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:Glc_0-2Man₉-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:Glc_0-2Man₉GlcNAc₂-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.