Lipid Rafts and the Regulation of Exocytosis

Authors

  • Christine Salaün,

    1. Sir Henry Wellcome Laboratory of Cell Biology, Division of Biochemistry & Molecular Biology, Davidson Building,
      University of Glasgow, Glasgow, G12 8QQ, United Kingdom
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  • Declan J. James,

    1. Sir Henry Wellcome Laboratory of Cell Biology, Division of Biochemistry & Molecular Biology, Davidson Building,
      University of Glasgow, Glasgow, G12 8QQ, United Kingdom
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  • Luke H. Chamberlain

    Corresponding author
    1. Sir Henry Wellcome Laboratory of Cell Biology, Division of Biochemistry & Molecular Biology, Davidson Building,
      University of Glasgow, Glasgow, G12 8QQ, United Kingdom
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Corresponding author: Luke H. Chamberlain, l.chamberlain@bio.gla.ac.uk

Abstract

Exocytosis is the process whereby intracellular fluid-filled vesicles fuse with the plasma membrane, incorporating vesicle proteins and lipids into the plasma membrane and releasing vesicle contents into the extracellular milieu. Exocytosis can occur constitutively or can be tightly regulated, for example, neurotransmitter release from nerve endings. The last two decades have witnessed the identification of a vast array of proteins and protein complexes essential for exocytosis. SNARE proteins fill the spotlight as probable mediators of membrane fusion, whereas proteins such as munc18/nsec1, NSF and SNAPs function as essential SNARE regulators. A central question that remains unanswered is how exocytic proteins and protein complexes are spatially regulated. Recent studies suggest that lipid rafts, cholesterol and sphingolipid-rich microdomains, enriched in the plasma membrane, play an essential role in regulated exocytosis pathways. The association of SNAREs with lipid rafts acts to concentrate these proteins at defined sites of the plasma membrane. Furthermore, cholesterol depletion inhibits regulated exocytosis, suggesting that lipid raft domains play a key role in the regulation of exocytosis. This review examines the role of lipid rafts in regulated exocytosis, from a passive role as spatial coordinator of exocytic proteins to a direct role in the membrane fusion reaction.

The intracellular transport of proteins and lipids relies to a large extent on their sorting into specific vesicle populations, the directional movement of the vesicles through the cell, and the subsequent fusion of the vesicles with specific cellular compartments. The fusion of vesicles with the plasma membrane (PM) occurs in a process called exocytosis; this membrane fusion event mediates the targeting of proteins and lipids to the PM and the secretion of molecules from the cell. Exocytosis can occur constitutively or can be tightly regulated.

Constitutive exocytosis events include the fusion of vesicles derived from the trans-Golgi network (TGN) with the PM, which is essential for the insertion of newly synthesized proteins and lipids into the PM. Polarized cells have developed specialized mechanisms for the targeting of these TGN-derived vesicles to specific regions of the PM, for example, apical vs. basolateral membrane in polarized epithelial cells (1). In addition, proteins that are constitutively recycled through the endosomal system, such as the transferrin receptor, are transported to the cell surface via the fusion of endosomal vesicles with the PM. These constitutive pathways operate in all cells. In addition, a number of cell types undergo a more specialized form of exocytosis known as regulated exocytosis. Exocytosis of regulated secretory vesicles only occurs upon receipt of a specific stimulus, such as exocytosis of synaptic vesicles in nerve cells. In the majority of cases, regulated exocytosis is stimulated by a local and transient increase in calcium levels (2).

Exocytosis can involve the full fusion of a vesicle with the PM or, in more specialized cases such as regulated exocytosis from neuronal and neuroendocrine cells, can also occur by a “kiss-and-run” mechanism. Kiss-and-run exocytosis involves the formation of a transient fusion pore that allows release of a limited amount of the vesicle content before the pore re-seals and the vesicle is released from the plasma membrane (3).

The study of regulated exocytosis at the molecular level has been driven by a number of key questions: How are vesicles recruited to the PM? What proteins anchor vesicles to the PM? What proteins sense and respond to elevated calcium levels? What proteins initiate and catalyze membrane fusion? Finally, how are all these proteins regulated? These questions have led to the identification of numerous proteins, each with their own intricate contribution to exocytosis. The high efficiency of the exocytosis machinery is exemplified in the ultra-fast response it can exhibit to appropriate stimuli; for example, synaptic vesicles can fuse with the presynaptic PM microseconds following calcium influx (4).

SNARE Proteins: Universally Conserved Fusion Proteins

All intracellular membrane fusion events in eukaryotic cells involve the interaction of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) proteins on one membrane with SNAREs present on the other membrane. Analysis of the human genome reveals that there are some 35 different SNARE proteins, with specific SNAREs being localized to specific membrane compartments (5). SNARE protein interactions may therefore contribute to the specificity of membrane fusion (6). The first SNARE complex identified was purified from rat brain, and consisted of syntaxin 1, SNAP-25 and VAMP (7,8). In brain, syntaxin and SNAP-25 are associated with the plasma membrane, whereas VAMP is a synaptic vesicle protein, hence the interaction of these three proteins provides a potential mechanism to mediate synaptic vesicle attachment to the presynaptic plasma membrane. This trimeric complex was called the “SNARE” complex as it provided a docking site for NSF (N-ethylmaleimide-sensitive factor) and SNAP (soluble NSF attachment protein), proteins that had previously been implicated in a number of intracellular trafficking steps (9,10). As stated above, whereas SNAP and NSF have a fairly ubiquitous function in membrane fusion pathways, the SNARE protein homologs that function in a particular membrane fusion event vary depending on the cellular location of membrane fusion and the cell type.

SNARE complex formation was originally suggested to mediate vesicle tethering/docking to the plasma membrane (7). However, later studies did not support this idea, as inhibition of SNARE protein function did not inhibit docking of regulated secretory vesicles at presynaptic terminals or in PC12 cells, but instead increased the number of morphologically docked vesicles (11,12). These data are consistent with a postdocking function for SNAREs in regulated exocytosis and, indeed, analysis of SNARE function in an in vitro assay demonstrated that the interaction of SNAREs on distinct vesicle populations promoted vesicle–vesicle fusion (13). Although current models support the view that SNAREs mediate membrane fusion (or function just upstream from fusion), it is clear that SNAREs are not sufficient for membrane fusion in vivo, and that a large number of other accessory proteins are required for effective exocytosis (14,15).

SNARE Complex Regulation

Because of their central role in membrane fusion and exocytosis, SNARE proteins are subject to strict regulation. One essential SNARE regulatory protein is munc18/nSec1 (16); this protein interacts with monomeric syntaxin 1, preventing the interaction of syntaxin with SNAP-25 and VAMP (17). The interaction of munc18 with syntaxin may be important for the controlled conversion of syntaxin to an “open” conformation that is compatible with SNARE complex formation. This model of munc18 function suggests that it acts as a key regulator of SNARE complex formation and membrane fusion. Indeed a munc18 mutant with a reduced affinity for syntaxin modified the kinetics of regulated exocytosis when expressed in adrenal chromaffin cells (18). Munc18 is also required for docking of regulated secretory vesicles in chromaffin cells and Caenorhabditis elegans (19,20).

Following membrane fusion, the ATPase NSF is recruited to the cis-SNARE complex by the adapter protein α/βSNAP (21). NSF couples ATP hydrolysis to SNARE complex disassembly (7), allowing recycling of SNAREs and subsequent rounds of exocytosis. Thus, the assembly and disassembly of the SNARE complex is tightly controlled, and a vast array of other proteins have also been implicated in the regulation of SNARE function.

As numerous different proteins regulate SNARE protein function, questions arise as to how the cell regulates these protein–protein interactions. How does the cell ensure that SNARE protein interactions are specific and appropriate for efficient exocytosis? What mechanisms exist to ensure the physical separation of proteins and protein complexes within the membrane? Are all SNARE proteins active for membrane fusion?

Lipid Rafts: Membrane Platforms Regulating Intracellular Pathways

The lipids that compose cellular membranes are diverse, and as such have different affinities towards proteins and other lipids. The lipid “raft” hypothesis suggests that sphingolipids and cholesterol cluster into discrete regions of the cell membrane (22). These sphingolipid- and cholesterol-rich domains have been termed lipid “rafts” because they exist in a less fluid and more ordered state than glycerophospholipid-rich domains of the membrane. Studies on model membranes clearly demonstrate clustering and segregation of sphingolipids, cholesterol and certain types of glycerophospholipids, and there is now also evidence that raft-type domains exist in living cells (23–26).

Lipid rafts are resistant to solubilization by cold nonionic detergents; this resistance has been used as the criterion for raft purification from numerous cell types, and has allowed a detailed analysis of raft function in various cellular pathways (27). To avoid confusion, we reserve the use of the term “lipid raft” to describe membrane domains that are resistant to detergent extraction. In addition to the characterization of detergent-insoluble lipid rafts, fluorescent imaging techniques such as fluorescence energy transfer and patching/co-patching of membrane proteins have provided essential data on the domain structure of the plasma membrane in fixed and living cells (28,29).

The ability of lipid rafts to sequester specific proteins and to exclude others makes them ideally suited to spatially organize cellular pathways. Rafts have been implicated in the regulation of a range of signal transduction pathways, where raft association of components of a signaling cascade likely facilitates protein–protein interactions and signal amplification (30). Rafts have also been implicated in membrane traffic pathways, such as the formation of regulated and constitutive secretory vesicles (31).

SNAREs, Lipid Rafts and Cholesterol-Rich Domains

Evidence that lipid rafts play a key role in regulated exocytosis has emerged from a number of recent studies examining the membrane domain distribution of SNARE proteins. SNARE association with detergent-insoluble lipid rafts was first documented in polarized Madin-Darby canine kidney (MDCK) cells, where apically targeted SNAREs cofractionated with lipid rafts (32). Following this, two independent studies demonstrated that SNAREs are localized in raft-type domains in PC12 cells. Lang et al. (33) observed that syntaxin 1A and SNAP-25 were present in clusters at the plasma membrane in PC12 cells; these clusters dispersed following cholesterol extraction. This group could not isolate SNAREs in detergent-insoluble raft fractions, and suggested that SNAREs were localized in cholesterol-rich membrane domains that were distinct from lipid rafts. However, Chamberlain et al. (34) demonstrated that a fraction (∼ 20%) of syntaxin 1A and SNAP-25 copurified with detergent-insoluble rafts from PC12 cells in a cholesterol-dependent manner. Importantly, both of these studies demonstrated that cholesterol depletion inhibited regulated exocytosis, implicating lipid rafts or similar cholesterol-rich membrane domains as essential regulators of exocytic events.

In addition to the analyses of SNARE association with lipid rafts in PC12 cells, more recent work has shown that exocytic SNAREs also interact with detergent-insoluble lipid rafts in 3T3-L1 adipocytes, RBL mast cells, HeLa cells and brain synaptosomes (35–38). These analyses demonstrate that exocytic SNAREs are raft-associated in diverse cell types, and also that SNARE homologs exhibit different levels of raft association. Whereas around 20% of SNAP-25 was raft associated in PC12 cells, greater than 70% of SNAP-23 was raft associated in adipocytes, and SNAP-23 was similarly enriched in mast cell rafts (34–36). Syntaxin isoforms also display different levels of raft association: syntaxin 2 was excluded from rafts in mast cells, whereas syntaxin 4 was equally present in raft and nonraft domains, and syntaxin 3 was selectively enriched in rafts (36).

The above studies suggest the following:

  • • SNARE proteins are clustered in the plasma membrane in a cholesterol-dependent manner.
  • • A proportion of these cholesterol-rich clusters correspond to lipid raft domains − the amount of SNAREs in lipid raft domains depends upon the specific SNARE isoform and the cell type.
  • • The integrity of lipid raft domains is important for exocytosis.

Targeting of SNAREs to Lipid Rafts

Palmitoylation is a major raft-targeting signal, and around 50% of detergent-insoluble raft proteins in MDCK cells could be labeled with palmitate (39). SNAP-25 is multiply palmitoylated and this probably accounts for its accumulation in lipid raft domains. However, there are likely to be other elements within the SNAP-25 protein that regulate the level of lipid raft association. This is suggested because the SNAP-25 homolog SNAP-23 is also palmitoylated but yet is present in raft fractions at 3-fold higher levels than SNAP-25 (34–36). It will be fascinating to identify regions outside of the palmitoylation domain of SNAP-25 that regulate the level of raft association; mutant proteins with a different affinity for rafts will provide essential tools to understand the importance of SNARE raft association for effective exocytosis.

Whereas SNAP-25/23 contain readily identifiable putative raft-targeting signals, the mechanism underlying syntaxin accumulation in rafts is not obvious. Syntaxin 1A has been shown to interact with cholesterol (33), providing a potential mechanism for raft accumulation of this protein. However, a recent study demonstrated that syntaxin 1A was largely excluded from sphingolipid/cholesterol-rich domains in model membranes (40), suggesting that syntaxin 1A may not have an intrinsic affinity for lipid rafts, and that raft accumulation of this protein depends upon its interaction with another raft protein. Indeed, syntaxin 1A and SNAP-25 have recently been shown to extensively codistribute in clusters at the PM of chromaffin cells. This coclustering was abolished by disrupting the syntaxin/SNAP-25 interaction, suggesting that the microdomain localization of these SNAREs is dependent upon their interaction with each other (41). Another possible explanation as to why syntaxin 1A was largely excluded from raft domains in an in vitro assay (40) is the absence of raft lipids such as phosphoinositides, which may be important for syntaxin accumulation in raft domains in vivo (42,43). In addition, it should be noted that such in vitro studies do not adequately reflect the asymmetry of cell membranes and, as such, are a very simplistic model of membrane architecture.

Lipid Rafts and Viral Fusion

The involvement of rafts in viral entry into cells is an area of intensive research, and lipid raft domains are clearly important for the productive infection of certain enveloped and nonenveloped viruses (44). Perhaps most relevant to this review is the infection cycle of Influenza. Hemagglutinin (HA), an Influenza coat protein, binds to a receptor on the surface of the host cell, leading to internalization of the virus particle. The acidic pH of the endosomal compartment activates HA, which then inserts into the endosomal membrane and catalyzes the fusion of virus and endosome. This fusion step leads to the release of viral components into the cytoplasm, a key step for Influenza infectivity.

The mechanism of HA-mediated membrane fusion has been suggested to share a number of close similarities with SNARE-mediated fusion (45). In this respect it is worth noting that HA has an intrinsic affinity for lipid rafts and is associated with raft domains on the viral membrane (46). Lipid raft association of HA has recently been reported to be important for efficient virus budding (47). In addition, raft incorporation of HA promoted fusion activity in a cell–cell fusion assay, probably as a result of enhanced clustering of the protein (47). Thus, raft association is yet another similarity between HA and SNARE proteins, and implies that lipid rafts play a central role not only in HA function, but also in SNARE protein function.

Rafts and the Regulation of Membrane Fusion and Exocytosis

If SNAREs are localized in different domains at the plasma membrane, what does this mean for membrane fusion? Specifically, do the SNARE clusters in lipid rafts or nonrafts mark the sites of exocytosis? In PC12 cells, regulated secretory vesicles dock and fuse at sites corresponding to syntaxin 1A clusters (33); this is not surprising given that the majority of syntaxin 1A is clustered. Further analyses will hopefully address whether regulated exocytosis occurs at specific SNARE clusters and determine the molecular characteristics of these fusion-competent domains.

The different protein and lipid composition of lipid rafts and nonraft domains is likely to impact directly on membrane fusion and exocytosis. There are a number of possibilities for how lipid rafts may function in regulated exocytosis (Figure 1):

Figure 1.

Figure 1.

Possible models for the spatial segregation of exocytic events in raft and nonraft domains of the plasma membrane.A) Rafts are the preferred site for all exocytic events. Fusion may occur within a raft domain as shown or alternatively, rafts may surround the fusion site. B) Exocytosis predominates in nonraft domains. C) Lipid rafts mark the site for kiss-and-run and similar transient exocytic events, whereas full fusion of the vesicle and plasma membrane occurs in nonraft domains. D) Kiss-and-run and similar types of exocytosis occur predominantly in nonraft domains, whereas full fusion of the vesicle and plasma membrane occurs in lipid rafts. E) Raft and nonraft domains may regulate the docking and fusion of specific pools of vesicles (the Figure shows one vesicle pool with blue content, the other with green content).

  • • The protein/lipid composition of rafts is conducive to exocytosis, whereas membrane fusion in nonraft domains is prevented (Figure 1A). The figure depicts fusion in the middle of the lipid raft domain; alternatively, membrane fusion may occur at the edges of raft domains, or rafts may surround the fusion site.
  • • Fusion occurs exclusively in nonraft domains (Figure 1B).
  • • Lipid rafts and nonraft domains support different types of exocytosis, for example full fusion vs. kiss-and-run exocytosis. In the latter form of exocytosis, a transient fusion pore is formed, allowing release of vesicle contents; this fusion pore then quickly seals and the vesicle is released from the plasma membrane. The lipid composition of rafts and nonraft domains may favor one of these exocytic events (Figure 1C,D).
  • • Raft and nonraft domains may also regulate the fusion of specific vesicle pools (Figure 1E).

Another possibility, not depicted in Figure 1, is that exocytosis involves lipid raft domains present on exocytic vesicles. This is suggested because VAMP2 and tetanus toxin-insensitive VAMP are associated with rafts present on exocytic vesicles (32,35).

What are the molecular mechanisms that could account for differences in fusion at raft and nonraft domains? The following models are suggested for the regulation of exocytosis by lipid rafts: “lipid specificity of fusion”, “protein accumulation/exclusion”, and “regulation of SNARE conformation”. These three models are discussed below. However, it should be emphasized that these models are not exclusive.

Lipid specificity of fusion

In this model the lipid species present in rafts and nonrafts directly regulate membrane fusion and exocytosis. Many observations are in agreement with a lipidic nature of the fusion reaction, and the addition of exogenous lipids affects the efficiency of various biological fusion events, including SNARE-mediated exocytosis (48). It is therefore likely that the intrinsic lipid composition of raft and nonraft domains modulates the efficiency of membrane fusion and exocytosis.

Figure 2 shows the lipidic intermediates described by the popular stalk-pore model for membrane fusion (reviewed in 45,49). The merging of the cis contacting monolayers gives rise to a negatively curved lipid structure called a stalk. The structure of the stalk depends on the composition of the cis monolayers (the outer leaflet of the vesicle membrane and the inner leaflet of the plasma membrane). The addition of cone-shaped lipids which have a negative intrinsic curvature, such as unsaturated phosphatidylethanolamines and diacylglycerol, to the cis leaflets of contacting bilayers enhances membrane fusion (49) (Figure 2). The tight organization of lipids in raft domains may limit the association of such cone-shaped lipids with the inner leaflet of these domains. However, the exact composition and especially the degree of saturation of the acyl chains of the lipids composing the inner leaflet of plasma membrane raft domains is still unclear (50). Interestingly, phosphatidylinositol 4,5-bisphosphate (PIP2) has been suggested to associate with the inner leaflet of lipid raft domains (51,52). This is intriguing, as phosphorylated phosphoinositides promote calcium-dependent fusion in vitro (53), and PIP2 is required for calcium-dependent exocytosis (54). Syntaxin 1A and SNAP-25 have been suggested to interact with PIP2 (42), and syntaxin 1A colocalizes with PIP2 in PC12 cell membranes (43). Thus, interaction of syntaxin 1A and SNAP-25 with PIP2 may regulate stalk formation and membrane fusion in raft domains.

Figure 2.

Figure 2.

Stalk-pore model of membrane fusion highlighting the importance of lipid geometry for stalk and fusion pore formation. A) Plasma membrane and vesicle membrane prior to merger. B) Merger of the cis leaflets of the bilayers leads to formation of a stalk. Cone-shaped lipids in the cis leaflets favor stalk formation (shown in yellow). Contact between the trans leaflets then promotes hemifusion (C) and pore formation (D). Inverted-cone shaped lipids (shown in blue) in the trans leaflets facilitate hemifusion and pore formation.

A possible remodeling of the inner leaflet of the plasma membrane at the site of exocytosis may also create the lipid packing defects required for the formation of the stalk. The interaction of calcium with PIP2 has been proposed to convert this inverted cone-shaped lipid to a cone (55); this reversal of PIP2 geometry would favor stalk formation, and explains the positive effect of this lipid on calcium-stimulated membrane fusion (53). Similarly, a breakdown of cylindrical phosphatidylcholine to the cone-shaped phosphatidic acid by phospholipase D1 (PLD1) has been proposed to be important for regulated exocytosis pathways (56,57). PLD1 activity depends on phosphorylated phosphoinositides (58) and is suggested to reside in lipid rafts (59). Figure 3 depicts calcium and PLD1-induced changes in lipid geometry in raft domains which may promote membrane fusion at these sites. Interestingly, endophilin-mediated conversion of lysophosphatidic acid (inverted cone) to phosphatidic acid (cone) is important for synaptic vesicle invagination from the PM and fission (60), suggesting that changes in lipid geometry may play a role in numerous membrane trafficking pathways.

Figure 3.

Figure 3.

Lipid geometry that may facilitate fusion in raft domains. A) Putative lipid geometry in lipid rafts and nonraft domains. Enrichment of inverted cone-shaped lipids in the outer leaflet of lipid rafts may facilitate pore formation in these domains. B) Calcium- and PLD1-induced changes in inner leaflet lipid raft geometry that may promote stalk formation in these domains. GSL, glycosphingolipid; PIP2, phosphatidylinositol 4,5-bisphosphate; PC, phosphatidylcholine; SM, sphingomyelin; PE, phosphatidylethanolamine; PA, phosphatidic acid; DAG, diacylglycerol.

Following stalk formation a hemifusion state evolves where there is contact between the trans fusing monolayer (inner leaflet of vesicle, outer leaflet of plasma membrane). Formation and expansion of a fusion pore then completes fusion (Figure 2). The overall curvature of these structures is positive and depends on the composition of the trans fusing leaflets. Fusion is enhanced by inverted cone shaped lipids in the trans monolayers (48,49) (Figure 2). Because of their bulky polar heads and the saturation of their acyl chains, glycosphingolipids (which are enriched in raft domains) have a positive spontaneous curvature, and could thus stimulate fusion pore formation. Moreover, the specific composition of the outer leaflet of lipid raft could be the basis for differentiating full fusion from kiss-and-run fusion, as the composition of the outer monolayers affects the opening, flickering and expansion of fusion pores (49).

Another aspect of fusion that deserves mention is the restriction of lipid flow during the opening of the fusion pore. In the model of fusion mediated by the HA fusion peptide of Influenza, the fusion pore formed by HA develops before the lipid pore becomes visible. A scaffold of HA protein has been proposed to explain the lipid flow retardation in this fusion system (61) but one can also imagine that liquid ordered lipid raft domains at the boundaries of fusion sites may also be involved in restricting lipid flow.

Protein accumulation/exclusion

Lipid rafts and nonraft domains have distinct profiles of exocytic proteins and protein complexes. Efficient exocytosis most likely requires spatial regulation of the exocytosis machinery. A simple example makes this point obvious: how does SNARE complex formation between the vesicle and plasma membrane promote exocytosis if SNARE regulators such as munc18 and αSNAP prevent their assembly or disrupt complex formation before membrane fusion? This scenario could be prevented by spatially separating fusion-competent SNARE proteins from SNARE regulators. In PC12 cells, munc18 was not detected in lipid rafts and the syntaxin 1A–munc18 complex was only detected in nonraft fractions (34). A similar situation exists in RBL mast cells, where munc18-2/syntaxin 3 complexes were only detected in nonraft domains (36). In contrast, syntaxin 1A/SNAP-25 complexes were present in both raft and nonraft domains in PC12 cells (34), and SNAP-23/syntaxin 3-containing protein complexes were enriched in raft domains in mast cells (36). These results imply that association of SNARE proteins with raft and nonraft domains of the plasma membrane facilitates spatial separation of protein complexes essential for exocytosis. Interestingly, other SNARE regulators such as αSNAP, NSF and complexin are also excluded from lipid raft domains (34). Thus, by excluding SNARE regulators, lipid rafts may provide a suitable environment for effective SNARE-mediated membrane fusion. The interaction of SNAREs with lipid raft domains also provides a mechanism to concentrate SNARE proteins, an essential requirement for effective exocytosis (62).

Interestingly, a very recent study has shown that P/Q-type calcium channels are raft associated in presynaptic membranes; furthermore, the calcium channels were complexed with the SNARE proteins syntaxin 1A and SNAP-25 in these raft domains (38). The coaccumulation of SNAREs and presynaptic calcium channels in raft domains may contribute to the coupling of calcium influx and neurotransmitter release (38).

Proteins can also directly control the mode of regulated exocytosis; for example, recent work showed isoform-specific effects of the vesicle protein synaptotagmin on kiss-and-run exocytosis and full fusion (63,64). Thus, the protein accumulation/exclusion model for raft function in exocytosis offers a possible mechanism to regulate and spatially separate different forms of exocytosis. It is interesting to note that munc18 has been reported to affect the kinetics of regulated exocytosis (18). The exclusive presence of this protein in nonraft domains may therefore influence the dynamics of membrane fusion at these sites.

Regulation of SNARE conformation

The SNARE proteins present in raft and nonraft domains may have distinct conformations. This is particularly relevant to the transmembrane domain (TMD) of syntaxin, which plays a critical role in SNARE-mediated fusion (48,65). As sphingolipid-rich bilayers are significantly thicker than phospholipid-rich bilayers (66), the TMD of syntaxin is likely to have a distinct orientation in raft and nonraft domains. In support of this idea, a recent molecular dynamics study suggested that the transmembrane domain of syntaxin 1A has a 15° tilt relative to a phospholipid bilayer as a result of the TMD having a greater diameter than the bilayer (67). However, as the thickness of sphingolipid-rich membranes is greater than that of phospholipid membranes, incorporation of syntaxin 1A into sphingolipid-rich rafts would be predicted to significantly reduce the tilt of the TMD of syntaxin (67). The TMD of syntaxin is essential for effective membrane fusion and exocytosis (48,65), and thus the different orientation of the TMD in raft and nonraft domains will undoubtedly affect the efficiency of SNARE-mediated membrane fusion. One model of SNARE-mediated fusion suggests that the interaction of SNARE proteins on different membranes transduces force to the SNARE membrane anchors, pulling up on the TMDs (65). The force applied to SNARE TMDs may be important for initial lipid mixing. The reduced tilt of the syntaxin 1A TMD in rafts may have a positive effect on membrane fusion by generating a greater and more concentrated force on the lipid bilayer (67).

Perspectives and Future Direction

The ideas and models put forward in this review are intended to stimulate research and discussion on the role of lipid rafts in regulated exocytosis and other membrane fusion events. At this stage we have no direct evidence that lipid rafts influence the fusion of regulated exocytic vesicles. However, the described raft association of both SNARE proteins and Influenza HA suggests that lipid rafts may play an important role in membrane fusion reactions. Rafts have the potential to regulate many facets of exocytosis pathways; the selective concentration of specific proteins and lipids in these domains is likely to impinge directly on membrane fusion and exocytosis. Although this review has focused largely on the role of lipid rafts in regulated exocytosis pathways, the high degree of conservation of eukaryotic membrane fusion events suggests that rafts may also be involved in constitutive exocytosis and other types of intracellular membrane fusion. The lipid symmetry of most in vitro fusion assays makes it difficult to draw conclusions about the influence of a particular lipid species on the fusion of asymmetric membranes in vivo. Similarly, it is difficult to draw direct parallels between exocytosis and other membrane fusion pathways, such as viral fusion with the outside of a cell. Thus, a detailed picture of lipid raft function in exocytosis will come only from direct studies in vivo addressing pertinent questions such as: Do vesicles fuse at sites which have the molecular architecture of lipid rafts? Does selective neutralization of SNARE proteins in raft or nonraft domains affect exocytosis? Can different modes of exocytosis be monitored at distinct domains of the PM? Answering these questions should lead to a more detailed molecular picture of lipid raft function in membrane fusion and exocytosis.

Acknowledgments

We would like to thank Gwyn Gould and Nia Bryant (University of Glasgow), and Bob Burgoyne and Alan Morgan (University of Liverpool) for critical reading of the prepared manuscript and helpful discussions. Work in the authors laboratory is funded by the Wellcome Trust, the Biotechnology and Biological Sciences Research Council, and the Diabetes Research and Wellness Foundation.

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