Identification of detergent-resistant plasma membrane microdomains in dictyostelium: enrichment of signal transduction proteins.
Unlike most other cellular proteins, the chemoattractant receptor, cAR1, of Dictyostelium is resistant to extraction by the zwitterionic detergent, CHAPS. We exploited this property to isolate a subcellular fraction highly enriched in cAR1 by flotation of CHAPS lysates of cells in sucrose density gradients. Immunogold electron microscopy studies revealed a homogeneous preparation of membrane bilayer sheets. This preparation, designated CHAPS-insoluble floating fraction (CHIEF), also contained a defined set of 20 other proteins and a single uncharged lipid. Cell surface biotinylation and preembedding immunoelectron microscopy 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 structures, 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-subunits were absent. Overall, CHIFF represents about 3-5% of cell externally exposed membrane proteins. All of these results indicate that CHIFF is derived from specialized microdomains of the plasma membrane. The method of isolation is analogous to that of caveolae. However, we were unable to detect distinct caveolae-like structures on the cell surface associated with cAR1, which showed a diffuse staining profile. The discovery of CHIFF facilitates the purification of cAR1 and related signaling proteins and the biochemical characterization of receptor-mediated processes such as G-protein activation and desensitization. It also has important implications for the "fluid mosaic" model of the plasma membrane structures.
Xiao
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Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205-2185, USA.
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D000818:Animals; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002793:Cholic Acids / Q000494:pharmacology; D003902:Detergents / Q000494:pharmacology; D004023:Dictyostelium / Q000378:metabolism*; D006031:Glycosylation; D008565:Membrane Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism* / Q000648:ultrastructure; D011953:Receptors, Cyclic AMP / Q000302:isolation & purification / Q000378:metabolism* / Q000648:ultrastructure; D015398:Signal Transduction; D016622:Silver Staining; D012995:Solubility; D013265:Stigmasterol
D002793:Cholic Acids; D003902:Detergents; D008565:Membrane Proteins; D011953:Receptors, Cyclic AMP; C068125:cyclic AMP receptor cAR1; D013265:Stigmasterol; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate