Functional reconstitution into proteoliposomes and partial purification of a rat liver ER transport system for a water-soluble analogue of mannosylphosphoryldolichol.
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.
Rush
Jeffrey S
JS
Department of Molecular and Cellular Biochemistry, University of Kentucky College of Medicine, Lexington, Kentucky 40536, USA.
Waechter
C J
CJ
D000080462:Acyclic Monoterpenes; D000818:Animals; D001692:Biological Transport; D002852:Chromatography, Ion Exchange; D003902:Detergents / Q000737:chemistry / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000737:chemistry / Q000378:metabolism*; D006017:Glycolipids / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D008099:Liver / Q000166:cytology*; D017348:Microscopy, Electron, Scanning Transmission; D039821:Monoterpenes / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D010743:Phospholipids / Q000494:pharmacology; D011510:Proteolipids / Q000737:chemistry* / Q000378:metabolism* / Q000648:ultrastructure; D051381:Rats; D013237:Stereoisomerism; D014357:Trypsin / Q000378:metabolism; D014867:Water / Q000737:chemistry / Q000378:metabolism*
D000080462:Acyclic Monoterpenes; D003902:Detergents; D006017:Glycolipids; D039821:Monoterpenes; D010743:Phospholipids; D011510:Proteolipids; C429286:mannose-phosphate-citronellol; C020485:proteoliposomes; D014867:Water; D014357:Trypsin