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 Glc3Man9GlcNAc 2-P-P-Dol after being translocated to the lumenal leaflet. An assay, based on the transport of Man-P-citronellol (Man-P-Dol10), a water-soluble analogue of Man-P-Dol95, 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-Dol10 transporter, and as an initial step toward purification of the protein(s) involved in the transport of Man-P-Dol10, the activity has been solubilized from rat liver microsomes with n-octyl-β-D-glucoside and reconstituted into proteoliposomes (∼0.1 μm in diameter). The properties of the reconstituted Man-P-Dol10 transport system are similar to the Man-P-Dol10 uptake activity in microsomal vesicles from rat liver. Man-P-Dol10 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.
|Number of pages||10|
|State||Published - Jun 15 2004|
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