The Membrane Domains Occupied by Glycosylphosphatidylinositol-anchored Prion Protein and Thy-1 Differ in Lipid Composition

Abstract

Glycosylphosphatidylinositol-anchored prion protein and Thy-1, found in adjacent microdomains or "rafts" on the neuronal surface, traffic very differently and show distinctive differences in their resistance to detergent solubilization. Monovalent immunogold labeling showed that the two proteins were largely clustered in separate domains on the neuronal surface: 86% of prion protein was clustered in domains containing no Thy-1, although 40% of Thy-1 had a few molecules of prion protein associated with it. Only 1% of all clusters contained appreciable levels of both proteins (>3 immunogold label for both). In keeping with this distribution, immunoaffinity isolation of detergent-resistant membranes (DRMs) using the non-ionic detergent Brij 96 yielded prion protein DRMs with little Thy-1, whereas Thy-1 DRMs contained ϳ20% of prion protein. The lipid content of prion protein and Thy-1 DRMs was measured by quantitative nano-electrospray ionization tandem mass spectrometry. In four independent preparations, the lipid content was highly reproducible, with Thy-1 and prion protein DRMs differing markedly from each other and from the total DRM pool from which they were immunoprecipitated. Prion protein DRMs contained significantly more unsaturated, longer chain lipids than Thy-1 DRMs and had 5-fold higher levels of hexosylceramide. The different lipid compositions are in keeping with the different trafficking dynamics and solubility of the two proteins and show that, under the conditions used, DRMs can isolate individual membrane microenvironments. These results further identify unsaturation and glycosylation of lipids as major sources of diversity of raft structure.The separation of membrane lipids into different phases creates diverse microenvironments within a biological membrane (1, 2). In particular, cholesterol is believed to condense with saturated phosphatidylcholine (PC) 1 and sphingomyelin (SM) to form minute patches (40 -100 nm wide) of lipids in a liquid-ordered phase (3-7), creating specialized lipid microenvironments called "rafts" within the disordered fluid phase formed by unsaturated lipids (8). These ordered microdomains control the access and egress of subsets of membrane proteins, regulating signaling systems at the cell surface (9). liquidordered domains resist solubilization in non-ionic detergents (6, 7, 10 -13), enabling them to be isolated as detergent-resistant membranes (DRMs) that float at low density upon gradient centrifugation (14). Lipid-anchored proteins partition into both leaflets of these domains, the glycosylphosphatidylinositol (GPI)-anchored proteins into the outer (surface) layer and the diacylated cytoplasmic proteins into the inner layer (9, 15, 16). The membrane environment of GPI-anchored prion protein (PrP) is of particular interest since it is a candidate for chaperoning the conversion of PrP to the altered pathogenic conformation associated with prion disease (17-19). Immunolabeling shows PrP to be present on the neuronal surface in different, albeit often closely adjacent, d...

Association of Major Histocompatibility Complex II with Cholesterol- and Sphingolipid-rich Membranes Precedes Peptide Loading

“…1). Recent evidence for such subpopulations was obtained by electron microscopy on native plasma membrane sheets and by immunoisolation procedures (58,59) Our work also demonstrates that DRMs can be separated from tetraspanins with a Brij 98-based isolation procedure. With previously described fractionation protocols (27,33), it has been not possible to decide whether a given protein floats because of its affinity to cholesterol-and sphingolipid-rich membranes or to tetraspanins.…”

Rafts in oligodendrocytes: Evidence and structure–function relationship

“…These findings would favor the notion (Brügger et al, 2004;Madore et al, 1999) that the disparate results obtained by different detergents may relate to the co-existence of different domains, characterized by differences in composition. However, claims have been made that differential insolubility of proteins in different detergents is not sufficient to imply their association with distinct lipid rafts (Chamberlain, 2004;Pike, 2004).…”

Membrane lipids and vesicular traffic

“…The occurrence of two GPI-proteins in separate plasma membrane domains of different lipid composition [47] and the finding of lipid-anchored proteins in cholesterolindependent microdomains on the cytosolic surface (see [48]) illustrate that the reality of the organization of biomembranes is not explained by the mere notion of sphingolipid/cholesterol rafts. A specific function of sphingolipids on cytosolic surfaces is suggested by the fact that the cytosolic protein FAPP2, which contains a glycolipid binding domain, plays a role in regulating membrane flow between the Golgi and the plasma membrane [49].…”