
CJ
D000818:Animals; D001692:Biological Transport / Q000502:physiology; D001921:Brain / Q000201:enzymology / Q000378:metabolism*; D002240:Carbohydrate Sequence; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D004912:Erythrocytes / Q000378:metabolism; D005947:Glucose / Q000378:metabolism*; D005964:Glucosyltransferases / Q000378:metabolism; D007700:Kinetics; D008051:Lipid Bilayers / Q000378:metabolism*; D008081:Liposomes / Q000378:metabolism; D013058:Mass Spectrometry; D008862:Microsomes, Liver / Q000378:metabolism; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000096:biosynthesis / Q000737:chemistry; D011103:Polyisoprenyl Phosphate Monosaccharides / Q000378:metabolism*; D051381:Rats; D012995:Solubility; D013552:Swine
D008051:Lipid Bilayers; D008081:Liposomes; D009844:Oligosaccharides; D011103:Polyisoprenyl Phosphate Monosaccharides; C002968:dolichol-D-glucosylmonophosphate; D005964:Glucosyltransferases; D005947:Glucose