Physiology Cambridge Electronic Design Limited
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Physiology 21: 430-439, 2006; doi:10.1152/physiol.00032.2006
1548-9213/06 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (45)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Brown, D. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Brown, D. A.
Physiology, Vol. 21, No. 6, 430-439, December 2006
© 2006 Int. Union Physiol. Sci./Am. Physiol. Soc.

REVIEW

Lipid Rafts, Detergent-Resistant Membranes, and Raft Targeting Signals

Deborah A. Brown

Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, New York, deborah.brown{at}sunysb.edu


   Abstract
 
Lipid rafts are liquid-ordered (lo) phase microdomains proposed to exist in biological membranes. Rafts have been widely studied by isolating lo-phase detergent-resistant membranes (DRMs) from cells. Recent findings have shown that DRMs are not the same as preexisting rafts, prompting a major revision of the raft model. Nevertheless, raft-targeting signals identified by DRM analysis are often required for protein function, implicating rafts in a variety of cell processes.


   Introduction
Top
 Introduction
Detergent Insolubility and Lipid...
 A Current Picture of...
 DRM Association is Useful...
 Conclusions
 References
 
The liquid-ordered (lo) phase is a sterol-dependent state in which lipid acyl chains are tightly packed and highly extended but in which individual lipid molecules have a high degree of lateral diffusion in the bilayer (109). In model membranes, lo-phase domains can coexist with both liquid-disordered (ld)-phase domains, in which lipids are much less condensed, and with gel-phase domains, in which lipids are highly condensed and essentially immobile (44). lo-ld phase separation in model membranes is not controversial: micron-sized lo-phase domains can be seen by microscopy in giant unilamellar vesicles (GUVs) (109). The raft hypothesis proposes that lo-phase domains also form in sterol-rich cell membranes, especially the plasma membrane, where they coexist with disordered membrane domains (42, 61, 82).


   Detergent Insolubility and Lipid Rafts
Top
 Introduction
 Detergent Insolubility and Lipid...
A Current Picture of...
 DRM Association is Useful...
 Conclusions
 References
 
An important early indication that rafts may exist in cells came from the observation that cell membranes are not fully solubilized by non-ionic detergents such as Triton X-100 (TX100) or CHAPS at low temperatures. Instead, lo-phase DRMs remain and can be isolated on sucrose gradients (15, 95).

Detergent insolubility in model membranes
Model membrane studies explained this finding, showing that close packing of lipids in the lo or gel phases prevents detergent incorporation into the bilayer (115). Two studies showed that detergent insolubility is a useful assay of phase state in model membranes. In one, the phase state of a series of sterol-rich mixtures containing varying amounts of order- and disorder-preferring lipids was first determined by a fluorescence-quenching assay (1). This pinpointed the amount of order-preferring lipid required for phase separation. The same mixtures were then subjected to TX100 extraction. DRMs could only be detected in mixtures that contained lo-phase domains before the addition of TX100 (1). A second approach used two-phase supported lipid bilayers that contained separate ld and lo domains visible by microscopy (28). TX100 selectively solubilized ld-phase domains, leaving the lo-phase domains relatively unchanged as DRMs. Both of these approaches showed that TX100 did not substantially change the relative amounts of membrane in the two phases. By contrast, using calorimetry, Heerklotz and colleagues reported that TX100 can induce phase separation in previously uniform mixtures (45, 46). However, results of the other two studies described above suggest that any such effect may be relatively minor.

Development of the raft model in cells
These model membrane results initially suggested that DRMs isolated from cells were also derived from preexisting raft domains. This model was attractive because plasma membranes contain significant amounts of order-preferring sphingolipids and sterols, together with disorder-preferring phospho-lipids. Model membrane studies showed that lo-ld phase separation could occur at 37°C in lipid mixtures with physiological levels of each of these lipid classes, making phase separation in cell membrane plausible (1, 101). Furthermore, DRMs isolated from cells are enriched in a subset of proteins, mostly from the plasma membrane. DRM enrichment initially suggested that these proteins were concentrated in rafts in cells and that raft targeting might be important in function.

This idea was supported by the use of agents that deplete or sequester membrane cholesterol. ß-Cyclodextrins can rapidly and reversibly extract most sterol from cell membranes (25). Polyene antibiotics such as filipin and nystatin partition into membranes and bind sterols, sequestering them from other interactions. For convenience, I will refer to both classes of compound together as cholesterol modulators. These agents reduce or abolish DRM association of most proteins, suggesting that they disrupt rafts in cells. This idea is reasonable, since phase separation is often enhanced by sterols (1, 102) and could plausibly be abolished by sterol removal. (It is worth noting, however, that lo-phase disruption by cholesterol modulators has not been demonstrated directly using detergent-free assays in model membrane systems.)

DRMs are not rafts
Despite the encouraging precedent provided by model membranes, DRMs isolated from cells do not correspond precisely to preexisting rafts in living cells. This is clear from the fact that isolation of TX100-insoluble DRMs from mammalian cells requires extraction at low temperatures (69). Phase behavior is very temperature dependent. If rafts exist in cell membranes, chilling will certainly expand them, as additional proteins and lipids are recruited to rafts. If rafts do not exist in living cells, chilling could cause them to form by inducing phase separation.

DRMs can be isolated from cells at higher temperatures following extraction with other non-ionic detergents, such as Lubrol WX and detergents of the Brij series (73, 91). This has been taken to suggest that different classes of rafts are isolated by different detergents. However, there is no direct evidence to support this idea. Of concern, these detergents are less efficient than TX100 and may fail to fully solubilize moderately ordered ld-phase membranes. Lipids in the plasma membrane, with its high sterol and sphingolipid content, are expected to be more ordered than those in intracellular membranes, even if phase separation and raft formation do not occur. Thus weak detergents might not fully solubilize the plasma membrane even if no rafts were present (96). Detergent concentration, and the ratio of detergent to cellular proteins and lipids, can also affect the degree to which proteins associate with DRMs (7, 78). That is, even proteins with low inherent affinity for ordered membranes can be detected in DRMs prepared with low amounts of TX100 (7). It is important in DRM studies to include non-raft-loving proteins (such as the transferrin receptor) as negative controls.

Studies of glycosyl phosphatidylinositol-anchored proteins (GPI-APs) also show that DRMs are not direct descendants of rafts in living cells. GPI-APs have saturated, order-preferring acyl chains and are highly enriched in large (up to 1-µm diameter) DRMs (15). Nevertheless, analysis by fluorescence resonance energy transfer (FRET) shows that most GPI-APs are uniformly distributed on the cell surface or are present in at most very small (nanoscale) clusters, containing only a few proteins (37, 56, 97). Fluorescence recovery after photobleaching (FRAP) also shows that GPI-APs, as well as other DRM-enriched proteins and lipids, do not diffuse as components of large, stable rafts (55). By extension, DRM-association of proteins other than GPI-APs cannot be taken to show that they are present in rafts in living cells.

Likewise, the ability of cholesterol modulators to affect a process does not prove that rafts are involved. Agents that modulate membrane cholesterol are likely to affect cell function in ways that are unrelated to raft formation. Sterols have profound effects on bilayer structure, even in single-phase membranes (51, 66), and their removal should greatly affect even membranes that do not contain rafts. Thus inhibition of a process by cholesterol modulators suggests that it requires cholesterol but does not prove that it requires rafts. However, a few groups have strengthened the case for raft involvement by showing that functions inhibited by cholesterol depletion can be rescued by repletion of cells with sterols or sterol analogs that promote lo-phase formation in model membranes, but not by compounds that lack this property (16, 77, 90).

In summary, finding that cholesterol modulators affect a given process in cells is consistent with, but does not demonstrate, a role for rafts. Nevertheless, an intriguing report suggests that at least one pathogen may use a cholesterol modulator to avoid host defenses. Pathogenic Brucella bacteria, which are phagocytosed by host cells, secrete a cyclic ß-1,2-glucan that weakly extracts cholesterol from membranes. This compound was reported to inhibit phagosome-lysosome fusion, allowing replication of the pathogen in the host cell (6).


   A Current Picture of Raft Structure and Function in Cells
Top
 Introduction
 Detergent Insolubility and Lipid...
 A Current Picture of...
DRM Association is Useful...
 Conclusions
 References
 
Much of the evidence for raft function in cells comes from studies pairing DRM-association of key proteins with the effect of cholesterol modulators in disrupting function. Such studies have suggested several important roles for rafts, especially in signal transduction (30, 49, 82) and membrane trafficking (57). Even before their structure was understood, rafts were proposed to act in sorting in the trans-Golgi network of polarized epithelial cells (103). Further work supported roles for rafts in this process (54) and in sorting in endosomes (74). However, intracellular rafts have proven difficult to study because they cannot be directly accessed from the cell surface. In recent years, much attention has shifted to the plasma membrane. Because of the caveats outlined above, the areas in which the evidence for raft function is most compelling are those in which DRM analysis and cholesterol depletion have been combined with microscopy. Thus the cell-surface distribution of proteins and lipids suspected (usually from DRM analysis) of being in rafts is examined microscopically, with the aim of visualizing the putative rafts directly. This approach has been most fruitful in studies of signaling in hematopoietic cells, especially lymphocytes and mast cells. The success of these studies may reflect the fact that cell-surface signaling molecules can assemble into particularly large complexes in these cells, facilitating detection by microscopy. Thus DRM association of the T- and B-cell receptors (21, 73) and of Fc{varepsilon}RI (the IgE-binding signaling receptor on mast cells) (33) increases during signaling, and signaling in T cells (114) and mast cells (98) is sensitive to cholesterol depletion. In addition, order-preferring proteins and lipids can be observed by microscopic techniques to cluster together on receptor ligation in T cells (36, 52, 111) and mast cells (107), and FRET analysis implicates raft-dependent interactions of the Src-family kinase Lyn with the B-cell receptor (104). Recent studies have raised questions about the role of rafts in T-cell signaling (29, 38) whose resolution is not yet clear. Nevertheless, studies on hematopoietic cell signaling provide a useful paradigm for how rafts may function in other cell types where they have proven harder to detect.

Ironically, some of the strongest evidence for raft function in mammalian cells comes from the behavior of pathogens. A number of pathogenic bacteria (60, 63) and viruses (18, 80) bind order-preferring proteins and lipids on the surface of mammalian cells and appear to co-opt host cell rafts during infection. As an example of how rafts have been implicated in pathogenesis, Wolf and colleagues (113) compared two bacterial toxins that bind different gangliosides on the surface of cultured human intestinal cells. Cholera toxin, which binds the DRM-enriched ganglioside GM1, intoxicated the cells much more efficiently than the related Escherichia coli heat-labile type II entero-toxin LTIIb, which does not associate with DRMs (113). Most putative raft-targeting pathogenic agents seem to use rafts for internalization into mammalian cells, suggesting a role for rafts in normal endocytic pathways (22, 57, 80).

Any general model for raft structure and function must incorporate the finding that GPI-anchored proteins, highly enriched in DRMs, appear to be uniformly distributed on the surface of resting cells. An emerging paradigm in the field is that raft formation and/or stabilization may be highly regulated and occur only in response to stimulation, such as receptor clustering. According to this model, rafts in unstimulated cells are small (nanoscale) and unstable, if they exist at all. However, clustering of molecules with high inherent affinity for ordered lipids [helpfully termed raftophilicity by Kusumi and colleagues (59)] can greatly increase raft size and stability. Thus clustering of cell surface receptors, as often occurs during signaling, may increase the affinity of the clustered receptors for rafts and may also stabilize rafts or induce their formation. Proof of principle comes from an elegant recent study (41): clustering of gangliosides in single-phase model membranes close to a phase-separation boundary using cholera toxin induced dramatic large-scale phase separation (shown in FIGURE 1Go). This model suggests that the plasma membrane lipid composition is also maintained close to that required for phase separation, so that small changes in protein or lipid aggregation can have large effects on microdomain organization. Recruitment of raftophilic proteins to rafts may enhance their interactions with each other, for instance during signaling, while reducing their interactions with non-raftophilic proteins.


Figure 1
View larger version (28K):
[in this window]
[in a new window]
  		FIGURE 1. Clustering GM1 ganglioside with cholera toxin B subunit induces phase separation in GUVs

Giant unilamellar vesicles (GUVs), consisting of DOPC, DOPG, sphingomyelin, cholesterol, and GM1 with rhodamine-labeled LAT transmembrane peptide were prepared. Other analysis (see Ref. 41) showed that these GUVs were in a single ld phase. Rhodamine fluorescence of the peptide, which partitions into disordered domains, shown as bright staining, was examined before (A) or after (B) binding of pentavalent cholera toxin B subunit to GM1 in the GUVs. Additional analysis (see Ref. 41) showed that dark regions in B were in the lo phase. Scale bars, 5 µm. Reprinted with permission from Ref. 41.

 
An example of how such a model could work in practice, from the elegant work of Baird, Holowka, and colleagues on Fc{varepsilon}RI signaling in mast cells (49, 119), is shown in FIGURE 2Go. Ligand-induced Fc{varepsilon}RI clustering is proposed to stabilize rafts containing both the receptor and the raftophilic kinase Lyn. Lyn itself requires phosphorylation for activation, and raft clustering and stabilization activate Lyn by sequestering it from non-raftophilic phosphatases.


Figure 2
View larger version (109K):
[in this window]
[in a new window]
  		FIGURE 2. Model of the role of rafts in Fc{varepsilon}RI (IgE receptor) signaling

A: in unstimulated cells, monomeric IgE is bound to its receptor. The nonreceptor tyrosine kinase Lyn may constitutively associate with small, unstable rafts. Autophosphorylation is required for efficient Lyn activity. In unstimulated cells, Lyn activity is kept low by frequent encounters with non-raft associated phosphatases. B: stimulation by binding of multivalent antigen clusters IgE receptors and induces formation of larger, stabilized rafts that contain both IgE receptor and Lyn. The presence of Lyn in these rafts shields the kinase from non-raft-targeted phosphatases, increasing its activity and enhancing phosphorylation of the IgE receptor and other substrates, leading to downstream signaling. Reprinted from Ref. 33, with permission from Elsevier.

 

   DRM Association is Useful in Demonstrating Raftophilicity and Identifying Raft Targeting Signals
Top
 Introduction
 Detergent Insolubility and Lipid...
 A Current Picture of...
 DRM Association is Useful...
Conclusions
 References
 
In this context, what do detergent insolubility studies tell us? Enrichment of a protein in DRMs shows that it is raftophilic and is likely to associate with rafts when they form. DRM analysis can also help identify raftophilicity-conferring motifs in proteins, as detailed in the rest of this review. For convenience, I will refer to these as raft targeting signals, although it is important to note that, in most cases, only DRM targeting has been shown. The striking number of cases in which DRM-targeting correlates with function provides some of the strongest available evidence of a physiological role for rafts.

Discussing raft-targeting signals in proteins tacitly assumes that rafts are formed from lipids and that proteins partition into them based on their affinity for an ordered membrane environment. Indeed, lo-phase domains form spontaneously in model membranes of appropriate lipid composition, and lipid interactions are undoubtedly also crucial for raft formation in vivo. However, raft formation in cells is very likely to be regulated by proteins. In addition to the transient protein-clustering effects outlined above, proteins such as caveolins (79) and flotillins (88) probably organize rafts into specialized membrane domains. Furthermore, important protein-mediated links of rafts to the actin cytoskeleton are starting to be elucidated (20, 49, 68, 84).

Raft targeting of peripheral proteins
Raft targeting of proteins that lack transmembrane spans is best understood. GPI anchors and tandem or closely spaced acyl chains on these proteins are well-known raft-targeting signals (14). Unexpectedly, fluorescence quenching analysis showed that a tandem NH2-terminal myristate/palmitate motif targeted a peripheral peptide to rafts in model membranes more strongly than tandem palmitoylation on two internal sites (112). The myristate/palmitate motif, present in Src-family kinases and some G protein {alpha} subunits, appears to be an especially efficient raft-targeting signal. Microscopy analysis in cells supports this idea. A tandem myristoyl/palmitoyl motif can target GFP to clusters on the inner leaflet of the plasma membrane that coincide with rafts (visualized by experimental clustering of outer-leaflet raftophilic proteins) in the extracellular leaflet (40, 87, 120). It is notable that G{alpha}s is modified with an unusual dual-palmitate motif, in which the first palmitate is linked to an NH2-terminal Gly instead of the usual internal Cys (58). By analogy with the myristate/palmitate motif, the dual-palmitate motif of G{alpha}s might also confer high raftophilicity. However, this idea has not been tested, and it has been difficult to identify a role for raft targeting in G{alpha}s function (71).

A recent study shows that the matrix domain (MA) of HIV Gag, which is myristoylated, uses a novel variation of this motif (93). As illustrated in FIGURE 3Go, MA binds membrane PI(4,5)P2 in such a way that the polyunsaturated 2'-arachidonate chain is sequestered in a cleft in the protein, whereas the saturated 1'-stearate chain is exposed. PI(4,5)P2 binding also exposes the myristate chain of MA. As a result, MA may be targeted to rafts by membrane insertion of two saturated acyl chains: the covalently linked myristate and the stearate of the bound PI(4,5)P2. Use of a similar strategy by cellular proteins could explain the surprising enrichment of PI(4,5)P2 in DRMs (50), despite the presence of its order-disfavoring arachidonate chain.


Figure 3
View larger version (100K):
[in this window]
[in a new window]
  		FIGURE 3. Model of membrane binding of MA domain of HIV1-Gag protein

The predicted MA trimer (gray) is modeled bound to a bilayer. MA binding to membrane PI(4,5)P2 (yellow; phosphates red) sequesters the phosphoinositide’s 2' arachidonate chain in a cleft in the protein, leaving the 1' stearate chain inserted in the membrane. PI(4,5)P2 binding also exposes MA’s myristate chain (green), predicted to insert into the membrane. A basic surface adjacent to the bilayer (Lys and Arg groups shown in blue) also promotes membrane binding. Reprinted with permission from Ref. 93.

 
Prenyl groups, which are branched, should not fit well into rafts. Model membrane studies verified the expected low affinity of prenylated peptides for rafts (112), and DRMs isolated from cells contain few prenylated proteins (70). Some proteins are modified both tandem palmitoylation (expected to increase raft affinity) and prenylation (expected to reduce raft affinity), raising the possibility that overall raft affinity may be regulated. For heterotrimeric G proteins, this could occur by dissociation of acylated G{alpha} subunits from prenylated Gß{gamma} subunits during signaling. As expected, monomeric G{alpha}i associated better than the G{alpha}iß{gamma} trimeric complex with DRMs isolated from model membranes containing the purified proteins (72). H-Ras, modified by closely spaced dual palmitoylation and farnesylation, is also targeted to rafts, although it is solubilized by standard TX100 extraction (83). The prenyl group may bind reversibly to other proteins to regulate raft-disfavoring insertion of the moiety into the bilayer. Alternatively, other raft-favoring interactions may overcome raft-disfavoring tendency of the prenyl group.

The vexing question of transmembrane protein targeting to rafts
As schematized in FIGURE 4Go, transmembrane peptides have rough surfaces and should not fit well into rafts. Detergent-free model membrane studies, examining raft targeting by fluorescence quenching, have confirmed this expectation, showing that such peptides are excluded from rafts (32, 108). As expected, most transmembrane proteins associate poorly with DRMs. However, a few such proteins are found in DRMs, and raft targeting appears to be important in their function. It is not known how the strongly raft-disfavoring transmembrane domains are arranged with respect to rafts in the bilayer. Palmitoylated proteins might be expected to prefer domain boundaries, positioning their transmembrane domains in ld domains and their palmitate chains in adjacent lo domains. However, a dually palmitoylated transmembrane domain peptide corresponding to the T-cell scaffold protein LAT was mostly excluded from rafts in two-phase giant unilamellar vesicles, and showed no enrichment at domain boundaries (99).


Figure 4
View larger version (65K):
[in this window]
[in a new window]
  		FIGURE 4. Transmembrane proteins and lipid rafts

Rough-surfaced {alpha}-helical transmembrane domains of transmembrane proteins (C, D, E,) pack better with lipids in disordered non-raft domains (B) than in rafts (A) and are generally excluded from rafts. It is not known how a few transmembrane proteins associate with rafts. Rafts should be thicker than coexisting non-raft domains since lipid acyl chains are more extended. Proteins with long transmembrane domains have been proposed to partition into rafts to avoid hydrophobic mismatch. However, experimental evidence shows that this does not occur; unfavorable packing interactions outweigh any advantage of minimizing hydrophobic mismatch. Instead, long transmembrane spans might tilt in thinner disordered domains to avoid hydrophobic mismatch. Thus both protein D, with a short transmembrane domain, and protein E, with a long one, are in non-raft domains, but the transmembrane span of protein E may tilt. Enrichment of palmitoylated transmembrane proteins (protein B; palmitate chains red) at raft borders, positioning palmitate chains in rafts and transmembrane domains in non-raft domains, is an attractive model, but supporting evidence is lacking.

 
Because increases in lipid order increase bilayer thickness, rafts are generally expected to be thicker than coexisting non-raft domains. This has suggested that correspondingly longer transmembrane spans might target some proteins to rafts to avoid hydrophobic mismatch, an idea that has achieved textbook status (2). However, model membrane/peptide studies have shown that the raft-disfavoring difficulty of packing transmembrane spans into an ordered lipid environment outweighs any raft-favoring effect of improved hydrophobic match (67, 108, 110). Transmembrane span length is not known to affect raft affinity of any protein.

Palmitoylation and raft targeting
The best-studied raft targeting signal for transmembrane proteins is palmitoylation: usually linkage of two or more palmitoyl chains to the cytoplasmic domain, close to the membrane interface. Palmitoylation is also required for function of some of these proteins, suggesting a physiological role for raft targeting. Most reported cases of this behavior are in hematopoietic cells and (to a lesser extent) neurons. Thus palmitoylation of the tetraspanin CD81 is required to recruit the associated B-cell receptor to rafts in B cells, prolonging its signaling (23). Palmitoylation is also required for raft targeting and efficient function of the T-cell co-receptors CD4 (9, 34) and CD8 (4, 5). Palmitoylation of the T-cell scaffolding protein LAT is required for raft targeting (121), for signaling (65), and for recruitment of the protein to T-cell receptor-enriched immunoisolates on activation (43). [However, interpretation of these results might be complicated by another group’s findings that LAT palmitoylation was required for cell-surface trafficking, membrane insertion, and stability (106).] Palmitoylation of a LAT transmembrane domain peptide slightly increased DRM-association in model membranes (99). However, the intact protein was more highly enriched in DRMs than the acylated peptide, showing that the protein contained additional raft targeting information (99). Palmitoylation of the platelet glycoprotein GP Ib-IX-V complex is required for raft targeting, and the amount of DRM-associated GP Ib-IX-V increases dramatically upon activation (100). Palmitoylation of Fc{gamma}RIIa, an IgG receptor expressed on most hematopoietic cells, is required for raft targeting and signaling (10). In neurons, palmitoylation of NCAM is required for raft targeting and for signaling during neurite outgrowth (76). Similarly, the netrin receptor DCC requires palmitoylation for raft association and for function in axon guidance in developing neurons (47). Palmitoylation of the {alpha}6ß4 integrin in epithelial cells is required for raft targeting and for coupling to Src-family kinases and downstream signaling pathways on ligation (35). However, palmitoylation of the luteinizing hormone receptor is needed for raft targeting but not for function (62).

The glycoproteins of many enveloped viruses are palmitoylated, and some require this modification for raft targeting. Nevertheless, a role for acylation in viral life cycles has often been difficult to define. Thus, although palmitoylation of the murine leukemia virus envelope protein is required for raft targeting and enhances cell-surface transport, it does not affect fusion activity (64). Palmitoylation of the HIV-1 envelope glycoprotein (gp160; later cleaved to generate the transmembraneous gp41 and the peripheral protein gp120) was initially reported to be required for raft targeting and infectivity (92). However, loss of gp41 palmitoylation was later reported to affect infection only partially (11) or not at all (17). This issue may be complicated by the fact that HIV-1 Gag protein can recruit gp41 to rafts (12). Palmitoylation of influenza hemagglutinin is required for raft targeting (19, 70), but the role of the modification in protein function is controversial, as well summarized earlier (19), and may be subtype specific.

Not all palmitoylated transmembrane proteins associate with DRMs, and it is not known what distinguishes the two groups. Increasing the number of acyl chains may enhance DRM association, although protein-specific factors are also clearly important since different numbers of acyl chains are required for DRM association of different proteins. Thus LAT requires two chains (121), whereas influenza HA requires three (70) for DRM association. Many singly palmitoylated proteins, such as VSV G, are excluded from DRMs (15). Transferrin receptor, a widely used non-DRM marker protein, was originally reported to be either singly or doubly palmitoylated, depending on cell type (53), and this issue has not been investigated further.

Transmembrane domain raft targeting signals
Very little is known about how transmembrane domain amino acid sequence affects raft targeting. Several residues of influenza hemagglutinin that are predicted to contact the extracellular half of the bilayer are essential for raft targeting (94, 105). Gosse et al. examined hybrid proteins containing the extracellular domain of the IgE receptor Fc{varepsilon}RI, the intracellular domain of the {zeta} chain of the T-cell receptor, and variable transmembrane domains (39). On receptor clustering, the hybrid containing the TCR {zeta} chain transmembrane domain associated with DRMs, whereas hybrids containing transmembrane domains from two putative non-raft proteins (CD45 and PTP{alpha}) did not. In all cases examined, the ability of the hybrids to signal after clustering correlated with DRM association. Truncation and domain-swapping experiments have implicated transmembrane domains of CD44 (75, 81) and CD40 (13) in raft targeting. Signaling through the Epstein Barr virus latent membrane protein (LMP-1), which is similar to CD40, is essential for EBV-mediated B-cell immortalization (26). Transmembrane domains 1 and 2 of this polytopic protein (26), and in particular a motif (FWLY) in transmembrane domain 1 (118) are essential for raft targeting. The FWLY motif is also required for LMP1-mediated signaling (118). The COOH-terminal transmembrane domain of herpes virus saimiri tip (which induces downregulation of the T-cell receptor and CD4 in T cells) is also required for raft targeting (24).

Extracellular and cytoplasmic domain raft targeting signals
A few raft targeting signals have been identified in extracellular or cytoplasmic domains, although mechanisms have not been defined. Such motifs could act by partially inserting in the bilayer (as proposed below for caveolins), by inducing conformational changes that affect raftophilicity, or indirectly through binding to other raft-targeted proteins. Conformational changes in the extracellular domain affect raft targeting of the T-cell adhesion protein CD2 (117), and a 60-residue membrane-proximal sequence was reported to target the EGF receptor to rafts (116). Both studies showed that glycosylation played no role in raft targeting. The pentapeptide LWYIK, present in the juxtamembrane region of the HIV fusion protein gp41, was shown to bind cholesterol and induce formation of cholesterol-rich domains in vitro (31).

The cytoplasmic domain sequence LIRW aids in DRM association of the proteolipid MAL, although this effect may be secondary to altered targeting of the mutant protein from the TGN to an uncharacterized BFA-insensitive compartment (86). The RING finger domain of tumor necrosis factor receptor-associated factor (TRAF) 2 is required for DRM association of the protein on engagement of TNF receptors and for activation of downstream signaling (8). Replacement of the RING finger with another raft targeting signal can partially rescue signaling. PKA-mediated phosphorylation of the neurotrophin receptor p75NTR is required for both DRM association and signaling (48). A membrane-proximal signal (RHRRR) in the cytoplasmic domain of the T-cell co-receptor CD4 is required for DRM association (85). This effect is independent of palmitoylation, since mutation of the signal abolishes DRM association but not palmitoylation. Analysis of hybrids of the DRM-associated, Ca2+-sensitive adenylyl cyclase AC5 and the non-DRM-associated, Ca2+-insensitive adenylyl cyclase AC7 showed that DRM-targeting signals were present in cytoplasmic loops and not in transmembrane domains (27). An intriguing model membrane study suggests that a membrane-proximal peptide may target caveolins to DRMs through insertion of aromatic residue side-chains into the bilayer, favoring recruitment of cholesterol and presumably other small-head group lipids through steric effects (3). However, attempts to localize raft targeting signals by mutagenesis of intact caveolin-1 have been confused by the tendency of mutations throughout the protein to induce misfolding and loss of DRM association (89).


   Conclusions
Top
 Introduction
 Detergent Insolubility and Lipid...
 A Current Picture of...
 DRM Association is Useful...
 Conclusions
References
 
Two simple and widely used methods, DRM analysis and cholesterol depletion, have implicated rafts in a variety of cell functions. Furthermore, DRM association has been used to identify raftophilicity-conferring signals in many proteins. However, raft dynamics in cells remain poorly understood. An important challenge for the future will be to learn how and when proteins associate with rafts and precisely how rafts exist and function in cells. Development of improved methods for studying rafts (61) will be essential for further progress toward these goals.


   Acknowledgments
 
I thank Dr. Erwin London for valuable discussions.

This work was supported by National Institute of General Medical Sciences Grant GM-47897.


   References
Top
 Introduction
 Detergent Insolubility and Lipid...
 A Current Picture of...
 DRM Association is Useful...
 Conclusions
 References
 

  1. Ahmed SN, Brown DA, and London E. On the origin of sphingolipid-cholesterol rich detergent-insoluble domains in cell membranes: Physiological concentrations of cholesterol and sphingolipid induce formation of a detergent-insoluble, liquid-ordered lipid phase in model membranes. Biochemistry 36: 10944–10953, 1997.[CrossRef][Medline]
  2. Alberts B, Johnson A, Lewis J, Raff M, Roberts K, and Walter P. Molecular Biology of the Cell (4th ed.). New York: Garland Science, 2002.
  3. Arbuzova A, Wang L, Wang J, Hangyas-Mihalyne G, Murray D, Honig B, and McLaughlin S. Membrane binding of peptides containing both basic and aromatic residues. Experimental studies with peptides corresponding to the scaffolding region of caveolin and the effector region of MARCKS. Biochemistry 39: 10330–10339, 2000.[CrossRef][Medline]
  4. Arcaro A, Gregoire C, Bakker TR, Baldi L, Jordan M, Goffin L, Boucheron N, Wurm F, van der Merwe PA, Malissen B, and Luescher IF. CD8beta endows CD8 with efficient coreceptor function by coupling T cell receptor/CD3 to raft-associated CD8/p56(lck) complexes. J Exp Med 194: 1485–1495, 2001.[Abstract/Free Full Text]
  5. Arcaro A, Grégoire C, Boucheron N, Stotz S, Palmer E, Malissen B, and Luescher IF. Essential role of CD8 palmitoylation in CD8 coreceptor runction. J Immunol 165: 2068–2076, 2000.[Abstract/Free Full Text]
  6. Arellano-Reynoso B, Lapaque N, Salcedo S, Briones G, Ciocchini AE, Ugalde R, Moreno E, Moriyón I, and Gorvel J-P. Cyclic beta-1,2-glucan is a brucella virulence factor required for intracellular survival. Nat Immunol 6: 618–625, 2005.[CrossRef][ISI][Medline]
  7. Arni S, Keilbaugh SA, Ostermeyer AG, and Brown DA. Association of GAP-43 with detergent-resistant membranes requires two palmitoylated cysteine residues. J Biol Chem 273: 28478–28485, 1998.[Abstract/Free Full Text]
  8. Arron JR, Pewzner-Jung Y, Walsh MC, Kobayashi T, and Choi Y. Regulation of the subcellular localization of tumor necrosis factor receptor-associated factor (TRAF)2 by TRAF1 reveals mechanisms of TRAF2 signaling. J Exp Med 196: 923–934, 2002.[Abstract/Free Full Text]
  9. Balamuth F, Brogdon JL, and Bottomly K. CD4 raft association and signaling regulate molecular clustering at the immunological synapse site. J Immunol 172: 5887–5892, 2004.[Abstract/Free Full Text]
  10. Barnes NC, Powell MS, Trist HM, Gavin AL, Wines BD, and Hogarth PM. Raft localisation of FcgammaRIIa and efficient signaling are dependent on palmitoylation of cysteine 208. Immunol Lett 104: 118–123, 2006.[CrossRef][ISI][Medline]
  11. Bhattacharya J, Peters PJ, and Clapham PR. Human immunodeficiency virus type 1 envelope glycoproteins that lack cytoplasmic domain cysteines: impact on association with membrane lipid rafts and incorporation onto budding virus particles. J Virol 78: 5500–5506, 2004.[Abstract/Free Full Text]
  12. Bhattacharya J, Repik A, and Clapham PR. Gag regulates association of human immunodeficiency virus type 1 envelope with detergent-resistant membranes. J Virol 80: 5292–5300, 2006.[Abstract/Free Full Text]
  13. Bock J and Gulbins E. The transmembranous domain of CD40 determines CD40 partitioning into lipid rafts. FEBS Lett 534: 169–174, 2003.[CrossRef][ISI][Medline]
  14. Brown DA and London E. Functions of lipid rafts in biological membranes. Annu Rev Cell Devel Biol 14: 111–136, 1998.[CrossRef][ISI][Medline]
  15. Brown DA and Rose JK. Sorting of GPI-anchored proteins to glycolipid-enriched membrane subdomains during transport to the apical cell surface. Cell 68: 533–544, 1992.[CrossRef][ISI][Medline]
  16. Campbell S, Gaus K, Bittman R, Jessup W, Crowe S, and Mak J. The raft-promoting property of viri-on-associated cholesterol, but not the presence of virion-associated Brij 98 rafts, is a determinant of human immunodeficiency virus type 1 infectivity. J Virol 78: 10556–10565, 2004.[Abstract/Free Full Text]
  17. Chan WE, Lin HH, and Chen SS. Wild-type-like viral replication potential of human immunodeficiency virus type 1 envelope mutants lacking palmitoylation signals. J Virol 79: 8374–8387, 2005.[Abstract/Free Full Text]
  18. Chazal N and Gerlier D. Virus entry, assembly, budding, and membrane rafts. Microbiol. Mol. Biol. Rev. 67: 226–237, 2003.[Abstract/Free Full Text]
  19. Chen BJ, Takeda M, and Lamb RA. Influenza virus hemagglutinin (H3 subtype) requires palmitoylation of its cytoplasmic tail for assembly: M1 proteins of two subtypes differ in their ability to support assembly. J Virol 79: 13673–13684, 2005.[Abstract/Free Full Text]
  20. Chen Y, Thelin WR, Yang B, Milgram SL, and Jacobson K. Transient anchorage of cross-linked glycosyl-phosphatidylinositol-anchored proteins depends on cholesterol, Src family kinases, caveolin, and phosphoinositides. J Cell Biol 175: 169–178, 2006.[Abstract/Free Full Text]
  21. Cheng PC, Brown BK, Song W, and Pierce SK. Translocation of the B cell antigen receptor into lipid rafts reveals a novel step in signaling. J Immunol 166: 3693–3701, 2001.[Abstract/Free Full Text]
  22. Cheng ZJ, Deep Singh R, Marks DL, and Pagano RE. Membrane microdomains, caveolae, and caveolar endocytosis of sphingolipids (Review). Mol Membr Biol 23: 101–110, 2006.[CrossRef][ISI][Medline]
  23. Cherukuri A, Carter RH, Brooks S, Bornmann W, Finn R, Dowd CS, and Pierce SK. B cell signaling is regulated by induced palmitoylation of CD81. J Biol Chem 279: 31973–31982, 2004.[Abstract/Free Full Text]
  24. Cho NH, Kingston D, Chang HC, Kwon EK, Kim JM, Lee JH, Chu H, Choi MS, Kim IS, and Jung JU. Association of herpesvirus saimiri tip with lipid raft is essential for downregulation of T-cell receptor and CD4 coreceptor. J Virol 80: 108–118, 2006.[Abstract/Free Full Text]
  25. Christian AE, Haynes MP, Phillips MC, and Rothblat GH. Use of cyclodextrins for manipulating cellular cholesterol content. J Lipid Res 38: 2264–2272, 1997.[Abstract]
  26. Coffin WFI, Geiger TR, and Martin JM. Transmembrane domains 1 and 2 of the latent membrane protein 1 of Epstein-Barr virus contain a lipid raft targeting signal and play a critical role in cytostasis. J Virol 77: 3749–3758, 2003.[Abstract/Free Full Text]
  27. Crossthwaite AJ, Seebacher T, Masada N, Ciruela A, Dufraux K, Schultz JE, and Cooper DMF. The cytosolic domains of Ca2+-sensitive adenylyl cyclases dictate their targeting to plasma membrane lipid rafts. J Biol Chem 280: 6380–6391, 2005.[Abstract/Free Full Text]
  28. Dietrich C, Bagatolli LA, Volovyk ZN, Thompson NL, Levi M, Jacobson K, and Gratton E. Lipid rafts reconstituted in model membranes. Biophys J 80: 1417–1428, 2001.[ISI][Medline]
  29. Douglass AD and Vale RD. Single-molecule microscopy reveals plasma membrane microdomains created by protein-protein networks that exclude or trap signaling molecules in T cells. Cell 121: 937–950, 2005.[CrossRef][ISI][Medline]
  30. Dykstra M, Cherukuri A, Sohn HW, Tzeng SJ, and Pierce SK. Location is everything: lipid rafts and immune cell signaling. Annu Rev Immunol 21: 457–481, 2003.[CrossRef][ISI][Medline]
  31. Epand RF, Thomas A, Brasseur R, Vishwanathan SA, Hunter E, and Epand RE. Juxtamembrane protein segments that contribute to recruitment of cholesterol into domains. Biochemistry 45: 6105–6114, 2006.[CrossRef][Medline]
  32. Fastenberg ME, Shogomori H, Xu X, Brown DA, and London E. Exclusion of a transmembrane-type peptide from ordered-lipid domains (rafts) detected by fluorescence quenching: extension of quenching analysis to account for the effects of domain size and domain boundaries. Biochemistry 42: 12376–12390, 2003.[CrossRef][Medline]
  33. Field KA, Holowka D, and Baird B. Structural aspects of the association of Fc-epsilonRI with detergent-resistant membranes. J Biol Chem 274: 1753–1758, 1999.[Abstract/Free Full Text]
  34. Fragoso R, Ren D, Zhang X, Su MW, Burakoff SJ, and YJ J. Lipid raft distribution of CD4 depends on its palmitoylation and association with Lck, and evidence for CD4-induced lipid raft aggregation as an additional mechanism to enhance CD3 signaling. J Immunol 170: 913–921, 2003.[Abstract/Free Full Text]
  35. Gagnoux-Palacios L, Dans M, van’t Hof W, Mariotti A, Pepe A, Meneguzzi G, Resh MD, and Giancotti FG. Compartmentalization of integrin alpha6beta4 signaling in lipid rafts. J Cell Biol 162: 1189–1196, 2003.[Abstract/Free Full Text]
  36. Gaus K, Chklovskaia E, de St. Groth BF, Jessup W, and Harder T. Condensation of the plasma membrane at the site of T lymphocyte activation. J Cell Biol 171: 121–131, 2005.[Abstract/Free Full Text]
  37. Glebov OO and Nichols BJ. Distribution of lipid raft markers in live cells. Biochem Soc Trans 32: 673–675, 2004.[CrossRef][ISI][Medline]
  38. Glebov OO and Nichols BJ. Lipid raft proteins have a random distribution during localized activation of the T-cell receptor. Nat Cell Biol 6: 238–243, 2004.[ISI][Medline]
  39. Gosse JA, Wagenknecht-Wiesner A, Holowka D, and Baird B. Transmembrane sequences are determinants of immunoreceptor signaling. J Immunol 175: 2123–2131, 2005.[Abstract/Free Full Text]
  40. Gri G, Molon B, Manes S, Pozzan T, and Viola A. The inner side of T cell lipid rafts Immunol Lett 94: 247–252, 2004.[CrossRef][ISI][Medline]
  41. Hammond AT, Heberle FA, Baumgart T, Holowka D, Baird B, and Feigenson GW. Crosslinking a lipid raft component triggers liquid ordered-liquid disordered phase separation in model plasma membranes Proc Natl Acad Sci USA 102: 6320–6325, 2005.[Abstract/Free Full Text]
  42. Hancock JF. Lipid rafts: contentious only from simplistic standpoints. Nat Rev Mol Cell Biol 7: 456–462, 2006.[CrossRef][ISI][Medline]
  43. Harder T and Kuhn M. Selective accumulation of raft-associated membrane protein Lat in T cell receptor signaling assemblies. J Cell Biol 151: 199–208, 2000.[Abstract/Free Full Text]
  44. Heberle FA, Buboltz JT, Stringer D, and Feigenson GW. Fluorescence methods to detect phase boundaries in lipid bilayer mixtures. Biochim Biophys Acta 1746: 186–192, 2005.[Medline]
  45. Heerklotz H. Triton promotes domain formation in lipid raft mixtures. Biophys J 83: 2693–2701, 2002.[ISI][Medline]
  46. Heerklotz H, Szadkowska H, Anderson T, and Seelig J. The sensitivity of lipid domains to small perturbations demonstrated by the effect of Triton. J Mol Biol 329: 793–799, 2003.[CrossRef][ISI][Medline]
  47. Hérincs Z, Corset V, Cahuzac N, Furne C, Castellani V, Hueber A-O, and Mehlen P. DCC association with lipid rafts is required for netrin-1-mediated axon guidance J Cell Sci 118: 1687–1692, 2005.[Abstract/Free Full Text]
  48. Higuchi H, Yamashita T, Yoshikawa H, and Tohyama M. PKA phosphorylates the p75 receptor and regulates its localization to lipid rafts. EMBO J 22: 1790–1800, 2003.[CrossRef][ISI][Medline]
  49. Holowka D, Gosse JA, Hammond AT, Han X, Sengupta P, Smith NL, Wagenknecht-Wiesner A, Wu M, Young RM, and Baird B. Lipid segregation and IgE receptor signaling: a decade of progress. Biochim Biophys Acta 1746: 252–259, 2005.[Medline]
  50. Hope HR and Pike LJ. Phosphoinositides and phosphoinositide-utilizing enzymes in detergent-insoluble lipid domains. Mol Biol Cell 7: 843–851, 1996.[Abstract]
  51. Huang J and Feigenson GW. A microscopic interaction model of maximum solubility of cholesterol in lipid bilayers. Biophys J 76: 2142–2157, 1999.[ISI][Medline]
  52. Janes PW, Ley SC, and Magee AI. Aggregation of lipid rafts accompanies signaling via the T cell antigen receptor. J Cell Biol 147: 447–461, 1999.[Abstract/Free Full Text]
  53. Jing SQ and Trowbridge IS. Nonacylated human transferrin receptors are rapidly internalized and mediate iron uptake. J Biol Chem 265: 11555–11559, 1990.[Abstract/Free Full Text]
  54. Keller P and Simons K. Cholesterol is required for surface transport of influenza virus hemagglutinin. J Cell Biol 140: 1357–1367, 1998.[Abstract/Free Full Text]
  55. Kenworthy AK, Nichols BJ, Remmert CL, Hendrix GM, Kumar M, Zimmerberg J, and Lippincott-Schwartz J. Dynamics of putative raft-associated proteins at the cell surface. J Cell Biol 165: 735–746, 2004.[Abstract/Free Full Text]
  56. Kenworthy AK, Petranova N, and Edidin M. High-resolution FRET microscopy of cholera toxin B-subunit and GPI-anchored proteins in cell plasma membranes. Mol Biol Cell 11: 1645–1655, 2000.[Abstract/Free Full Text]
  57. Kirkham M and Parton RG. Clathrin-independent endocytosis: new insights into caveolae and non-caveolar lipid raft carriers. Biochim Biophys Acta 1745: 273–286, 2005.[Medline]
  58. Kleuss C and Krause E. G alpha-s is palmitoylated at the N-terminal glycine. EMBO J 22: 826–832, 2003.[CrossRef][ISI][Medline]
  59. Kusumi A and Suzuki K. Toward understanding the dynamics of membrane-raft-based molecular interactions. Biochim Biophys Acta 1746: 234–251, 2005.[Medline]
  60. Lafont F and van der Goot FG. Bacterial invasion via lipid rafts. Cell Microbiol 7: 613–620, 2005.[CrossRef][ISI][Medline]
  61. Lagerholm BC, Weinreb GE, Jacobson K, and Thompson NL. Detecting microdomains in intact cell membranes. Ann Rev Phys Chem 56: 309–336, 2005.[CrossRef][ISI][Medline]
  62. Lei Y, Hagen GM, Smith SM, Barisas BG, and Roess DA. Chimeric GnRH-LH receptors and LH receptors lacking C-terminus palmitoylation sites do not localize to plasma membrane rafts. Biochem Biophys Res Commun 337: 430–434, 2005.[CrossRef][ISI][Medline]
  63. Lencer WI and Saslowsky D. Raft trafficking of AB(5) subunit bacterial toxins. Biochim Biophys Acta 1746: 314–321, 2005.[Medline]
  64. Li M, Yang C, Tong S, Weidmann A, and Compans RW. Palmitoylation of the murine leukemia virus envelope protein is critical for lipid raft association and surface expression. J Virol 76: 11845–11852, 2002.[Abstract/Free Full Text]
  65. Lin J, Weiss A, and Finco TS. Localization of LAT in glycolipid-enriched microdomains is required for T cell activation. J Biol Chem 274: 28861–28864, 1999.[Abstract/Free Full Text]
  66. McConnell HM and Radhakrishnan A. Condensed complexes of cholesterol and phospholipids. Biochim Biophys Acta 1610: 159–173, 2003.[Medline]
  67. McIntosh TJ, Vidal A, and Simon SA. Sorting of lipids and transmembrane peptides between detergent-soluble bilayers and detergent-resistant rafts. Biophys J 85: 1656–1666, 2003.[Medline]
  68. Meiri KF. Lipid rafts and regulation of the cytoskeleton during T cell activation. Philos Trans R Soc Lond B Biol Sci 360: 1663–1672, 2005.[CrossRef][Medline]
  69. Melkonian KA, Chu T, Tortorella LB, and Brown DA. Characterization of proteins in detergent-resistant membrane complexes from Madin-Darby canine kidney epithelial cells. Biochemistry 34: 16161–16170, 1995.[CrossRef][Medline]
  70. Melkonian KA, Ostermeyer AG, Chen JZ, Roth MG, and Brown DA. Role of lipid modifications in targeting proteins to detergent-resistant membrane rafts: many raft proteins are acylated, while few are prenylated. J Biol Chem 274: 3910–3917, 1999.[Abstract/Free Full Text]
  71. Miura Y, Hanada K, and Jones TLZ. Gs signaling is intact after disruption of lipid rafts. Biochemistry 40: 15418–15423, 2001.[CrossRef][Medline]
  72. Moffett S, Brown DA, and Linder ME. Lipid-dependent targeting of G proteins into rafts. J Biol Chem 275: 2191–2198, 2000.[Abstract/Free Full Text]
  73. Montixi C, Langlet C, Bernard A-M, Thimonier J, Dubois C, Wurbel M-A, Chauvin J-P, Pierres M, and He H-T. Engagement of T cell receptor triggers its recruitment to low-density detergent-insoluble membrane domains. EMBO J 17: 5334–5348, 1998.[CrossRef][ISI][Medline]
  74. Mukherjee S, Soe TT, and Maxfield FR. Endocytic sorting of lipid analogues differing solely in the chemistry of their hydrophobic tails. J Cell Biol 144: 1271–1284, 1999.[Abstract/Free Full Text]
  75. Neame SJ, Uff CR, Sheikh H, Wheatley SC, and Isacke CM. CD44 exhibits a cell type dependent interaction with Triton X-100 insoluble, lipid rich, plasma membrane domains. J Cell Sci 108: 3127–3135, 1995.[Abstract]
  76. Niethammer P, Delling M, Sytnyk V, Dityatev A, Fukami K, and Schachner M. Cosignaling of NCAM via lipid rafts and the FGF receptor is required for neuritogenesis. J Cell Biol 157: 521–532, 2002.[Abstract/Free Full Text]
  77. Nimmo MR and Cross NL. Structural features of sterols required to inhibit human sperm capacitation. Biol Reprod 68: 1308–1317, 2003.[Abstract/Free Full Text]
  78. Ostermeyer AG, Beckrich BT, Ivarson KA, Grove KE, and Brown DA. Glycosphingolipids are not essential for formation of detergent-resistant membrane rafts in melanoma cells. Methyl-beta-cyclodextrin does not affect cell-surface transport of a GPI-anchored protein. J Biol Chem 274: 34459–34466, 1999.[Abstract/Free Full Text]
  79. Parton RG, Hanzal-Bayer M, and Hancock JF. Biogenesis of caveolae: a structural model for caveolin-induced domain formation. J Cell Sci 119: 787–796, 2006.[Abstract/Free Full Text]
  80. Pelkmans L. Secrets of caveolae- and lipid raft-mediated endocytosis revealed by mammalian viruses. Biochim Biophys Acta 1746: 295–304, 2005.[Medline]
  81. Perschl A, Lesley J, English N, Hyman R, and Trowbridge IS. Transmembrane domain of CD44 is required for its detergent insolubility in fibroblasts. J Cell Sci 108: 1033–1041, 1995.[Abstract]
  82. Pike LJ. Lipid rafts: bringing order to chaos. J Lipid Res 44: 655–667, 2003.[Abstract/Free Full Text]
  83. Plowman SJ and Hancock JF. Ras signaling from plasma membrane and endomembrane microdomains. Biochim Biophys Acta 1746: 274–283, 2005.[Medline]
  84. Plowman SJ, Muncke C, Parton RG, and Hancock JF. H-ras, K-ras, and inner plasma membrane raft proteins operate in nanoclusters with differential dependence on the actin cytoskeleton. Proc Natl Acad Sci USA 102: 15500–15505, 2005.[Abstract/Free Full Text]
  85. Popik W and Alce TM. CD4 receptor localized to non-raft membrane microdomains supports HIV-1 entry: Identification of a novel raft localization marker in CD4. J Biol Chem 279: 704–712, 2004.[Abstract/Free Full Text]
  86. Puertollano R and Alonso MA. A short peptide motif at the carboxyl terminus is required for incorporation of the integral membrane MAL protein to glycolipid-enriched membranes. J Biol Chem 273: 12740–12745, 1998.[Abstract/Free Full Text]
  87. Pyenta PS, Holowka D, and Baird B. Cross-correlation analysis of inner-leaflet-anchored green fluorescent protein co-redistributed with IgE receptors and outer leaflet lipid raft components. Biophys J 80: 2120–2132, 2001.[Medline]
  88. Rajendran L, Masilamani M, Solomon S, Tikkanen R, Stuermer CA, Plattner H, and Illges H. Asymmetric localization of flotillins/reggies in pre-assembled platforms confers inherent polarity to hematopoietic cells. Proc Natl Acad Sci USA 100: 8241–8246, 2003.[Abstract/Free Full Text]
  89. Ren X, Ostermeyer AG, Ramcharan LT, Zeng Y, Lublin DM, and Brown DA. Conformational defects slow Golgi exit, block oligomerization, and reduce raft affinity of caveolin-1 mutant proteins. Mol Biol Cell 15: 4556–4567, 2004.[Abstract/Free Full Text]
  90. Romanenko VG, Rothblat GH, and Levitan I. Sensitivity of volume-regulated anion current to cholesterol structural analogues. J Gen Physiol 123: 77–88, 2003.[Abstract/Free Full Text]
  91. Röper K, Corbeil D, and Huttner WB. Retention of prominin in microvilli reveals distinct cholesterol-based lipid microdomains in the apical plasma membrane. Nat Cell Biol 2: 582–592, 2000.[CrossRef][ISI][Medline]
  92. Rousso I, Mixon MB, Chen BK, and Kim PS. Palmitoylation of the HIV-1 envelope glycoprotein is critical for viral infectivity. Proc Natl Acad Sci USA 97: 13523–13525, 2000.[Abstract/Free Full Text]
  93. Saad JS, Miller J, Tai J, Kim A, Ghanam RH, and Summers MF. Structural basis for targeting HIV-1 Gag proteins to the plasma membrane for virus assembly. Proc Natl Acad Sci USA 103: 11364–11369, 2006.[Abstract/Free Full Text]
  94. Scheiffele P, Roth MG, and Simons K. Interaction of influenza virus haemagglutinin with sphin-golipid-cholesterol membrane domains via its transmembrane domain. EMBO J. 16: 5501–5508, 1997.[CrossRef][ISI][Medline]
  95. Schroeder R, London E, and Brown D. Interactions between saturated acyl chains confer detergent resistance on lipids and GPI-anchored proteins: GPI-anchored proteins in liposomes and cells show similar behavior. Proc Natl Acad Sci USA 91: 12130–12134, 1994.[Abstract/Free Full Text]
  96. Schuck S, Honsho M, Ekroos K, Shevchenko A, and Simons K. Resistance of cell membranes to different detergents. Proc Natl Acad Sci USA 100: 5795–5800, 2003.[Abstract/Free Full Text]
  97. Sharma P, Varma R, Sarasij RC, Ira, Gousset K, Krishnamoorthy G, Rao M, and Mayor S. Nanoscale organization of multiple GPI-anchored proteins in living cell membranes. Cell 116: 577–589, 2004.[CrossRef][ISI][Medline]
  98. Sheets ED, Holowka D, and Baird B. Critical role for cholesterol in Lyn-mediated tyrosine phosphorylation in Fc{varepsilon}RI and their association with detergent-resistant membranes. J Cell Biol 145: 877–887, 1999.[Abstract/Free Full Text]
  99. Shogomori H, Hammond AT, Ostermeyer AG, Barr DJ, Feigenson GW, Erwin London E, and Brown DA. Palmitoylation and intracellular-domain interactions both contribute to raft targeting of linker for activation of T cells (LAT). J Biol Chem 280: 18931–18942, 2005.[Abstract/Free Full Text]
  100. Shrimpton CN, Borthakur G, Larrucea S, Cruz MA, Dong JF, and Lopez JA. Localization of the adhesion receptor glycoprotein Ib-IX-V complex to lipid rafts is required for platelet adhesion and activation. J Exp Med 196: 1057–1066, 2002.[Abstract/Free Full Text]
  101. Silvius J. Fluorescence energy transfer reveals microdomain formation at physiological temperatures in lipid mixtures modeling the outer leaflet of the plasma membrane. Biophys J 85: 1034–1045, 2003.[ISI][Medline]
  102. Silvius JR, del Guidice D, and Lafleur M. Cholesterol at different bilayer concentrations can promote or antagonize lateral segregation of phospholipids of differing acyl chain length. Biochemistry 35: 15198–15208, 1996.[CrossRef][Medline]
  103. Simons K and van Meer G. Lipid sorting in epithelial cells. Biochemistry 27: 6197–6202, 1988.[CrossRef][Medline]
  104. Sohn HW, Tolar P, Jin T, and Pierce SK. Fluorescence resonance energy transfer in living cells reveals dynamic membrane changes in the initiation of B cell signaling. Proc Natl Acad Sci USA 103: 8143–8148, 2006.[Abstract/Free Full Text]
  105. Tall RD, Alonso MA, and Roth MG. Features of influenza HA required for apical sorting differ from those required for association with DRMs or MAL. Traffic 4: 838–849, 2003.[CrossRef][ISI][Medline]
  106. Tanimura N, Saitoh SI KS, Kosugi A,, and Miyake K. Palmitoylation of LAT contributes to its subcellular localization and stability. Biochem Biophys Res Commun 341: 1177–1183, 2006.[CrossRef][ISI][Medline]
  107. Thomas JL, Holowka D, Baird B, and Webb WW. Large scale co-aggregation of fluorescent lipid probes with cell surface proteins. J Cell Biol 125: 795–802, 1994.[Abstract/Free Full Text]
  108. van Duyl BY, Rijkers DT, de Kruijff B, and Killian JA. Influence of hydrophobic mismatch and palmitoylation on the association of transmembrane alpha-helical peptides with detergent-resistant membranes. FEBS Lett 523: 79–84, 2002.[CrossRef][ISI][Medline]
  109. Veatch SL and Keller SL. Seeing spots: complex phase behavior in simple membranes. Biochim Biophys Acta 1746: 172–185, 2005.[Medline]
  110. Vidal A and McIntosh TJ. Transbilayer peptide sorting between raft and nonraft bilayers: comparisons of detergent extraction and confocal microscopy. Biophys J 89: 1102–1108, 2005.[Medline]
  111. Viola A, Schroeder S, Sakakibara Y, and Lanzavecchia A. T lymphocyte costimulation mediated by reorganization of membrane microdomains. Science 283: 680–682, 1999.[Abstract/Free Full Text]
  112. Wang TY, Leventis R, and Silvius JR. Partitioning of lipidated peptide sequences into liquid-ordered lipid domains in model and biological membranes. Biochemistry 40: 13031–13040, 2001.[CrossRef][Medline]
  113. Wolf AA, Jobling MG, Wimer-Mackin S, Ferguson-Maltzman M, Madara JL, Holmes RK, and Lencer WI. Ganglioside structure dictates signal transduction by cholera toxin and association with caveolae-like membrane domains in polarized epithelia. J Cell Biol 141: 917–927, 1998.[Abstract/Free Full Text]
  114. Xavier R, Brennan T, Li Q, McCormack C, and Seed B. Membrane compartmentation is required for efficient T cell activation. Immunity 8: 723–732, 1998.[CrossRef][ISI][Medline]
  115. Xu X, Bittman R, Duportail G, Heissler D, Vilcheze C, and London E. Effect of the structure of natural sterols and sphingolipids on the formation of ordered sphingolipid/sterol domains (rafts). Comparison of cholesterol to plant, fungal, and disease-associated sterols and comparison of sphingomyelin, cerebrosides, and ceramide. J Biol Chem 276: 33540–33546, 2001.[Abstract/Free Full Text]
  116. Yamabhai M and Anderson RGW. Second cysteine-rich region of epidermal growth factor receptor contains targeting information for caveolae/rafts. J Biol Chem 277: 24843–24846, 2002.[Abstract/Free Full Text]
  117. Yang H and Reinherz EL. Dynamic recruitment of human CD2 into lipid rafts. Linkage to T cell signal transduction. J Biol Chem 276: 18775–18785, 2001.[Abstract/Free Full Text]
  118. Yasui T, Luftig M, Soni V, and Kieff E. Latent infection membrane protein transmembrane FWLY is critical for intermolecular interaction, raft localization, and signaling. Proc Natl Acad Sci USA 101: 278–283, 2004.[Abstract/Free Full&