Raft Affinity Research Articles

A comparative structural study in monolayers of GPI fragments and their binary mixtures

Glycosylphosphatidylinositols (GPIs), natural complex glycolipids essential for a range of biological functions, are poorly understood with regard to their interactions and arrangements in cellular membranes. To evaluate the role of the head group in the structure formation in 2D model membranes (monolayers formed at the soft air/liquid interface), we employed the highly surface sensitive grazing incidence X-ray diffraction technique to investigate three GPI-fragments bearing the same hydrophobic part but different head groups. Condensed monolayers of simple GPI fragments are defined only by ordered alkyl chains. The monolayers of more complex fragments are additionally characterized by highly ordered head groups. Due to the strong H-bond network formed by the head groups, GPI-fragment both segregates and induces order into a model membrane phospholipid (POPC) that mimics the liquid-disordered phase of cell membranes. Here, we show that the strong van der Waals interactions between hydrophobic chains overcome the head group interactions and dominate the structure formation in mixtures of GPI-fragment with lipids that form liquid-condensed phases. This behaviour can be linked to the GPIs affinity for the lipid rafts.

Oligomerization is required for normal endocytosis/transcytosis of a GPI-anchored protein in polarized hepatic cells

Targeting of glycosyl-phosphatidylinositol (GPI)-anchored proteins (GPI-APs) in polarized epithelial cells depends on their association with detergent-resistant membrane microdomains called rafts. In MDCK cells, GPI-APs associate with rafts in the trans-Golgi network and are directly delivered to the apical membrane. It has been shown that oligomerization is required for their stabilization in rafts and their apical targeting. In hepatocytes, GPI-APs are first delivered to the basolateral membrane and secondarily reach the apical membrane by transcytosis. We investigated whether oligomerization is required for raft association and apical sorting of GPI-APs in polarized HepG2 cells, and at which step of the pathway oligomerization occurs. Model proteins were wild-type GFP-GPI and a double cysteine GFP-GPI mutant, in which GFP dimerization was impaired. Unlike wild-type GFP-GPI, which was efficiently endocytosed and transcytosed to the apical surface, the double cysteine mutant was basolaterally internalized, but massively accumulated in early endosomes, and reached the bile canaliculi with delayed kinetics. The double cysteine mutant was less resistant to Triton X-100 extraction, and formed fewer high molecular weight complexes. We conclude from these results that, in hepatocytes, oligomerization plays a key role in targeting GPI-APs to the apical membrane, by increasing their affinity for rafts and allowing their transcytosis.

Molecular Dynamics and Concentration in Raft and Boundary Domains in Actin-Depleted Plasma Membrane Vesicles as Revealed by Single-Molecule Imaging

Compartmentalization of the cellular plasma membrane has been considered a critical mechanism for regulating molecular interactions occurring in the plane of the membrane, by varying local molecular concentration and dynamics within and across the compartments. Among various compartments, raft domains, enriched in sterols, sphingolipids, and proteins anchored by saturated aliphatic lipid tails, have been drawing extensive attention but remained elusive due to their nano-meso-scale sizes. Recently developed plasma membrane vesicles (PMVs), which are largely depleted of the actin filaments (actin-based membrane skeleton) but contain virtually the full complement of lipids and proteins of native membranes, provide a unique platform for investigating raft domains because, by lowering the temperature, micron-sized raft-like, liquid-ordered-phase (Lo)-like domains can be induced. Here, using these PMVs with coexisting domains and single-molecule imaging-tracking methods, we examined molecular dynamics and concentration of various molecules in raft and boundary domains. One of the most interesting findings is that GPI-APs, although they preferentially partition into the Lo-like raft domains, continually move back and forth between Lo-like domains and the bulk domain, showing very dynamic partitioning, without any particular concentration in the boundary domain. Their diffusion coefficient within the boundary region has been measured for the first time: it was in the middle of the values for the Lo-like domain and the bulk domain. Other observations using molecules with various levels of raft affinity will be reported and discussed in the context of signal transduction.

Altering Hydrophobic Sequence Lengths Shows That Hydrophobic Mismatch Controls Affinity for Ordered Lipid Domains (Rafts) in the Multitransmembrane Strand Protein Perfringolysin O

The hypothesis that mismatch between transmembrane (TM) length and bilayer width controls TM protein affinity for ordered lipid domains (rafts) was tested using perfringolysin O (PFO), a pore-forming cholesterol-dependent cytolysin. PFO forms a multimeric barrel with many TM segments. The properties of PFO mutants with lengthened or shortened TM segments were compared with that of PFO with wild type TM sequences. Both mutant and wild type length PFO exhibited cholesterol-dependent membrane insertion. Maximal PFO-induced pore formation occurred in vesicles with wider bilayers for lengthened TM segments and in thinner bilayers for shortened TM segments. In diC(18:0) phosphatidylcholine (PC)/diC(14:1) PC/cholesterol vesicles, which form ordered domains with a relatively thick bilayer and disordered domains with a relatively thin bilayer, affinity for ordered domains was greatest with lengthened TM segments and least with shortened TM segments as judged by FRET. Similar results were observed by microscopy in giant vesicles containing sphingomyelin in place of diC(18:0) PC. In contrast, in diC(16:0) PC/diC(14:0) PC/diC(20:1) PC/cholesterol vesicles, which should form ordered domains with a relatively thin bilayer and disordered domains with a relatively thick bilayer, relative affinity for ordered domains was greatest with shortened TM segments and least with lengthened TM segments. The inability of multi-TM segment proteins (unlike single TM segment proteins) to adapt to mismatch by tilting may explain the sensitivity of raft affinity to mismatch. The difference in width sensitivity for single and multi-TM helix proteins may link raft affinity to multimeric state and thus control the assembly of multimeric TM complexes in rafts.

Abstract 3495: The novel phospholipid ether analog CLR1404 decreases glioblastoma stem cell proliferation, suppresses GBM growth, and improves survival

Abstract Most patients treated for glioblastoma multiforme (GBM), the most common primary brain malignancy, have less than 2 years survival due to rapid recurrence. The GBM stem cell (GSC) sub-population exhibits therapeutic resistance and is hypothesized to drive tumor recurrence. Therefore, GSC-specific targeting is likely critical for improving outcome. Phospholipid ether analog, CLR1404 and its radioiodinated isosteres, exhibit cancer cell-specific uptake and prolonged retention in 57/61 cancer cell lines and xenografts (including GBM) due to affinity for cancer cell lipid rafts. In this study, we investigated the therapeutic potential of CLR1404 against GBM and its GSC subpopulation. Multiple sphere-forming GSC lines were isolated from patient specimens with IRB approval, and rigorously validated for self-renewal, multi-lineage potential, and high efficiency orthotopic tumor initiation in NOD-SCID mice. Proliferation assays were performed by addition of CLR1404 in serum-free medium for 24 hours. To test effects on stem cell properties, GSCs were treated with CLR1404, dissociated to single cells, plated at 500 cells in a 96-well plate, and allowed to form spheres (≥2-4 weeks). CLR1404 inhibition of the AKT oncogenic signaling pathway in GSCs was assayed with immunoblot analysis. In vivo, GSCs were pre-treated for 24 hours with CLR1404 prior to orthotopic injection of 200,000 live cells into immunodeficient mice. Subsequent survival curves were then constructed. CLR1404 anti-proliferative effects were seen on all 7 different GSC and GBM lines tested with IC50 values ranging between 5-10 µM using MTS assay; control normal differentiated neural cells exhibited an IC50 of approximately 40 µM. CLR1404 treatment also decreased sphere-forming ability of multiple GSC lines with IC50 values between 5-10 µM. CLR1404 inhibition of AKT activation was observed using immunoblot analysis. CLR1404 pre-treatment of GSCs significantly increased survival time in an orthotopic mouse model (Control: 59±6.1 days; CLR1404: 94±4.4 days), suggesting CLR1404 treatment of the GSC tumor initiating cells improves outcome. This data of CLR1404's therapeutic potential against GBM and its GSCs, combined with previously demonstrated tumor cell targeting specificity of CLR1404 and its radioactive isosteres, provides strong evidence for the potential of novel CLR1404-based therapies to improve GBM outcomes. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3495. doi:1538-7445.AM2012-3495

The Effect of Hydrophobic Match on Transmembrane Protein Raft Affinity

It is believed that lipid rafts control protein-protein and lipid-protein interactions within cell membranes. However, how transmembrane (TM) proteins associate with rafts remains poorly understood. One proposed mechanism is that hydrophobic match/mismatch drives TM proteins into or out of rafts. We tested this hypothesis using perfringolysin O (PFO), a member of the cholesterol-dependent cytolysin family. Upon interaction with cholesterol, PFO inserts into membranes and forms a rigid TM beta-barrel. PFO interacts with rafts, so is a useful model protein to study the origin of TM protein raft affinity. PFO mutants with different length TM segments were generated with each β-strand in each of the two TM β-hairpins lengthened (l -PFO) or shortened (s-PFO) by two residues. Similar to wildtype PFO, these mutants showed cholesterol-dependent membrane binding and pore-formation activities, although with somewhat different properties. A pore detection assay carried out to determine if the pore-forming efficiency of PFO mutants was correlated to membrane width found that l-PFO shows higher pore formation activity in vesicles with thicker bilayers than did s-PFO. Microscopy studies in giant unilamellar vesicles confirmed that both PFO mutants could bind to vesicles with co-existing Lo/Ld domains: l-PFO tends to locate within thick Lo domains, while s-PFO appears at thin Ld domains. Energy transfer (FRET) experiments were also used to compare the raft affinity of wildtype and mutated PFO in vesicles containing ordered domains. Preliminary studies indicate in cholesterol-containing vesicles having wide Lo domains (distearoylphosphatidylcholine) and thin Ld domains (dimyristoleoylphosphatidylcholine), l-PFO showed higher raft affinity, whereas s-PFO shows little raft affinity. The raft affinity of l-PFO and s-PFO was reversed in vesicles containing thinner Lo domains (dipalmitoylphosphatidylcholine/dimyristoylphosphatidylcholine) and thick Ld domains (dieicosenoylphosphatidylcholine). These studies suggest that hydrophobic match may have an important role in TM protein raft affinity.

1Q1624 ロドプシン2量体のラフト親和性とロドプシンのパルミトイル修飾(光生物_視覚・光受容1,第49回日本生物物理学会年会)

1Q1624 ロドプシン2量体のラフト親和性とロドプシンのパルミトイル修飾(光生物_視覚・光受容1,第49回日本生物物理学会年会)

Dietary free fatty acids form alkaline phosphatase-enriched microdomains in the intestinal brush border membrane

Free fatty acids released during intralumenal digestion of dietary fat must pass through the enterocyte brush border membrane before triacylglycerol reassembly and subsequent chylomicron delivery to the lymph system. In the present work fluorescent BODIPY fatty acid analogs were used to study this membrane passage in organ cultured intestinal mucosal explants. We found that in addition to a rapid uptake into the cytoplasm, a fraction of the fatty acid analogs were inserted directly into the brush border membrane. Furthermore, a brief exposure of microvillar membrane vesicles to a fat mixture mimicking a physiological solution of dietary mixed micelles, rearranged the lipid raft microdomain organization of the membranes. Thus, the fat mixture generated a low-density subpopulation of microvillar detergent resistant membranes (DRMs) highly enriched in alkaline phosphatase (AP). Since this GPI-linked enzyme is the membrane protein in the brush border with the highest affinity for lipid rafts, this implies that free fatty acids selectively insert stably into these membrane microdomains. We have previously shown that absorption of dietary lipids transiently induce a selective endocytosis of AP from the brush border, and from work by others it is known that fat absorption is accompanied by a rise in serum AP and secretion of surfactant-like particles from enterocytes. We propose that these physiological processes may be triggered by the sequestering of dietary free fatty acids in lipid raft microdomains of the brush border.

Palmitoylation regulates raft affinity for the majority of integral raft proteins

The physical basis for protein partitioning into lipid rafts remains an outstanding question in membrane biology that has previously been addressed only through indirect techniques involving differential solubilization by nonionic detergents. We have used giant plasma membrane vesicles, a plasma membrane model system that phase separates to include an ordered phase enriching for raft constituents, to measure the partitioning of the transmembrane linker for activation of T cells (LAT). LAT enrichment in the raft phase was dependent on palmitoylation at two juxtamembrane cysteines and could be enhanced by oligomerization. This palmitoylation requirement was also shown to regulate raft phase association for the majority of integral raft proteins. Because cysteine palmitoylation is the only lipid modification that has been shown to be reversibly regulated, our data suggest a role for palmitoylation as a dynamic raft targeting mechanism for transmembrane proteins.

Perfringolysin O Association with Ordered Lipid Domains: Implications for Transmembrane Protein Raft Affinity

Upon interaction with cholesterol, perfringolysin O (PFO) inserts into membranes and forms a rigid transmembrane (TM) β-barrel. PFO is believed to interact with liquid ordered lipid domains (lipid rafts). Because the origin of TM protein affinity for rafts is poorly understood, we investigated PFO raft affinity in vesicles having coexisting ordered and disordered lipid domains. Fluorescence resonance energy transfer (FRET) from PFO Trp to domain-localized acceptors indicated that PFO generally has a raft affinity between that of LW peptide (low raft affinity) and cholera toxin B (high raft affinity) in vesicles containing ordered domains rich in brain sphingomyelin or distearoylphosphatidylcholine. FRET also showed that ceramide, which increases exposure of cholesterol to water and thus displaces it from rafts, does not displace PFO from ordered domains. This can be explained by shielding of PFO-bound cholesterol from water. Finally, FRET showed that PFO affinity for ordered domains was higher in its non-TM (prepore) form than in its TM form, demonstrating that the TM portion of PFO interacts unfavorably with rafts. Microscopy studies in giant unilamellar vesicles confirmed that PFO exhibits intermediate raft affinity, and showed that TM PFO (but not non-TM PFO) concentrated at the edges of liquid ordered domains. These studies suggest that a combination of binding to raft-associating molecules and having a rigid TM structure that is unable to pack well in a highly ordered lipid environment can control TM protein domain localization. To accommodate these constraints, raft-associated TM proteins in cells may tend to locate within liquid disordered shells encapsulated within ordered domains.

Greasing Their Way: Lipid Modifications Determine Protein Association with Membrane Rafts

Increasing evidence suggests that biological membranes can be laterally subdivided into domains enriched in specific lipid and protein components and that these domains may be involved in the regulation of a number of vital cellular processes. An example is membrane rafts, which are lipid-mediated domains dependent on preferential association between sterols and sphingolipids and inclusive of a specific subset of membrane proteins. While the lipid and protein composition of rafts has been extensively characterized, the structural details determining protein partitioning to these domains remain unresolved. Here, we review evidence suggesting that post-translation modification by saturated lipids recruits both peripheral and transmembrane proteins to rafts, while short, unsaturated, and/or branched hydrocarbon chains prevent raft association. The most widely studied group of raft-associated proteins are glycophosphatidylinositol-anchored proteins (GPI-AP), and we review a variety of evidence supporting raft-association of these saturated lipid-anchored extracellular peripheral proteins. For transmembrane and intracellular peripheral proteins, S-acylation with saturated fatty acids mediates raft partitioning, and the dynamic nature of this modification presents an exciting possibility of enzymatically regulated raft association. The other common lipid modifications, that is, prenylation and myristoylation, are discussed in light of their likely role in targeting proteins to nonraft membrane regions. Finally, although the association between raft affinity and lipid modification is well-characterized, we discuss several open questions regarding regulation and remodeling of these post-translational modifications as well as their role in transbilayer coupling of membrane domains.

Protein-Lipid Interaction and Domain Formation in Asymmetric Membranes

The lateral organization of lipids and receptors in cellular membranes is connected to a multitude of biological and pathological processes. The role of sphingolipid- and cholesterol-rich lipid “rafts” as membrane domains that control protein-protein interaction has recently attracted much attention. While the model membrane approach has yielded fundamental insights into cell membrane structure and function, the majority of these studies did not take into account the very important asymmetry between the composition of the inner and the outer leaflets of cell membranes. Therefore, certain key questions remain still unanswered: for example, how is the partition of membrane components in raft domains affected by the presence of non-raft lipids in the inner leaflet? To answer questions of this type we have developed a new method based upon cyclodextrin-induced lipid exchange to create giant unilamellar vesicles (GUVs) featuring the same type of asymmetry encountered in biological membranes. These vesicles can be produced with a very large yield, while avoiding use of organic solvents. The asymmetry of the bilayer can be confirmed by leaflet-targeted Fluorescence Correlation Spectroscopy (FCS). The use of asymmetric GUVs as novel model bilayers should allow the investigation of the principles underlying communication between raft-like domains in opposite leaflets of the bilayer, as well as investigation of the origin of lipid and membrane protein affinity for rafts.

Palmitoyl acyltransferases, their substrates, and novel assays to connect them (Review)

Thio-palmitoylation is the post-translational addition of the 16-carbon fatty acid, palmitate, to the thiol side chain of cysteine residues by a labile thioester bond. Palmitoylation increases the lipophilicity of a protein resulting in dramatic changes in its subcellular distribution such as moving from the endoplasmic reticulum to the plasma membrane or in subtle changes like an increased affinity for cholesterol-rich lipid rafts in membranes. Palmitoylation is also dynamic, making it unique among post-translational protein lipid modifications. Discovering the molecular identity of palmitoyl acyltransferases (PATs) was a watershed event that dramatically accelerated the pace of discovery in the field. Likewise, there has been increased interest in palmitoylation partly because many of the genes encoding PATs have been linked to cancer and other diseases. Now, with a greater understanding of how palmitate is enzymatically attached to proteins, some of the most interesting questions include: What are the substrates of each PAT?; how does a PAT recognize and palmitoylate a substrate?; how are PATs regulated?; and, how is depalmitoylation regulated? The answers to these questions are beginning to unfold due to the recent development of novel assays as well as the expansion and refinement of existing assays. Our ability to understand palmitoylation and its importance to human health and disease is only as good as the methods we use to test our hypotheses. The continued development of methods with increased sensitivity and selectivity is critical to this venture.

Apical Cargo Traverses Endosomal Compartments on the Passage to the Cell Surface

Epithelial polarity is based on intracellular sorting machinery that maintains the asymmetric distribution of lipids and proteins to the cell surface. Dependent on their lipid raft affinity, newly synthesized apical polypeptides are segregated into distinct vesicle populations subsequent to the passage through the Golgi apparatus. Using a combined fluorescence microscopic and biochemical approach, we found that lipid raft-associated sucrase-isomaltase (SI) as well as non-raft-associated lactase-phlorizin hydrolase (LPH) traverse endosomal compartments before entering the apical membrane. Fluorescent fusion proteins of both hydrolases were co-stained with Rab4-, Rab8- and Rab11-positive endosomes in polarized Madin-Darby canine kidney and non-polarized COS-1 cells. Immunoisolation of post-Golgi vesicles subsequent to different times of TGN release revealed that LPH and SI navigate in chronological order through Rab4-, Rab8- and Rab11-positive endosomes. Thereafter, the two hydrolases are segregated into distinct vesicle populations. In addition, apical membrane traffic could be significantly inhibited by RNA interference-mediated depletion of these guanosine triphosphatases. These results suggest that in epithelial cells, lipid raft-dependent and -independent apical cargo follow a transendosomal route.

How Interaction of Perfringolysin O with Membranes Is Controlled by Sterol Structure, Lipid Structure, and Physiological Low pH: INSIGHTS INTO THE ORIGIN OF PERFRINGOLYSIN O-LIPID RAFT INTERACTION

Perfringolysin O (PFO) is a sterol-dependent, pore-forming cytolysin. To understand the molecular basis of PFO membrane interaction, we studied its dependence upon sterol and lipid structure and aqueous environment. PFO interacted with diverse sterols, although binding was affected by double bond location in the sterol rings, sterol side chain structure, and sterol polar group structure. Importantly, a sterol structure promoting formation of ordered membrane domains (lipid rafts) was not critical for interaction. PFO membrane interaction was also affected by phospholipid acyl chain structure, being inversely related to tight acyl chain packing with cholesterol. Experiments using the pre-pore Y181A mutant demonstrated that sterol binding strength and specificity was not affected by whether PFO forms a transmembrane beta-barrel. Combined, these observations are consistent with a model in which the strength and specificity of sterol interaction arises from both sterol interactions with domain 4 and sterol chemical activity within membranes. The lipid raft-binding portions of sterol bound to PFO may remain largely exposed to the lipid bilayer. These results place important constraints upon the origin of PFO raft affinity. Additional experiments demonstrated that the structure of membrane-inserted PFO at low and neutral pH was similar as judged by the effect of phospholipid and sterol structure upon PFO properties and membrane interaction. However, low pH enhanced PFO membrane binding, oligomerization, and pore formation. In lipid vesicles mimicking the exofacial (outer) membrane leaflet, PFO-membrane binding was maximal at pH 5.5-6. This is consistent with the hypothesis that PFO function involves acidic vacuoles.

Single-Molecule Diffusion Reveals Similar Mobility for the Lck, H-Ras, and K-Ras Membrane Anchors

Recent evidence on the occurrence of small (5–700 nm diameter) lipid microdomains in the exoplasmic leaflet of the plasma membrane has evoked interest in the possibility that similar domains may also be present in the cytoplasmic leaflet of the plasma membrane. However, current knowledge about these “lipid rafts”, in live cells is limited. One way to obtain insight into the occurrence and the size of lipid rafts is the use of single-molecule microscopy, which allows one to study the diffusive motion of individual molecules with high positional and temporal accuracy. Using this technique, we compared the diffusion behavior of the Lck membrane anchor, which has a high affinity for lipid rafts, to the diffusion behavior of the K-Ras membrane anchor, which has negligible affinity for rafts and compared the results with those of the H-Ras membrane anchor. Surprisingly, we found only minor differences in the diffusion behavior of the various lipid anchors, indicating that putative cytoplasmic leaflet lipid rafts would have to be small (<137 nm diameter) and do not affect the mobility of membrane-anchored molecules much on timescales up to 60 ms.

Palmitoylation and Intracellular Domain Interactions Both Contribute to Raft Targeting of Linker for Activation of T Cells

Some transmembrane proteins must associate with lipid rafts to function. However, even if acylated, transmembrane proteins should not pack well with ordered raft lipids, and raft targeting is puzzling. Acylation is necessary for raft targeting of linker for activation of T cells (LAT). To determine whether an acylated transmembrane domain is sufficient, we examined raft association of palmitoylated and nonpalmitoylated LAT transmembrane peptides in lipid vesicles by a fluorescence quenching assay, by microscopic examination, and by association with detergent-resistant membranes (DRMs). All three assays detected very low raft association of the nonacylated LAT peptide. DRM association was the same as a control random transmembrane peptide. Acylation did not measurably enhance raft association by the first two assays but slightly enhanced DRM association. The palmitoylated LAT peptide and a FLAG-tagged LAT transmembrane domain construct expressed in cells showed similar DRM association when both were reconstituted into mixed vesicles (containing cell-derived proteins and lipids and excess artificial raft-forming lipids) before detergent extraction. We conclude that the acylated LAT transmembrane domain has low inherent raft affinity. Full-length LAT in mixed vesicles associated better with DRMs than the peptide. However, cells appeared to contain two pools of LAT, with very different raft affinities. Since some LAT (but not the transmembrane domain construct) was isolated in a protein complex, and the Myc- and FLAG-tagged forms of LAT could be mutually co-immunoprecipitated, oligomerization or interactions with other proteins may enhance raft affinity of one pool of LAT. We conclude that both acylation and other factors, possibly protein-protein interactions, target LAT to rafts.

Lipid Raft Association of SNARE Proteins Regulates Exocytosis in PC12 Cells

SNAP25 and SNAP23 are plasma membrane SNARE proteins essential for regulated exocytosis in diverse cell types. Several recent studies have shown that these proteins are partly localized in lipid rafts, domains of the plasma membrane enriched in sphingolipids, and cholesterol. Here, we have employed cysteine mutants of SNAP25/SNAP23, which have modified affinities for raft domains, to examine whether raft association of these proteins is important for the regulation of exocytosis. PC12 cells were engineered that express the light chain of botulinum neurotoxin; in these cells all of the SNAP25 was cleaved to a lower molecular weight form, and regulated exocytosis was essentially absent. Exocytosis was rescued by expressing toxin-resistant SNAP25 or wild-type SNAP23, which is naturally toxin-resistant. Remarkably, a mutant SNAP25 protein with an increased affinity for rafts displayed a reduced ability to support exocytosis, whereas SNAP23 mutants with a decreased affinity for rafts displayed an enhancement of exocytosis when compared with wild-type SNAP23. The effects of the mutant proteins on exocytosis were dependent upon the integrity of the plasma membrane and lipid rafts. These results provide the first direct evidence that rafts regulate SNARE function and exocytosis and identify the central cysteine-rich region of SNAP25/23 as an important regulatory domain.

Differential insertion of GPI‐anchored GFPs into lipid rafts of live cells

Partitioning of proteins in cholesterol and sphingolipid enriched plasma membrane microdomains, called lipid rafts, is critical for many signal transduction and protein sorting events. Although raft partitioning of many signaling molecules remains to be determined, glycosylphosphatidyl-inositol (GPI)-anchored proteins possess high affinity for lipid rafts and are currently exploited as markers to investigate fundamental mechanisms in protein sorting and signal transduction events. In this study, we demonstrate that two recombinant GPI-anchored green fluorescent proteins (GFP-GPIs) that differ in their GPI signal sequence confer distinct localization in plasma membrane microdomains. GFP fused to the GPI signal of the decay accelerating factor GFP-GPI(DAF) partitioned exclusively in lipid rafts, whereas GFP fused to the GPI signal of TRAIL-R3, GFP-GPI(TRAIL-R3), associated only minimally with microdomains. In addition, we investigated the unique ability of purified GFP-GPIs to insert into membrane microdomains of primary lymphocytes. This cell surface painting allows rapid, stable, and functional association of the GPI-anchored proteins with the target cell plasma membrane. The distinct membrane localization of the two GFP-GPIs was observed irrespective of whether the GPI-anchored molecules were painted or transfected. Furthermore, we show that painted GFP-GPI(DAF) was totally dependent on the GPI anchor and that the membrane insertion was increased by the addition of raft-associated lipids such as cholesterol, sphingomyelin, and dipalmitoyl-phosphatidylethanolamine. Thus, this study provides evidence that different GPI signal sequences lead to distinct membrane microdomain localization and that painted GFP-GPI(DAF) serves as an excellent fluorescent marker for lipid rafts in live cells.

Three separable domains regulate GTP-dependent association of H-ras with the plasma membrane.

The microlocalization of Ras proteins to different microdomains of the plasma membrane is critical for signaling specificity. Here we examine the complex membrane interactions of H-ras with a combination of FRAP on live cells to measure membrane affinity and electron microscopy of intact plasma membrane sheets to spatially map microdomains. We show that three separable forces operate on H-ras at the plasma membrane. The lipid anchor, comprising a processed CAAX motif and two palmitic acid residues, generates one attractive force that provides a high-affinity interaction with lipid rafts. The adjacent hypervariable linker domain provides a second attractive force but for nonraft plasma membrane microdomains. Operating against the attractive interaction of the lipid anchor for lipid rafts is a repulsive force generated by the N-terminal catalytic domain that increases when H-ras is GTP loaded. These observations lead directly to a novel mechanism that explains how H-ras lateral segregation is regulated by activation state: GTP loading decreases H-ras affinity for lipid rafts and allows the hypervariable linker domain to target to nonraft microdomains, the primary site of H-ras signaling.