[38] Biosynthesis of glycosylphosphatidylinositol anchors

This chapter describes radiolabeling techniques for studying glycosylphosphatidylinositol (GPI) biosynthesis by cell lysates, subcellular fractions, and permeabilized cells. GPIs are synthesized by all eukaryotic cells and are typically found covalently linked to cell surface glycoproteins. GPIs serve as an important alternative mechanism for anchoring proteins to cell membranes and a wide spectrum of functionally diverse proteins rely on a GPI anchor for membrane association. The GPI moiety is synthesized in the endoplasmic reticulum and then transferred to proteins containing a carboxyl-terminal GPI-attachment signal sequence. The methods described in the chapter are used to generate well-characterized radiolabeled GPI biosynthetic intermediates and determine the topological arrangement of the lipids in the membrane bilayer. The completed GPI structure in bloodstream trypanosomes undergoes fatty acid remodeling reactions in which the glycerol-linked fatty acids are replaced by myristic acid.