Assay to Measure Scrambling of Dolichol-Linked Oligosaccharide in Reconstituted Vesicles.

The oligosaccharide needed for N-glycoprotein biosynthesis is assembled in two stages on the lipid carrier dolichyl phosphate. First, the key glycolipid intermediate Mannose5N-acetylglucosamine2-PP-dolichol (M5-DLO) is synthesized on the cytoplasmic face of the endoplasmic reticulum (ER). Next, M5-DLO is translocated (scrambled) across the ER membrane, where it is converted to mature DLO. Scrambling is facilitated by specific membrane proteins, some of which remain to be identified. Here, we describe an assay to measure scrambling of M5-DLO in synthetic phospholipid vesicles reconstituted with detergent-solubilized ER membrane proteins, or purified scramblase candidates. This assay will aid in the discovery and mechanistic characterization of M5-DLO scramblases.