Functional reconstitution into proteoliposomes and partial purification of a rat liver ER transport system for a water-soluble analogue of mannosylphosphoryldolichol.

Abstract

Mannosylphosphoryldolichol (Man-P-Dol) is synthesized on the cytosolic leaflet of the rough endoplasmic reticulum (ER), and functions as a mannosyl donor in the biosynthesis of Glc(3)Man(9)GlcNAc(2)-P-P-Dol after being translocated to the lumenal leaflet. An assay, based on the transport of Man-P-citronellol (Man-P-Dol(10)), a water-soluble analogue of Man-P-Dol(95), into sealed microsomal vesicles, has been devised to identify protein(s) (flippases) that could mediate the thermodynamically unfavorable movement of Man-P-Dol between the two leaflets of the ER. To develop a defined system for the systematic investigation of the properties of the Man-P-Dol(10) transporter, and as an initial step toward purification of the protein(s) involved in the transport of Man-P-Dol(10), the activity has been solubilized from rat liver microsomes with n-octyl-beta-D-glucoside and reconstituted into proteoliposomes (approximately 0.1 microm in diameter). The properties of the reconstituted Man-P-Dol(10) transport system are similar to the Man-P-Dol(10) uptake activity in microsomal vesicles from rat liver. Man-P-Dol(10) transport into reconstituted proteoliposomes is time-dependent, reversible, saturable, and stereoselective. The direct role of ER proteins in the functionally reconstituted transport system is supported by the inhibitory effects of trypsin treatment, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), or diethylpyrocarbonate (DEPC). Solubilization and functional reconstitution are shown to provide an experimental approach to the partial purification of the protein(s) mediating the transport process.