H-Ras Distribution and Signaling in Plasma Membrane Microdomains Are Regulated by Acylation and Deacylation Events

H-Ras must adhere to the plasma membrane to be functional. This is accomplished by posttranslational modifications, including palmitoylation, a reversible process whereby H-Ras traffics between the plasma membrane and the Golgi complex. At the plasma membrane, H-Ras has been proposed to occupy distinct sublocations, depending on its activation status: lipid rafts/detergent-resistant membrane fractions when bound to GDP, diffusing to disordered membrane/soluble fractions in response to GTP loading. Herein, we demonstrate that H-Ras sublocalization is dictated by its degree of palmitoylation in a cell type-specific manner. Whereas H-Ras localizes to detergent-resistant membrane fractions in cells with low palmitoylation activity, it locates to soluble membrane fractions in lineages where it is highly palmitoylated. Interestingly, in both cases GTP loading results in H-Ras diffusing away from its original sublocalization. Moreover, tilting the equilibrium between palmitoylation and depalmitoylation processes can substantially alter H-Ras segregation and, subsequently, its biochemical and biological functions. Thus, the palmitoylation/depalmitoylation balance not only regulates H-Ras cycling between endomembranes and the plasma membrane but also serves as a key orchestrator of H-Ras lateral diffusion between different types of plasma membrane and thereby of H-Ras signaling.     

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.     

Transient palmitoylation supports H-Ras membrane binding but only partial biological activity.

H-Ras is >95% membrane-bound when modified by farnesyl and palmitate, but <10% membrane-bound if only farnesyl is present, implying that palmitate provides major support for membrane interaction. However the direct contribution of palmitate to H-Ras membrane interaction or the extent of its cooperation with farnesyl is unknown, because in the native protein the isoprenoid must be present before palmitate can be attached. To examine if palmitates can maintain H-Ras membrane association despite multiple cycles of turnover, a nonfarnesylated H-Ras(Cys186Ser) was constructed, with an N-terminal palmitoylation signal, derived from the GAP-43 protein. Although 40% of the GAP43:Ras(61Leu,186Ser) protein (G43:Ras61L) partitioned with membranes, the chimera had less than 10% of the transforming activity of fully lipidated H-Ras(61Leu) in NIH 3T3 cells. Poor focus formation was not due to incorrect targeting or gross structural changes, because G43:Ras61L localized specifically to plasma membranes and triggered differentiation of PC12 cells as potently as native H-Ras61L. Proteolytic digestion indicated that in G43:Ras61L both the N-terminal and the two remaining C-terminal cysteines of G43:Ras61L were palmitoylated. A mutant lacking all three C-terminal Cys residues had decreased membrane binding and differentiating activity. Therefore, even with correct targeting and palmitates at the C-terminus, G43:Ras61L was only partially active. These results indicate that although farnesyl and palmitate share responsibility for H-Ras membrane binding, each lipid also has distinct functions. Farnesyl may be important for signaling, especially transformation, while palmitates may provide potentially dynamic regulation of membrane binding.     

FKBP12 binds to acylated H-ras and promotes depalmitoylation

A cycle of palmitoylation/depalmitoylation of H-Ras mediates bidirectional trafficking between the Golgi apparatus and the plasma membrane, but nothing is known about how this cycle is regulated. We show that the prolyl isomerase (PI) FKBP12 binds to H-Ras in a palmitoylation-dependent fashion and promotes depalmitoylation. A variety of inhibitors of the PI activity of FKBP12, including FK506, rapamycin, and cycloheximide, increase steady-state palmitoylation. FK506 inhibits retrograde trafficking of H-Ras from the plasma membrane to the Golgi in a proline 179-dependent fashion, augments early GTP loading of Ras in response to growth factors, and promotes H-Ras-dependent neurite outgrowth from PC12 cells. These data demonstrate that FKBP12 regulates H-Ras trafficking by promoting depalmitoylation through cis-trans isomerization of a peptidyl-prolyl bond in proximity to the palmitoylated cysteines.     

Differential membrane localization of ERas and Rheb, two Ras-related proteins involved in the phosphatidylinositol 3-kinase/mTOR pathway

Two Ras-related proteins, ERas and Rheb, which are involved in the phosphatidylinositol 3-kinase pathway, display high GTP affinity and have atypical CAAX motifs. The factors governing the intracellular localization of ERas and Rheb are incompletely understood. In the current study, we show by confocal microscopy that ERas is localized to the plasma membrane, whereas Rheb is confined to the endomembranes. Membrane localization of the two proteins was abolished by mutation of the cysteine of the CAAX motif. Membrane targeting was also abolished by a farnesyltransferase inhibitor but not by a geranylgeranyltransferase inhibitor. In mouse fibroblasts deficient in either Rce1 (Ras converting enzyme 1) or Icmt (isoprenylcysteine carboxyl methyltransferase), ERas was mislocalized mainly to the Golgi apparatus, whereas Rheb showed diffuse localization. Mutation of cysteines in the hypervariable region of ERas prevented the plasma membrane localization of ERas, very strongly suggesting that palmitoylation of the cysteines is essential for membrane targeting. The hypervariable region of Rheb does not contain cysteines or polybasic residues, and when it was replaced with the hypervariable region of H-Ras, Rheb displayed plasma membrane localization. These data indicate that ERas shares the same posttranslational modifications with H-Ras and N-Ras and is localized at the plasma membrane. Rheb also shares the same membrane-targeting pathway but because of the absence of palmitoylation is located on endomembranes.     

Dynamic Palmitoylation Links Cytosol-Membrane Shuttling of Acyl-protein Thioesterase-1 and Acyl-protein Thioesterase-2 with That of Proto-oncogene H-Ras Product and Growth-associated Protein-43

Acyl-protein thioesterase-1 (APT1) and APT2 are cytosolic enzymes that catalyze depalmitoylation of membrane-anchored, palmitoylated H-Ras and growth-associated protein-43 (GAP-43), respectively. However, the mechanism(s) of cytosol-membrane shuttling of APT1 and APT2, required for depalmitoylating their substrates H-Ras and GAP-43, respectively, remained largely unknown. Here, we report that both APT1 and APT2 undergo palmitoylation on Cys-2. Moreover, blocking palmitoylation adversely affects membrane localization of both APT1 and APT2 and that of their substrates. We also demonstrate that APT1 not only catalyzes its own depalmitoylation but also that of APT2 promoting dynamic palmitoylation (palmitoylation-depalmitoylation) of both thioesterases. Furthermore, shRNA suppression of APT1 expression or inhibition of its thioesterase activity by palmostatin B markedly increased membrane localization of APT2, and shRNA suppression of APT2 had virtually no effect on membrane localization of APT1. In addition, mutagenesis of the active site Ser residue to Ala (S119A), which renders catalytic inactivation of APT1, also increased its membrane localization. Taken together, our findings provide insight into a novel mechanism by which dynamic palmitoylation links cytosol-membrane trafficking of APT1 and APT2 with that of their substrates, facilitating steady-state membrane localization and function of both.     

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.     

Endomembrane H-Ras Controls Vascular Endothelial Growth Factor-induced Nitric-oxide Synthase-mediated Endothelial Cell Migration

We demonstrate for the first time that endomembrane-delimited H-Ras mediates VEGF-induced activation of endothelial nitric-oxide synthase (eNOS) and migratory response of human endothelial cells. Using thiol labeling strategies and immunofluorescent cell staining, we found that only 31% of total H-Ras is S-palmitoylated, tethering the small GTPase to the plasma membrane but leaving the function of the large majority of endomembrane-localized H-Ras unexplained. Knockdown of H-Ras blocked VEGF-induced PI3K-dependent Akt (Ser-473) and eNOS (Ser-1177) phosphorylation and nitric oxide-dependent cell migration, demonstrating the essential role of H-Ras. Activation of endogenous H-Ras led to recruitment and phosphorylation of eNOS at endomembranes. The loss of migratory response in cells lacking endogenous H-Ras was fully restored by modest overexpression of an endomembrane-delimited H-Ras palmitoylation mutant. These studies define a newly recognized role for endomembrane-localized H-Ras in mediating nitric oxide-dependent proangiogenic signaling.     

Functional and structural roles of conserved cysteine residues in the carboxyl-terminal domain of the follicle-stimulating hormone receptor in human embryonic kidney 293 cells

The carboxyl-terminal segment of G protein-coupled receptors has one or more conserved cysteine residues that are potential sites for palmitoylation. This posttranslational modification contributes to membrane association, internalization, and membrane targeting of proteins. In contrast to other members of the glycoprotein hormone receptor family (the LH and thyroid-stimulating hormone receptors), it is not known whether the follicle-stimulating hormone receptor (FSHR) is palmitoylated and what are the effects of abolishing its potential palmitoylation sites. In the present study, a functional analysis of the FSHR carboxyl-terminal segment cysteine residues was carried out. We constructed a series of mutant FSHRs by substituting cysteine residues with alanine, serine, or threonine individually and together at positions 629 and 655 (conserved cysteines) and 627 (nonconserved). The results showed that all three cysteine residues are palmitoylated but that only modification at Cys629 is functionally relevant. The lack of palmitoylation does not appear to greatly impair coupling to G(s) but, when absent at position 629, does significantly impair cell surface membrane expression of the partially palmitoylated receptor. All FSHR Cys mutants were capable of binding agonist with the same affinity as the wild-type receptor and internalizing on agonist stimulation. Molecular dynamics simulations at a time scale of approximately 100 nsec revealed that replacement of Cys629 resulted in structures that differed significantly from that of the wild-type receptor. Thus, deviations from wild-type conformation may potentially contribute to the severe impairment in plasma membrane expression and the modest effects on signaling exhibited by the receptors modified in this particular position.     

H-Ras Nanocluster Stability Regulates the Magnitude of MAPK Signal Output

H-Ras is a binary switch that is activated by multiple co-factors and triggers several key cellular pathways one of which is MAPK. The specificity and magnitude of downstream activation is achieved by the spatio-temporal organization of the active H-Ras in the plasma membrane. Upon activation, the GTP bound H-Ras binds to Galectin-1 (Gal-1) and becomes transiently immobilized in short-lived nanoclusters on the plasma membrane from which the signal is propagated to Raf. In the current study we show that stabilizing the H-Ras-Gal-1 interaction, using bimolecular fluorescence complementation (BiFC), leads to prolonged immobilization of H-Ras.GTP in the plasma membrane which was measured by fluorescence recovery after photobleaching (FRAP), and increased signal out-put to the MAPK module. EM measurements of Raf recruitment to the H-Ras.GTP nanoclusters demonstrated that the enhanced signaling observed in the BiFC stabilized H-Ras.GTP nanocluster was attributed to increased H-Ras immobilization rather than to an increase in Raf recruitment. Taken together these data demonstrate that the magnitude of the signal output from a GTP-bound H-Ras nanocluster is proportional to its stability.     

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.     

The C-terminus of H-Ras as a target for the covalent binding of reactive compounds modulating Ras-dependent pathways

Ras proteins are crucial players in differentiation and oncogenesis and constitute important drug targets. The localization and activity of Ras proteins are highly dependent on posttranslational modifications at their C-termini. In addition to an isoprenylated cysteine, H-Ras, but not other Ras proteins, possesses two cysteine residues (C181 and C184) in the C-terminal hypervariable domain that act as palmitoylation sites in cells. Cyclopentenone prostaglandins (cyPG) are reactive lipidic mediators that covalently bind to H-Ras and activate H-Ras dependent pathways. Dienone cyPG, such as 15-deoxy-Δ(12,14)-PGJ(2) (15d-PGJ(2)) and Δ(12)-PGJ(2) selectively bind to the H-Ras hypervariable domain. Here we show that these cyPG bind simultaneously C181 and C184 of H-Ras, thus potentially altering the conformational tendencies of the hypervariable domain. Based on these results, we have explored the capacity of several bifunctional cysteine reactive small molecules to bind to the hypervariable domain of H-Ras proteins. Interestingly, phenylarsine oxide (PAO), a widely used tyrosine phosphatase inhibitor, and dibromobimane, a cross-linking agent used for cysteine mapping, effectively bind H-Ras hypervariable domain. The interaction of PAO with H-Ras takes place in vitro and in cells and blocks modification of H-Ras by 15d-PGJ(2). Moreover, PAO treatment selectively alters H-Ras membrane partition and the pattern of H-Ras activation in cells, from the plasma membrane to endomembranes. These results identify H-Ras as a novel target for PAO. More importantly, these observations reveal that small molecules or reactive intermediates interacting with spatially vicinal cysteines induce intramolecular cross-linking of H-Ras C-terminus potentially contributing to the modulation of Ras-dependent pathways.     

Palmitoylation plays a role in targeting Vac8p to specific membrane subdomains

Vac8p is a multifunctional yeast protein involved in several distinct vacuolar events including vacuole inheritance, vacuole homotypic fusion, nucleus-vacuole junction formation and the cytoplasm to vacuole protein targeting pathway. Vac8p associates with the vacuole membrane via myristoylation and palmitoylation. Vac8p has three putative palmitoylation sites, at Cys 4, 5 and 7. Here, we show that each of these cysteines may serve as a palmitoylation site. Palmitoylation at Cys 7 alone provides partial function of Vac8p, whereas palmitoylation at either Cys 4 or Cys 5 alone is sufficient for Vac8p function. In the former mutant, there is a severe defect in the localization of Vac8p to the vacuole membrane, while in the latter mutants, there is a partial defect in the localization of Vac8p. In addition, our studies provide evidence that palmitoylation targets Vac8p to specific membrane subdomains.     

Mass spectrometric analysis of GAP-43/neuromodulin reveals the presence of a variety of fatty acylated species

GAP-43 (neuromodulin) is a protein kinase C substrate that is abundant in developing and regenerating neurons. Thioester-linked palmitoylation at two cysteines near the GAP-43 N terminus has been implicated in directing membrane binding. Here, we use mass spectrometry to examine the stoichiometry of palmitoylation and the molecular identity of the fatty acid(s) attached to GAP-43 in vivo. GAP-43 expressed in either PC12 or COS-1 cells was acetylated at the N-terminal methionine. Approximately 35% of the N-terminal GAP-43 peptides were also modified by palmitate and/or stearate on Cys residues. Interestingly, a variety of acylated species was detected, in which one of the Cys residues was acylated by either palmitate or stearate, or both Cys residues were acylated by palmitates or stearates or a combination of palmitate and stearate. Depalmitoylation of membrane-bound GAP-43 did not release the protein from the membrane, implying that additional forces function to maintain membrane binding. Indeed, mutation of four basic residues within the N-terminal domain of GAP-43 dramatically reduced membrane localization of GAP-43 without affecting palmitoylation. These data reveal the heterogeneous nature of S-acylation in vivo and illustrate the power of mass spectrometry for identification of key regulatory protein modifications.     

Fluorescence-based methods to image palmitoylated proteins

A well known function of palmitoylation is to promote protein binding to cell membranes. Until recently, it was unclear what additional roles, if any, palmitoylation has in controlling protein localization in cells. Recent studies of palmitoylated forms of the small GTPase Ras have now revealed that palmitoylation plays multiple roles in the regulation of protein trafficking, including targeting proteins into the secretory pathway and recycling proteins between the plasma membrane and Golgi complex. We here describe how quantitative fluorescence microscopy and photobleaching approaches can be used to study the intracellular targeting and trafficking of GFP-tagged palmitoylated proteins in living cells. We discuss (1) general considerations for fluorescence recovery after photobleaching (FRAP) measurements of GFP-tagged proteins; (2) FRAP-based assays to test the strength of binding of palmitoylated proteins to cell membranes; (3) methods to establish the kinetics and mechanisms of recycling of palmitoylated proteins between the Golgi complex and the plasma membrane; (4) the use of the palmitoylation inhibitor 2-bromo-palmitate as a tool to study the dynamic regulation of protein targeting and trafficking by palmitate turnover.     

Palmitoylation of the TPbeta isoform of the human thromboxane A2 receptor. Modulation of G protein: effector coupling and modes of receptor internalization

Palmitoylation is a prevalent feature amongst G protein-coupled receptors. In this study we sought to establish whether the TPalpha and TPbeta isoforms of the human prostanoid thromboxane (TX) A2 receptor (TP) are palmitoylated and to assess the functional consequences thereof. Consistent with the presence of three cysteines within its unique carboxyl-terminal domain, metabolic labelling and site-directed mutagenesis confirmed that TPbeta is palmitoylated at Cys347 and, to a lesser extent, at Cys373,377 whereas TPalpha is not palmitoylated. Impairment of palmitoylation did not affect TPbeta expression or its ligand affinity. Conversely, agonist-induced [Ca2+]i mobilization by TPbetaC347S and the non-palmitoylated TPbetaC347,373,377S, but not by TPbetaC373S or TPbetaC373,377S, was significantly reduced relative to the wild type TPbeta suggesting that palmitoylation at Cys347 is specifically required for efficient Gq/phospholipase Cbeta effector coupling. Furthermore, palmitoylation at Cys373,377 is critical for TPbeta internalization with TPbetaC373S, TPbetaC373,377S and TPbetaC347,373,377S failing to undergo either agonist-induced or temperature-dependent tonic internalization. On the other hand, whilst TPbetaC347S underwent reduced agonist-induced internalization, it underwent tonic internalization to a similar extent as TPbeta. The deficiency in agonist-induced internalization by TPbetaC347S, but not by TPbetaC373,377 nor TPbeta(C347,373,377S), was overcome by over-expression of either beta-arrestin1 or beta-arrestin2. Taken together, data herein suggest that whilst palmitoylation of TPbeta at Cys373,377 is critical for both agonist- and tonic-induced internalization, palmitoylation at Cys347 has a role in determining which pathway is followed, be it by the beta-arrestin-dependent agonist-induced pathway or by the beta-arrestin-independent tonic internalization pathway.     

DHHC17 Palmitoylates ClipR-59 and Modulates ClipR-59 Association with the Plasma Membrane

ClipR-59 interacts with Akt and regulates Akt compartmentalization and Glut4 membrane trafficking in a plasma membrane association-dependent manner. The association of ClipR-59 with plasma membrane is mediated by ClipR-59 palmitoylation at Cys534 and Cys535. To understand the regulation of ClipR-59 palmitoylation, we have examined all known mammalian DHHC palmitoyltransferases with respect to their ability to promote ClipR-59 palmitoylation. We found that, among 23 mammalian DHHC palmitoyltransferases, DHHC17 is the major ClipR-59 palmitoyltransferase, as evidenced by the fact that DHHC17 interacted with ClipR-59 and palmitoylated ClipR-59 at Cys534 and Cys535. By palmitoylating ClipR-59, DHHC17 directly regulates ClipR-59 plasma membrane association, as ectopic expression of DHHC17 increased whereas silencing of DHHC17 reduced the levels of ClipR-59 associated with plasma membrane. We have also examined the role of DHHC17 in Akt signaling and found that silencing of DHHC17 in 3T3-L1 adipocytes decreased the levels of Akt as well as ClipR-59 on the plasma membrane and impaired insulin-dependent Glut4 membrane translocation. We suggest that DHHC17 is a ClipR-59 palmitoyltransferase that modulates ClipR-59 plasma membrane binding, thereby regulating Akt signaling and Glut4 membrane translocation in adipocytes.     

Amino acid residues 24-31 but not palmitoylation of cysteines 30 and 45 are required for membrane anchoring of glutamic acid decarboxylase, GAD65

The smaller isoform of the GABA synthesizing enzyme glutamic acid decarboxylase, GAD65, is synthesized as a soluble protein that undergoes post-translational modification(s) in the NH2-terminal region to become anchored to the membrane of small synaptic-like microvesicles in pancreatic beta cells, and synaptic vesicles in GABA-ergic neurons. A soluble hydrophilic form, a soluble hydrophobic form, and a hydrophobic firmly membrane-anchored form have been detected in beta cells. A reversible and hydroxylamine sensitive palmitoylation has been shown to distinguish the firmly membrane-anchored form from the soluble yet hydrophobic form, suggesting that palmitoylation of cysteines in the NH2-terminal region is involved in membrane anchoring. In this study we use site-directed mutagenesis to identify the first two cysteines in the NH2-terminal region, Cys 30 and Cys 45, as the sites of palmitoylation of the GAD65 molecule. Mutation of Cys 30 and Cys 45 to Ala results in a loss of palmitoylation but does not significantly alter membrane association of GAD65 in COS-7 cells. Deletion of the first 23 amino acids at the NH2 terminus of the GAD65 30/45A mutant also does not affect the hydrophobicity and membrane anchoring of the GAD65 protein. However, deletion of an additional eight amino acids at the NH2 terminus results in a protein which is hydrophilic and cytosolic. The results suggest that amino acids 24-31 are required for hydrophobic modification and/or targeting of GAD65 to membrane compartments, whereas palmitoylation of Cys 30 and Cys 45 may rather serve to orient or fold the protein at synaptic vesicle membranes.     

Characterization of the Palmitoylation Domain of SNAP-25

Abstract: SNAP-25 (synaptosomal associated protein of 25 kDa) is a neural specific protein that has been implicated in the synaptic vesicle docking and fusion process. It is tightly associated with membranes, and it is one of the major palmitoylated proteins found in neurons. The functional role of palmitoylation for SNAP-25 is unclear. In this report, we show that the palmitate of SNAP-25 is rapidly turned over in PC12 cells, with a half-life of ∼3 h, and the half-life for the protein is 8 h. Mutation of Cys to Ser at positions 85, 88, 90, and 92 reduced the palmitoylation to 9, 21, 42, and 35% of the wild-type protein, respectively. Additional mutations of either Cys^85,88 or Cys^90,92 nearly abolished palmitoylation of the protein. A similar effect on membrane binding for the mutant SNAP-25 was observed, which correlated with the degree of palmitoylation. These results suggest that all four Cys residues are involved in palmitoylation and that membrane association of SNAP-25 may be regulated through dynamic palmitoylation.