IDENTIFICATION OF DETERGENT-RESISTANT PLASMA-MEMBRANE MICRODOMAINS INDICTYOSTELIUM - ENRICHMENT OF SIGNAL-TRANSDUCTION PROTEINS

Unlike most other cellular proteins, the chemoattractant receptor, cAR 1, of Dictyostelium is resistant to extraction by the zwitterionic det ergent, CHAPS. We exploited this property to isolate a subcellular fra ction highly enriched in cAR1 by flotation of CHAPS lysates of cells i n sucrose density gradients. Immunogold electron microscopy studies re vealed a homogeneous preparation of membrane bilayer sheets. This prep aration, designated CHAPS-insoluble floating fraction (CHIFF), also co ntained a defined set of 20 other proteins and a single uncharged lipi d. Cell surface biotinylation and preembedding immunoelectron microsco py both confirmed the plasma membrane origin of this preparation. The cell surface phosphodiesterase (PDE) and a downstream effector of cAR1 , adenylate cyclase (ACA), were specifically localized in these struct ures, whereas the cell adhesion molecule gp80, most of the major cell surface membrane proteins, cytoskeletal components, the actin-binding integral membrane protein ponticulin, and G-protein alpha- and beta-su bunits were absent. Overall, CHIFF represents about 3-5% of cell exter nally exposed membrane proteins. All of these results indicate that CH IFF is derived from specialized microdomains of the plasma membrane. T he method of isolation is analogous to that of caveolae. However, we w ere unable to detect distinct caveolae-like structures on the cell sur face associated with cAR1, which showed a diffuse staining profile. Th e discovery of CHIFF facilitates the purification of cAR1 and related signaling proteins and the biochemical characterization of receptor-me diated processes such as G-protein activation and desensitization. It also has important implications for the ''fluid mosaic'' model of the plasma membrane structures.