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

Jeffrey S. Rush, C. J. Waechter

Research output: Contribution to journalArticlepeer-review

18 Scopus citations

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 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.

Original languageEnglish
Pages (from-to)7643-7652
Number of pages10
JournalBiochemistry
Volume43
Issue number23
DOIs
StatePublished - Jun 15 2004

ASJC Scopus subject areas

  • Biochemistry

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