Trafficking of endothelial nitric-oxide synthase in living cells. Quantitative evidence supporting the role of palmitoylation as a kinetic trapping mechanism limiting membrane diffusion.

To examine endothelial nitric-oxide synthase (eNOS) trafficking in living endothelial cells, the eNOS-deficient endothelial cell line ECV304 was stably transfected with an eNOS-green fluorescent protein (GFP) fusion construct and characterized by functional, biochemical, and microscopic analysis. eNOS-GFP was colocalized with Golgi and plasma membrane markers and produced NO in response to agonist challenge. Localization in the plasma membrane was dependent on the palmitoylation state, since the palmitoylation mutant of eNOS (C15S/C26S eNOS-GFP) was excluded from the plasma membrane and was concentrated in a diffuse perinuclear pattern. Fluorescence recovery after photobleaching (FRAP) revealed eNOS-GFP in the perinuclear region moving 3 times faster than the plasmalemmal pool, suggesting that protein-lipid or protein-protein interactions are different in these two cellular domains. FRAP of the palmitoylation mutant was two times faster than that of wild-type eNOS-GFP, indicating that palmitoylation was influencing the rate of trafficking. Interestingly, FRAP of C15S/C26S eNOS-GFP but not wild-type eNOS-GFP fit a model of protein diffusion in a lipid bilayer. These data suggest that the regulation of eNOS trafficking within the plasma membrane and Golgi are probably different mechanisms and not due to simple diffusion of the protein in a lipid bilayer.     

Selective accumulation of raft-associated membrane protein LAT in T cell receptor signaling assemblies

Activation of T cell antigen receptor (TCR) induces tyrosine phosphorylations that mediate the assembly of signaling protein complexes. Moreover, cholesterol-sphingolipid raft membrane domains have been implicated to play a role in TCR signal transduction. Here, we studied the assembly of TCR with signal transduction proteins and raft markers in plasma membrane subdomains of Jurkat T leukemic cells. We employed a novel method to immunoisolate plasma membrane subfragments that were highly concentrated in activated TCR-CD3 complexes and associated signaling proteins. We found that the raft transmembrane protein linker for activation of T cells (LAT), but not a palmitoylation-deficient non-raft LAT mutant, strongly accumulated in TCR-enriched immunoisolates in a tyrosine phosphorylation-dependent manner. In contrast, other raft-associated molecules, including protein tyrosine kinases Lck and Fyn, GM1, and cholesterol, were not highly concentrated in TCR-enriched plasma membrane immunoisolates. Many downstream signaling proteins coisolated with the TCR/LAT-enriched plasma membrane fragments, suggesting that LAT/TCR assemblies form a structural scaffold for TCR signal transduction proteins. Our results indicate that TCR signaling assemblies in plasma membrane subdomains, rather than generally concentrating raft-associated membrane proteins and lipids, form by a selective protein-mediated anchoring of the raft membrane protein LAT in vicinity of TCR.     

Amino-terminal Cysteine Residues Differentially Influence RGS4 Protein Plasma Membrane Targeting, Intracellular Trafficking, and Function

Regulator of G-protein signaling (RGS) proteins are potent inhibitors of heterotrimeric G-protein signaling. RGS4 attenuates G-protein activity in several tissues. Previous work demonstrated that cysteine palmitoylation on residues in the amino-terminal (Cys-2 and Cys-12) and core domains (Cys-95) of RGS4 is important for protein stability, plasma membrane targeting, and GTPase activating function. To date Cys-2 has been the priority target for RGS4 regulation by palmitoylation based on its putative role in stabilizing the RGS4 protein. Here, we investigate differences in the contribution of Cys-2 and Cys-12 to the intracellular localization and function of RGS4. Inhibition of RGS4 palmitoylation with 2-bromopalmitate dramatically reduced its localization to the plasma membrane. Similarly, mutation of the RGS4 amphipathic helix (L23D) prevented membrane localization and its G(q) inhibitory function. Together, these data suggest that both RGS4 palmitoylation and the amphipathic helix domain are required for optimal plasma membrane targeting and function of RGS4. Mutation of Cys-12 decreased RGS4 membrane targeting to a similar extent as 2-bromopalmitate, resulting in complete loss of its G(q) inhibitory function. Mutation of Cys-2 did not impair plasma membrane targeting but did partially impair its function as a G(q) inhibitor. Comparison of the endosomal distribution pattern of wild type and mutant RGS4 proteins with TGN38 indicated that palmitoylation of these two cysteines contributes differentially to the intracellular trafficking of RGS4. These data show for the first time that Cys-2 and Cys-12 play markedly different roles in the regulation of RGS4 membrane localization, intracellular trafficking, and G(q) inhibitory function via mechanisms that are unrelated to RGS4 protein stabilization.     

Palmitoylation-dependent estrogen receptor alpha membrane localization: regulation by 17beta-estradiol

A fraction of the nuclear estrogen receptor alpha (ERalpha) is localized to the plasma membrane region of 17beta-estradiol (E2) target cells. We previously reported that ERalpha is a palmitoylated protein. To gain insight into the molecular mechanism of ERalpha residence at the plasma membrane, we tested both the role of palmitoylation and the impact of E2 stimulation on ERalpha membrane localization. The cancer cell lines expressing transfected or endogenous human ERalpha (HeLa and HepG2, respectively) or the ERalpha nonpalmitoylable Cys447Ala mutant transfected in HeLa cells were used as experimental models. We found that palmitoylation of ERalpha enacts ERalpha association with the plasma membrane, interaction with the membrane protein caveolin-1, and nongenomic activities, including activation of signaling pathways and cell proliferation (i.e., ERK and AKT activation, cyclin D1 promoter activity, DNA synthesis). Moreover, E2 reduces both ERalpha palmitoylation and its interaction with caveolin-1, in a time- and dose-dependent manner. These data point to the physiological role of ERalpha palmitoylation in the receptor localization to the cell membrane and in the regulation of the E2-induced cell proliferation.     

Protein acylation and localization in T cell signaling (Review)

Many proteins with pivotal roles in T cell activation are modified by fatty acylation. Examples of these include transmembrane proteins such as the co-receptors CD4 and CD8, the adaptors LAT and Cbp/PAG, the pre-TCR as well as proteins synthesized on free cytosolic ribosomes, such as the Src-related tyrosine kinases Lck and Fyn. The two main types of fatty acylations in eukaryotic cells are N-myristoylation and S-acylation, the latter being more commonly referred to as palmitoylation. N-Myristoylation occurs exclusively on proteins synthesized on soluble ribosomes and provides substrates with an affinity for membranes. Palmitoylation modifies a wide range of substrates that includes both cytosolic and transmembrane proteins, its functions are diverse and in many cases not yet understood. Like myristoylation, palmitoylation promotes membrane-binding of cytosolic proteins, but it has also been implicated in protein targeting, trafficking, stability and activity. In addition, many palmitoylated proteins are insoluble in cold non-ionic detergent, and have therefore been proposed to localize to lipid rafts. The organization of receptors and signaling proteins into microdomains such as lipid rafts provides an attractive model for the initiation and propagation of T cell signaling, although many aspects of this are still poorly understood. This review will discuss the current evidence for the involvement of acylations in the localizations and functions of T cell signaling proteins.     

Global analysis of protein palmitoylation in yeast

Protein palmitoylation is a reversible lipid modification that regulates membrane tethering for key proteins in cell signaling, cancer, neuronal transmission, and membrane trafficking. Palmitoylation has proven to be a difficult study: Specifying consensuses for predicting palmitoylation remain unavailable, and first-example palmitoylation enzymes--i.e., protein acyltransferases (PATs)--were identified only recently. Here, we use a new proteomic methodology that purifies and identifies palmitoylated proteins to characterize the palmitoyl proteome of the yeast Saccharomyces cerevisiae. Thirty-five new palmitoyl proteins are identified, including many SNARE proteins and amino acid permeases as well as many other participants in cellular signaling and membrane trafficking. Analysis of mutant yeast strains defective for members of the DHHC protein family, a putative PAT family, allows a matching of substrate palmitoyl proteins to modifying PATs and reveals the DHHC family to be a family of diverse PAT specificities responsible for most of the palmitoylation within the cell.     

Palmitoylation targets CD39/endothelial ATP diphosphohydrolase to caveolae

Ectonucleotidases influence purinergic receptor function by the hydrolysis of extracellular nucleotides. CD39 is an integral membrane protein that is a prototype member of the nucleoside 5'-triphosphate diphosphohydrolase family. The native CD39 protein has two intracytoplasmic and two transmembrane domains. There is a large extracellular domain that undergoes extensive glycosylation and can be post-translationally modified by limited proteolysis. We have identified a potential thioester linkage site for S-acylation within the N-terminal region of CD39 and demonstrate that this region undergoes palmitoylation in a constitutive manner. The covalent lipid modification of this region of the protein appears to be important both in plasma membrane association and in targeting CD39 to caveolae. These specialized plasmalemmal domains are enriched in G protein-coupled receptors and appear to integrate cellular activation events. We suggest that palmitoylation could modulate the function of CD39 in regulating cellular signal transduction pathways.     

Palmitoylation of carboxypeptidase D. Implications for intracellular trafficking

Covalent lipid modifications mediate protein-membrane and protein-protein interactions and are often essential for function. The purposes of this study were to examine the Cys residues of the transmembrane domain of metallocarboxypeptidase D (CPD) that could be a target for palmitoylation and to clarify the function of this modification. CPD is an integral membrane protein that cycles between the trans Golgi network and the plasma membrane. We constructed AtT-20 cells stably expressing various constructs carrying a reporter protein (albumin) fused to a transmembrane domain and the CPD cytoplasmic tail. Some of the constructs contained the three Cys residues present in the CPD transmembrane region, while other constructs contained Ala in place of the Cys. Constructs carrying Cys residues were palmitoylated, while those constructs lacking the Cys residues were not. Because palmitoylation of several proteins affects their association with cholesterol and sphingolipid-rich membrane domains or caveolae, we tested endogenous CPD and several of the reporter constructs for resistance to extraction with Triton X-100. A construct containing the Cys residues of the CPD transmembrane domain was soluble in Triton X-100 as was endogenous palmitoylated CPD, indicating that palmitoylation does not target CPD to detergent-resistant membrane rafts. Interestingly, constructs of CPD that lack palmitoylation sites have an increased half-life, a slightly more diffuse steady-state localization, and a slower rate of exit from the Golgi as compared with constructs containing palmitoylation sites. Thus, the covalent attachment of palmitic acid to the Cys residues of CPD has a functional significance in the trafficking of the protein.     

Altered localization of H-Ras in caveolin-1-null cells is palmitoylation-independent

Caveolin-1 is a palmitoylated protein involved in the formation of plasma membrane subdomains termed caveolae, intracellular cholesterol transport, and assembly and regulation of signaling molecules in caveolae. Caveolin-1 interacts via a consensus binding motif with several signaling proteins, including H-Ras. Ras oncogene products function as molecular switches in several signal transduction pathways regulating cell growth and differentiation. Post-translational modifications, including palmitoylation, are critical for the membrane targeting and function of H-Ras. Subcellular localization regulates the signaling pathways engaged by H-Ras activation. We show here that H-Ras is localized at the plasma membrane in caveolin-1-expressing cells but not in caveolin-1-deficient cells. Since palmitoylation is required for trafficking of H-Ras from the endomembrane system to the plasma membrane, we tested whether the altered localization of H-Ras in caveolin-1-null cells is due to decreased H-Ras palmitoylation. Although the palmitoylation profiles of cultured embryo fibroblasts isolated from wild type and caveolin-1 gene-disrupted mice differed, suggesting that caveolin-1, or caveolae, play a role in the palmitate incorporation of a subset of palmitoylated proteins, the palmitoylation of H-Ras was not decreased in caveolin-1-null cells. We conclude that the altered localization of H-Ras in caveolin-1-deficient cells is palmitoylation-independent. This article shows two important new mechanisms by which loss of caveolin-1 expression may perturb intracellular signaling, namely the mislocalization of signaling proteins and alterations in protein palmitoylation.     

Altered localization of H-Ras in caveolin-1-null cells is palmitoylation-independent

Caveolin-1 is a palmitoylated protein involved in the formation of plasma membrane subdomains termed caveolae, intracellular cholesterol transport, and assembly and regulation of signaling molecules in caveolae. Caveolin-1 interacts via a consensus binding motif with several signaling proteins, including H-Ras. Ras oncogene products function as molecular switches in several signal transduction pathways regulating cell growth and differentiation. Post-translational modifications, including palmitoylation, are critical for the membrane targeting and function of H-Ras. Subcellular localization regulates the signaling pathways engaged by H-Ras activation. We show here that H-Ras is localized at the plasma membrane in caveolin-1-expressing cells but not in caveolin-1-deficient cells. Since palmitoylation is required for trafficking of H-Ras from the endomembrane system to the plasma membrane, we tested whether the altered localization of H-Ras in caveolin-1-null cells is due to decreased H-Ras palmitoylation. Although the palmitoylation profiles of cultured embryo fibroblasts isolated from wild type and caveolin-1 gene-disrupted mice differed, suggesting that caveolin-1, or caveolae, play a role in the palmitate incorporation of a subset of palmitoylated proteins, the palmitoylation of H-Ras was not decreased in caveolin-1-null cells. We conclude that the altered localization of H-Ras in caveolin-1-deficient cells is palmitoylation-independent. This article shows two important new mechanisms by which loss of caveolin-1 expression may perturb intracellular signaling, namely the mislocalization of signaling proteins and alterations in protein palmitoylation.     

The dually acylated NH2-terminal domain of gi1alpha is sufficient to target a green fluorescent protein reporter to caveolin-enriched plasma membrane domains. Palmitoylation of caveolin-1 is required for the recognition of dually acylated g-protein alpha subunits in vivo

Here we investigate the molecular mechanisms that govern the targeting of G-protein alpha subunits to the plasma membrane. For this purpose, we used Gi1alpha as a model dually acylated G-protein. We fused full-length Gi1alpha or its extreme NH2-terminal domain (residues 1-32 or 1-122) to green fluorescent protein (GFP) and analyzed the subcellular localization of these fusion proteins. We show that the first 32 amino acids of Gi1alpha are sufficient to target GFP to caveolin-enriched domains of the plasma membrane in vivo, as demonstrated by co-fractionation and co-immunoprecipitation with caveolin-1. Interestingly, when dual acylation of this 32-amino acid domain was blocked by specific point mutations (G2A or C3S), the resulting GFP fusion proteins were localized to the cytoplasm and excluded from caveolin-rich regions. The myristoylated but nonpalmitoylated (C3S) chimera only partially partitioned into caveolin-containing fractions. However, both nonacylated GFP fusions (G2A and C3S) no longer co-immunoprecipitated with caveolin-1. Taken together, these results indicate that lipid modification of the NH2-terminal of Gi1alpha is essential for targeting to its correct destination and interaction with caveolin-1. Also, a caveolin-1 mutant lacking all three palmitoylation sites (C133S, C143S, and C156S) was unable to co-immunoprecipitate these dually acylated GFP-G-protein fusions. Thus, dual acylation of the NH2-terminal domain of Gi1alpha and palmitoylation of caveolin-1 are both required to stabilize and perhaps regulate this reciprocal interaction at the plasma membrane in vivo. Our results provide the first demonstration of a functional role for caveolin-1 palmitoylation in its interaction with signaling molecules.     

Dynamic changes in the mobility of LAT in aggregated lipid rafts upon T cell activation

Lipid rafts are known to aggregate in response to various stimuli. By way of raft aggregation after stimulation, signaling molecules in rafts accumulate and interact so that the signal received at a given membrane receptor is amplified efficiently from the site of aggregation. To elucidate the process of lipid raft aggregation during T cell activation, we analyzed the dynamic changes of a raft-associated protein, linker for activation of T cells (LAT), on T cell receptor stimulation using LAT fused to GFP (LAT-GFP). When transfectants expressing LAT-GFP were stimulated with anti-CD3-coated beads, LAT-GFP aggregated and formed patches at the area of bead contact. Photobleaching experiments using live cells revealed that LAT-GFP in patches was markedly less mobile than that in nonpatched regions. The decreased mobility in patches was dependent on raft organization supported by membrane cholesterol and signaling molecule binding sites, especially the phospholipase C gamma 1 binding site in the cytoplasmic domain of LAT. Thus, although LAT normally moves rapidly at the plasma membrane, it loses its mobility and becomes stably associated with aggregated rafts to ensure organized and sustained signal transduction required for T cell activation.     

The effects of HIV-1 Nef on CD4 surface expression and viral infectivity in lymphoid cells are independent of rafts

The HIV-1 Nef protein is a critical virulence factor that exerts multiple effects during viral replication. Nef modulates surface expression of various cellular proteins including CD4 and MHC-I, enhances viral infectivity, and affects signal transduction pathways. Nef has been shown to partially associate with rafts, where it can prime T cells for activation. The contribution of rafts during Nef-induced CD4 down-regulation and enhancement of viral replication remains poorly understood. We show here that Nef does not modify the palmitoylation state of CD4 or its partition within rafts. Moreover, CD4 mutants lacking palmitoylation or unable to associate with rafts are efficiently down-regulated by Nef. In HIV-infected cells, viral assembly and budding occurs from rafts, and Nef has been suggested to increase this process. However, using T cells acutely infected with wild-type or nef-deleted HIV, we did not observe any impact of Nef on raft segregation of viral structural proteins. We have also designed a palmitoylated mutant of Nef (NefG3C), which significantly accumulates in rafts. Interestingly, the efficiency of NefG3C to down-regulate CD4 and MHC-I, and to promote viral replication was not increased when compared with the wild-type protein. Altogether, these results strongly suggest that rafts are not a key element involved in the effects of Nef on trafficking of cellular proteins and on viral replication.     

T-Cell Receptor Microclusters Critical for T-Cell Activation Are Formed Independently of Lipid Raft Clustering

We studied the function of lipid rafts in generation and signaling of T-cell receptor microclusters (TCR-MCs) and central supramolecular activation clusters (cSMACs) at immunological synapse (IS). It has been suggested that lipid raft accumulation creates a platform for recruitment of signaling molecules upon T-cell activation. However, several lipid raft probes did not accumulate at TCR-MCs or cSMACs even with costimulation and the fluorescence resonance energy transfer (FRET) between TCR or LAT and lipid raft probes was not induced at TCR-MCs under the condition of positive induction of FRET between CD3ζ and ZAP-70. The analysis of LAT mutants revealed that raft association is essential for the membrane localization but dispensable for TCR-MC formation. Careful analysis of the accumulation of raft probes in the cell interface revealed that their accumulation occurred after cSMAC formation, probably due to membrane ruffling and/or endocytosis. These results suggest that lipid rafts control protein translocation to the membrane but are not involved in the clustering of raft-associated molecules and therefore that the lipid rafts do not serve as a platform for T-cell activation.Lipid rafts are specialized liquid-ordered membrane microdomains that are enriched in cholesterol and sphingolipids. Many studies using various methodologies have shown that lipid rafts exist as leaflets less than 200 nm in size and float on the plasma membrane (6, 10, 24, 28, 32). They have been implied to play a role in protein sorting and cell activation as a platform by recruiting various signaling molecules such as Src family kinases, G proteins, and adaptor molecules. Because of size limitation, all of the raft-associated molecules could not be accommodated on the same lipid raft, and heterogeneity of lipid rafts both in size and in the repertoire of resident molecules has been suggested (22). The functional importance of lipid rafts in signal transduction has been particularly appreciated in T-cell activation through the T-cell receptor (TCR). Some of the initial observations in this area included the findings that cross-linking of the raft-associated ganglioside GM1 induces T-cell activation (12) and that a mutant of LAT, a membrane adaptor protein, that was unable to localize to rafts failed to induce activation signals (33). Since then, increasing data have demonstrated that lipid raft accumulation creates a platform to stabilize the signaling complex for T-cell activation (13, 29).T cells are activated upon recognition of peptide-major histocompatibility complex (MHC) complexes expressed on antigen-presenting cells (APC). An immunological synapse (IS) is formed at the interface between the T cell and the APC where a specialized segregated structure of T-cell surface receptors is generated. This supramolecular activation cluster (SMAC) contains the TCR in the central region (cSMAC) and lymphocyte function-associated antigen 1 (LFA-1) in the peripheral region (pSMAC). The accumulation of lipid rafts at this interface, particularly in the cSMAC, has been suggested to create a transient structure to mediate signal transduction (13, 17). In addition, CD28-mediated costimulation has been suggested to enhance lipid raft accumulation and TCR activation (29). However, the idea that lipid rafts accumulated in the cSMAC serve as the platform for T-cell activation has been controversial; the accumulation of the lipid raft was only partial in the contact area (3), or the concentration of lipid raft was constant even in the area of T-cell activation (5, 8, 28, 32). These variations could be partly attributed to differences in experimental approaches such as the cell systems being analyzed, stimulation conditions, and detection methods, including imaging and biochemical fractionation. The idea that the cSMAC is the site responsible for inducing signals for T-cell activation has been recently revised based on analysis of the dynamic assembly of signaling complexes upon TCR stimulation. Analysis of T-cell activation using a planar membrane system containing glycosylphosphatidylinositol (GPI)-anchored MHC-peptide complexes and the LFA-1 ligand intercellular adhesion molecule 1 (ICAM-1) revealed that small clusters containing approximately a hundred TCRs, kinases, and adaptors, which we termed TCR microclusters (MCs), were generated at the initial contact sites. This was followed by translocation of the MCs to the center of the interface to generate a cSMAC (31). Since protein phosphorylation, including that of ZAP-70, was induced in the TCR-MCs and Ca²⁺ mobilization was induced in parallel with the formation of TCR-MCs, these MCs appear to be the very first and minimum unit for generating TCR activation signals (31). Furthermore, a major costimulatory receptor, CD28, forms clusters which are also colocalized in TCR-MCs to regulate costimulatory signals (30).Among these TCR proximal signaling molecules, LAT is a well-studied raft-associated membrane adaptor protein that is indispensable for TCR activation. LAT is phosphorylated by ZAP-70 and then behaves as a signal scaffold, recruiting various signaling adaptors and effector molecules such as phospholipase Cγ (PLCγ), SLP-76, and Grb2/Gads. Because mutation of LAT palmitoylation sites (C26,29A) resulted in its dislocation from lipid rafts and defective signaling, it was concluded that the association with lipid rafts is essential for the function of LAT (33). However, a recent study showed that this mutant LAT has impaired trafficking to the plasma membrane in the Jurkat T-cell line (27), raising the question of whether the impaired signaling resulting from this LAT mutation was due to dislocation from the raft or defective trafficking to the membrane.Here, we analyzed the role of lipid rafts in T-cell activation, particularly their relationship with immunological synapse formation (9). Provided that lipid raft functions as a platform for T-cell activation, the new idea that TCR-MCs serve as the signal unit for activation would predict that lipid raft could be accumulated in or interact with TCR-MCs (29).Utilizing several lipid raft probes, which retain the capability of raft localization but lack signaling capacity, we found that the full-length LAT generated MCs, but none of the raft probes formed visible clusters at TCR-MCs or cSMAC, even in conjunction with CD28-mediated costimulation. Furthermore, no significant interaction between lipid rafts and TCR-MCs was revealed by fluorescence resonance energy transfer (FRET) analysis. Conversely, the non-raft-localizing LAT mutant showed MC formation upon TCR stimulation. These results suggest that lipid rafts do not serve as a platform for TCR signaling but rather regulate the traffic/recruitment of proteins to the plasma membrane. Furthermore, our data indicate that the previous observation of lipid raft accumulation at the cSMAC may reflect membrane ruffling and endocytosis rather than active formation of signal platform.     

Oxidized high-density lipoprotein promotes CD36 palmitoylation and increases lipid uptake in macrophages

Oxidized high-density lipoprotein (oxHDL) reduces the ability of cells to mediate reverse cholesterol transport and also shows atherogenic properties. Palmitoylation of cluster of differentiation 36 (CD36), an important receptor mediating lipoprotein uptake, is required for fatty acid endocytosis. However, the relationship between oxHDL and CD36 has not been described in mechanistic detail. Here, we demonstrate using acyl-biotin exchange analysis that oxHDL activates CD36 by increasing CD36 palmitoylation, which promotes efficient uptake in macrophages. This modification increased CD36 incorporation into plasma lipid rafts and activated downstream signaling mediators, such as Lyn, Fyn, and c-Jun N-terminal kinase, which elicited enhanced oxHDL uptake and foam cell formation. Furthermore, blocking CD36 palmitoylation with the pharmacological inhibitor 2-bromopalmitate decreased cell surface translocation and lowered oxHDL uptake in oxHDL-treated macrophages. We verified these results by transfecting oxHDL-induced macrophages with vectors expressing wildtype or mutant CD36 (mCD36) in which the cytoplasmic palmitoylated cysteine residues were replaced. We show that cells containing mCD36 exhibited less palmitoylated CD36, disrupted plasma membrane trafficking, and reduced protein stability. Moreover, in ApoE^−/−CD36^−/− mice, lipid accumulation at the aortic root in mice receiving the mCD36 vector was decreased, suggesting that CD36 palmitoylation is responsible for lipid uptake in vivo. Finally, our data indicated that palmitoylation of CD36 was dependent on DHHC6 (Asp-His-His-Cys) acyltransferase and its cofactor selenoprotein K, which increased the CD36/caveolin-1 interaction and membrane targeting in cells exposed to oxHDL. Altogether, our study uncovers a causal link between oxHDL and CD36 palmitoylation and provides insight into foam cell formation and atherogenesis.     

Prediction and analysis of protein palmitoylation sites

Palmitoylation is a universal and important lipid modification, involving a series of basic cellular processes, such as membrane trafficking, protein stability and protein aggregation. With the avalanche of new protein sequences generated in the post genomic era, it is highly desirable to develop computational methods for rapidly and effectively identifying the potential palmitoylation sites of uncharacterized proteins so as to timely provide useful information for revealing the mechanism of protein palmitoylation. By using the Incremental Feature Selection approach based on amino acid factors, conservation, disorder feature, and specific features of palmitoylation site, a new predictor named IFS-Palm was developed in this regard. The overall success rate thus achieved by jackknife test on a newly constructed benchmark dataset was 90.65%. It was shown via an in-depth analysis that palmitoylation was intimately correlated with the feature of the upstream residue directly adjacent to cysteine site as well as the conservation of amino acid cysteine. Meanwhile, the protein disorder region might also play an import role in the post-translational modification. These findings may provide useful insights for revealing the mechanisms of palmitoylation.     

Differential palmitoylation of the endosomal SNAREs syntaxin 7 and syntaxin 8

Palmitoylation is a posttranslational modification that regulates protein trafficking and stability. In this study we investigated whether the endosomal soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNARE) proteins syntaxin 7 and syntaxin 8 are modified with palmitate. Using metabolic labeling and site-directed mutagenesis, we show that human syntaxins 7 and 8 are modified with palmitate through a thioester linkage. Palmitoylation is dependent upon cysteine 239 of human syntaxin 7 and cysteine 214 of syntaxin 8, residues that are located on the cytoplasmic face of the transmembrane domain (TMD). Palmitoylation of syntaxin 8 is minimally affected by the Golgi-disturbing agent brefeldin A (BFA), whereas BFA dramatically inhibits palmitoylation of syntaxin7. The differential effect of BFA suggests that palmitoylation of syntaxins 7 and 8 occurs in distinct subcellular compartments. Palmitoylation does not affect the rate of protein turnover of syntaxins 7 and 8 nor does it influence the steady-state localization of syntaxin 8 in late endosomes. Syntaxin 7 actively cycles between endosomes and the plasma membrane. Palmitoylation-defective syntaxin 7 is selectively retained on the plasma membrane, suggesting that palmitoylation is important for intercompartmental transport of syntaxin 7.     

Effect of Redox Balance Alterations on Cellular Localization of LAT and Downstream T-Cell Receptor Signaling Pathways

The integral membrane protein linker for activation of T cells (LAT) is a central adapter protein in the T-cell receptor (TCR)-mediated signaling pathways. The cellular localization of LAT is extremely sensitive to intracellular redox balance alterations. Reduced intracellular levels of the antioxidant glutathione (GSH), a hallmark of chronic oxidative stress, resulted in the membrane displacement of LAT, abrogated TCR-mediated signaling and consequently hyporesponsiveness of T lymphocytes. The membrane displacement of LAT is accompanied by a considerable difference in the mobility of LAT upon native and nonreducing denaturing polyacrylamide gel electrophoresis analysis, a finding indicative of a conformational change. Targeted mutation of redox-sensitive cysteine residues within LAT created LAT mutants which remain membrane anchored under conditions of chronic oxidative stress. The expression of redox-insensitive LAT mutants allows for restoration of TCR-mediated signal transduction, whereas CD28-mediated signaling pathways remained impaired. These results are indicative that the membrane displacement of LAT as a result of redox balance alterations is a consequence of a conformational change interfering with the insertion of LAT into the plasma membrane. Conclusively, the data suggest a role for LAT as a crucial intermediate in the sensitivity of TCR signaling and hence T lymphocytes toward chronic oxidative stress.     

Mutation of juxtamembrane cysteines in the tetraspanin CD81 affects palmitoylation and alters interaction with other proteins at the cell surface

Palmitoylation of tetraspanins affects protein-protein interactions, suggesting a key role in the assembly of the tetraspanin web. Since palmitoylation occurs on intracellular cysteine residues, we examined whether mutating these residues in the human tetraspanin CD81 would affect the association of CD81 with other surface membrane proteins. Mutation of at least six of the eight juxtamembrane cysteines was required to completely eliminate detectable CD81 palmitoylation, indicating that several sites can be palmitoylated. Interestingly, these mutated proteins exhibited reduced cell surface detection by antibody compared to wild-type CD81, but this was not due to differences in the level of protein expression, trafficking to the cell surface, protein stability, or anti-CD81 antibody binding affinity. Instead, the mutant CD81 proteins appeared to be partially hidden from detection by anti-CD81 antibody, presumably due to altered interactions with other proteins at the cell surface. Associations with the known CD81-interacting proteins CD9 and EWI-2 were also impaired with the mutant CD81 proteins. Taken together, these findings indicate that mutation of juxtamembrane cysteines alters the interaction of CD81 with other proteins, either because of reduced palmitoylation, structural alterations in the mutant proteins, or a combination of both factors, and this affects the CD81 microenvironment on the cell surface.