GPI-ANCHORED PROTEINS ARE ORGANIZED IN SUBMICRON DOMAINS AT THE CELL-SURFACE

Lateral heterogeneities in the classical fluid-mosaic model of cell me mbranes are now envisaged as domains or 'rafts' that are enriched in ( glyco)sphingolipids, cholesterol, specific membrane proteins and glyco sylphosphatidylinositol (GPI)-anchored proteins(1). These rafts dictat e the sorting of associated proteins and/or provide sites for assembli ng cytoplasmic signalling molecules(2). However, there is no direct ev idence that rafts exist in living cells(3,4). We have now measured the extent of energy transfer between isoforms of the folate receptor bou nd to a fluorescent analogue of folic acid, in terms of the dependence of fluorescence polarization on fluorophore densities in membranes(5) . We find that the extent of energy transfer for the GPI-anchored fola te-receptor isoform is density-independent, which is characteristic of organization in sub-pixel-sized domains at the surface of living cell s; however, the extent of energy transfer for the transmembrane-anchor ed folate-receptor isoform was density-dependent, which is consistent with a random distribution. These domains are likely to be less than 7 0 nm in diameter and are disrupted by removal of cellular cholesterol. These results indicate that lipid-linked proteins are organized in ch olesterol-dependent submicron-sized domains. Our methodology offers a new way of monitoring nanometre-scale association between molecules in living cells.