MICRODOMAINS OF GPI-ANCHORED PROTEINS IN LIVING CELLS REVEALED BY CROSS-LINKING

There is some discussion as to whether glycosyl-phosphatidylinositol(G PI)-anchored proteins occur in microdomains in the cell membrane(.1,2) These putative microdomains have been implicated in processes such as sorting in polarized cells(3-5) and signal transduction(6-8), Complex es enriched in GPI-anchored proteins, cholesterol and glycosphingolipi ds have been isolated from cell membranes by using non-ionic detergent s: these complexes were thought to represent a clustered arrangement o f GPI-anchored proteins(9,10). However, results obtained when clusteri ng of GPI-anchored proteins induced by antibodies or by detergents was prevented support the idea of a dispersed surface distribution of GPI -anchored proteins at steady state(11-13). Here we use chemical crossl inking to show that membrane microdomains of a GPI-anchored protein ex ist at the surface in living cells. This clustering is specific for th e GPI-anchored form, as two transmembrane forms bearing the same ectod omain do not form oligomers. Depletion of membrane cholesterol causes the clustering of GPI-anchored proteins to break up, whereas treatment of cells with detergent substantially increases the size of the compl exes. We find that in living cells these GPI-anchored proteins reside in microdomains consisting of at least 15 molecules, which are much sm aller than those seen after detergent extraction.