Eukaryotic cell membranes contain microdomains called lipid rafts, which are cholesterol-rich domains in which lipid acyl chains are tightly packed and highly extended. A variety of proteins associate preferentially with rafts, and this raft association is important in a wide range of functions. A powerful and widely-used method for studying lipid rafts takes advantage of their insolubility in non-ionic detergents. Here we describe the basis of detergent insolubility, and review strengths, limitations, and unresolved puzzles regarding this method.

A number of studies have demonstrated that cholera toxin (CT) is found in detergent-insoluble, cholesterolenriched domains (rafts) in various cells, including neurons. We now demonstrate that even though CT is associated with these domains at the cell surface of cultured hippocampal neurons, it is internalized via a raft-independent mechanism, at both early and late stages of neuronal development. CT transport to the Golgi apparatus, and its subsequent degradation, is inhibited by hypertonic medium (sucrose), and by chlorpromazine; the former blocks clathrin recruitment, and the latter causes aberrant endosomal accumulation of clathrin. Moreover, both internalization of the transferrin receptor (Tf-R), which occurs via a clathrin-dependent mechanism, and CT internalization, are inhibited to a similar extent by sucrose. In contrast, the cholesterol-binding agents filipin and methyl-␤-cyclodextrin have no effect on the rate of CT or Tf-R internalization. Finally, once internalized, CT becomes more detergent-soluble, and chlorpromazine treatment renders internalized CT completely detergent-soluble. We propose two models to explain how, despite being detergent-insoluble at the cell surface, CT is nevertheless internalized via a raft-independent mechanism in hippocampal neurons. Cholera toxin (CT)1 consists of a pentameric B subunit that binds with high affinity to ganglioside GM1 and an A subunit comprising two peptides, A1 and A2, linked by a disulfide bond. The A1 subunit is responsible for activation of adenylate cyclase via the stimulatory G protein, G s . Electron microscopy analysis in A431 cells, and in cultured liver cells, demonstrated that CT does not bind uniformly over the plasma membrane but is rather concentrated in membrane invaginations (1) identified as caveolae (2). Caveolae contain the coat protein, caveolin (3), and are enriched in glycosphingolipids (GSLs) and cholesterol (4). Biochemical analysis has shown that the GSLs and cholesterol found in caveolae are insoluble in nonionic detergents at low temperature (4). However, not all cells contain caveolin or morphologically distinct caveolae. Smooth muscle cells, fibroblasts, adipocytes, endothelial cells, and epithelial cells express caveolin/caveolae, but lymphocytes and neurons do not (3). Even in cells lacking caveolae, a significant fraction of cellular cholesterol and GSLs are found in detergent-insoluble complexes (5-7), sometime known as rafts (8); and these complexes are indistinguishable, using the criteria of detergent insolubility, from those associated with caveolae (9).Due to its association with caveolae and/or detergent-insoluble domains, it is normally assumed that CT is internalized by the pinching off of caveolae from the plasma membrane (10), followed by transport to the Golgi apparatus and endoplasmic reticulum (11,12). Recent studies in A431 cells, which express high levels of caveolin, in CaCo-2 cells, which express low levels of caveolin, and in Jurkat cells, which express no caveolin, demonstrated that the cholesterol-bind...

Some transmembrane proteins must associate with lipid rafts to function. However, even if acylated, transmembrane proteins should not pack well with ordered raft lipids, and raft targeting is puzzling. Acylation is necessary for raft targeting of linker for activation of T cells (LAT). To determine whether an acylated transmembrane domain is sufficient, we examined raft association of palmitoylated and nonpalmitoylated LAT transmembrane peptides in lipid vesicles by a fluorescence quenching assay, by microscopic examination, and by association with detergent-resistant membranes (DRMs). All three assays detected very low raft association of the nonacylated LAT peptide. DRM association was the same as a control random transmembrane peptide. Acylation did not measurably enhance raft association by the first two assays but slightly enhanced DRM association. The palmitoylated LAT peptide and a FLAG-tagged LAT transmembrane domain construct expressed in cells showed similar DRM association when both were reconstituted into mixed vesicles (containing cell-derived proteins and lipids and excess artificial raft-forming lipids) before detergent extraction. We conclude that the acylated LAT transmembrane domain has low inherent raft affinity. Full-length LAT in mixed vesicles associated better with DRMs than the peptide. However, cells appeared to contain two pools of LAT, with very different raft affinities. Since some LAT (but not the transmembrane domain construct) was isolated in a protein complex, and the Myc-and FLAG-tagged forms of LAT could be mutually co-immunoprecipitated, oligomerization or interactions with other proteins may enhance raft affinity of one pool of LAT. We conclude that both acylation and other factors, possibly proteinprotein interactions, target LAT to rafts.Recent years have seen an explosion of interest in membrane microdomains called lipid rafts (1-3). Rafts have been implicated in processes as diverse as signal transduction (1, 4), membrane trafficking (5, 6), and apoptosis (7). In addition, many pathogenic viruses and bacteria hijack host cell rafts during infection (8 -10). In all of these cases, function depends on selective enrichment of a subset of membrane proteins in rafts. For this reason, it is important to determine how proteins are targeted to rafts.A key feature of raft structure is the tight packing of lipid acyl chains. Raft lipids are probably in the liquid-ordered (l o ) 1 phase, in which lipid acyl chains are extended and ordered (11,12). Many proteins are targeted to rafts by their favorable association with these ordered lipids. For example, raft proteins such as glycosylphosphatidylinositol-anchored proteins, Src family kinases, and heterotrimeric G protein ␣ subunits are linked to saturated acyl chains, which partition well into rafts. Because of this tight acyl chain packing, raft lipids and proteins are insoluble in nonionic detergents and can be isolated from cell lysates as DRMs. Although DRM association of a protein may not provide a quantitative measure of its asso...

Sphingolipid/cholesterol-rich rafts are membrane domains thought to exist in the liquid-ordered state. To understand the rules governing the association of proteins with rafts, the behavior of a model membrane-inserted hydrophobic polypeptide (LW peptide, acetyl-K(2)W(2)L(8)AL(8)W(2)K(2)-amide) was examined. The distribution of LW peptide between coexisting ordered and disordered lipid domains was probed by measuring the amount of LW Trp fluorescence quenched by a nitroxide-labeled phospholipid that concentrated in disordered lipid domains. Strong quenching of the Trp fluorescence (relative to quenching in model membranes lacking domains) showed that LW peptide was concentrated in quencher-rich disordered domains and was largely excluded from ordered domains. Exclusion of LW peptide from the ordered domains was observed both in the absence and in the presence of 25-33 mol % cholesterol, indicating that the peptide is relatively excluded both from gel-state domains (which form in the absence of cholesterol) and from liquid-ordered-state domains (which form at high cholesterol concentrations). Because exclusion was also observed when ordered domains contained sphingomyelin in place of DPPC, or ergosterol in place of cholesterol, it appeared that this behavior was not strongly dependent on lipid structure. In both the absence and the presence of 25 mol % cholesterol, exclusion was also not strongly dependent upon the fraction of the bilayer in the form of ordered domains. To evaluate LW peptide behavior in more detail, an analysis of the effects of domain size and edges upon quenching was formulated. This analysis showed that quenching can be affected both by domain size and by whether a fluorescent molecule localized at domain edges. Its application to the quenching of LW peptide indicated that the peptide did not preferentially reside at the boundaries between ordered and disordered domains.