Assay for the transbilayer movement of polylsoprenoid-linked saccharides based on the transport of water-soluble analogues

Flippases are a class of membrane proteins that are proposed to facilitate the transbilayer movement of amphipathic polar lipids that are required for membrane biogenesis and the assembly of many diverse complex glycoconjugates in eukaryotic and prokaryotic cells. Despite their crucial roles in membrane biology, very little is known about their structures and the precise mechanism(s) by which they overcome the biophysical barriers of the hydrophobic core, and allow polar head groups to traverse membrane bilayers. This chapter presents methods based on the transport of water-soluble analogues that can be applied to investigate membrane proteins mediating the transverse diffusion of polyisoprenoid-linked glycolipid inter-mediates involved in the biosynthesis of N-linked glycoproteins, glycosylphosphatidylinositol anchors and bacterial poly saccharides. (c) 2005 Published by Elsevier Inc.