Palmitoylation of the Immunity Related GTPase,Irgm1: Impact on Membrane Localization and Ability to Promote Mitochondrial Fission

The Immunity-Related GTPases (IRG) are a family of large GTPases that mediate innate immune responses. Irgm1 is particularly critical for immunity to bacteria and protozoa, and for inflammatory homeostasis in the intestine. Although precise functions for Irgm1 have not been identified, prior studies have suggested roles in autophagy/mitophagy, phagosome remodeling, cell motility, and regulating the activity of other IRG proteins. These functions ostensibly hinge on the ability of Irgm1 to localize to intracellular membranes, such as those of the Golgi apparatus and mitochondria. Previously, it has been shown that an amphipathic helix, the αK helix, in the C-terminal portion of the protein partially mediates membrane binding. However, in absence of αK, there is still substantial binding of Irgm1 to cellular membranes, suggesting the presence of other membrane binding motifs. In the current work, an additional membrane localization motif was found in the form of palmitoylation at a cluster of cysteines near the αK. An Irgm1 mutant possessing alanine to cysteine substitutions at these amino acids demonstrated little residual palmitoylation, yet it displayed only a small decrease in localization to the Golgi and mitochondria. In contrast, a mutant containing the palmitoylation mutations in combination with mutations disrupting the amphipathic character of the αK displayed a complete loss of apparent localization to the Golgi and mitochondria, as well as an overall loss of association with cellular membranes in general. Additionally, Irgm1 was found to promote mitochondrial fission, and this function was undermined in Irgm1 mutants lacking the palmitoylation domain, and to a greater extent in those lacking the αK, or the αK and palmitoylation domains combined. Our data suggest that palmitoylation together with the αK helix firmly anchor Irgm1 in the Golgi and mitochondria, thus facilitating function of the protein.     

Rapid Plasma Membrane Anchoring of Newly Synthesized p59 fyn: Selective Requirement for NH2-Terminal Myristoylation and Palmitoylation at Cysteine-3

The trafficking of Src family proteins after biosynthesis is poorly defined. Here we studied the role of dual fatty acylation with myristate and palmitate in biosynthetic transport of p59^fyn. Metabolic labeling of transfected COS or NIH 3T3 cells with [³⁵S]methionine followed by analysis of cytosolic and total membrane fractions showed that Fyn became membrane bound within 5 min after biosynthesis. Newly synthesized Src, however, accumulated in the membranes between 20– 60 min. Northern blotting detected Fyn mRNA specifically in soluble polyribosomes and soluble Fyn protein was only detected shortly (1–2 min) after radiolabeling. Use of chimeric Fyn and Src constructs showed that rapid membrane targeting was mediated by the myristoylated NH₂-terminal sequence of Fyn and that a cysteine at position 3, but not 6, was essential. Examination of Gα_o-, Gα_s-, or GAP43-Fyn fusion constructs indicated that rapid membrane anchoring is exclusively conferred by the combination of N-myristoylation plus palmitoylation of cysteine-3. Density gradient analysis colocalized newly synthesized Fyn with plasma membranes. Interestingly, a 10–20-min lag phase was observed between plasma membrane binding and the acquisition of non-ionic detergent insolubility. We propose a model in which synthesis and myristoylation of Fyn occurs on soluble ribosomes, followed by rapid palmitoylation and plasma membrane anchoring, and a slower partitioning into detergent-insoluble membrane subdomains. These results serve to define a novel trafficking pathway for Src family proteins that are regulated by dual fatty acylation.     

Plasma Membrane Localization of Gαz Requires Two Signals

Three covalent attachments anchor heterotrimeric G proteins to cellular membranes: the α subunits are myristoylated and/or palmitoylated, whereas the γ chain is prenylated. Despite the essential role of these modifications in membrane attachment, it is not clear how they cooperate to specify G protein localization at the plasma membrane, where the G protein relays signals from cell surface receptors to intracellular effector molecules. To explore this question, we studied the effects of mutations that prevent myristoylation and/or palmitoylation of an epitope-labeled α subunit, α_z. Wild-type α_z (α_z-WT) localizes specifically at the plasma membrane. A mutant that incorporates only myristate is mistargeted to intracellular membranes, in addition to the plasma membrane, but transduces hormonal signals as well as does α_z-WT. Removal of the myristoylation site produced a mutant α_z that is located in the cytosol, is not efficiently palmitoylated, and does not relay the hormonal signal. Coexpression of βγ with this myristoylation defective mutant transfers it to the plasma membrane, promotes its palmitoylation, and enables it to transmit hormonal signals. Pulse-chase experiments show that the palmitate attached to this myristoylation-defective mutant turns over much more rapidly than does palmitate on α_z-WT, and that the rate of turnover is further accelerated by receptor activation. In contrast, receptor activation does not increase the slow rate of palmitate turnover on α_z-WT. Together these results suggest that myristate and βγ promote stable association with membranes not only by providing hydrophobicity, but also by stabilizing attachment of palmitate. Moreover, palmitoylation confers on α_z specific localization at the plasma membrane.     

SNAP-25 Palmitoylation and Plasma Membrane Targeting Require a Functional Secretory Pathway

Synaptosomal-associated protein of 25 kDa (SNAP-25) is a palmitoylated membrane protein essential for neurotransmitter release from synaptic terminals. We used neuronal cell lines to study the biosynthesis and posttranslational processing of SNAP-25 to investigate how palmitoylation contributes to the subcellular localization of the protein. SNAP-25 was synthesized as a soluble protein that underwent palmitoylation approximately 20 min after synthesis. Palmitoylation of the protein coincided with its stable membrane association. Treatment of cells with brefeldin A or other disrupters of transport inhibited palmitoylation of newly synthesized SNAP-25 and abolished membrane association. These results demonstrate that the processing of SNAP-25 and its targeting to the plasma membrane depend on an intact transport mechanism along the exocytic pathway. The kinetics of SNAP-25 palmitoylation and membrane association and the sensitivity of these parameters to brefeldin A suggest a novel trafficking pathway for targeting proteins to the plasma membrane. In vitro, SNAP-25 stably associated with membranes was not released from the membrane after chemical deacylation. We propose that palmitoylation of SNAP-25 is required for initial membrane targeting of the protein but that other interactions can maintain membrane association in the absence of fatty acylation.     

EM Structure of the Ectodomain of Integrin CD11b/CD18 and Localization of Its Ligand-Binding Site Relative to the Plasma Membrane

One-half of the integrin α-subunit Propeller domains contain and extra vWFA domain (αA domain), which mediates integrin binding to extracellular physiologic ligands via its metal-ion-dependent adhesion site (MIDAS). We used electron microscopy to determine the 3D structure of the αA-containing ectodomain of the leukocyte integrin CD11b/CD18 (αMβ2) in its inactive state. A well defined density for αA was observed within a bent ectodomain conformation, while the structure of the ectodomain in complex with the Fab fragment of mAb107, which binds at the MIDAS face of CD11b and stabilizes the inactive state, further revealed that αA is restricted to a relatively small range of orientations relative to the Propeller domain. Using Fab 107 as probe in fluorescent lifetime imaging microscopy (FLIM) revealed that αA is positioned relatively far from the membrane surface in the inactive state, and a systematic orientation search revealed that the MIDAS face would be accessible to extracellular ligand in the inactive state of the full-length cellular integrin. These studies are the first to define the 3D EM structure of an αA-containing integrin ectodomain and to position the ligand-binding face of αA domain in relation to the plasma membrane, providing new insights into current models of integrin activation.     

Structural determinants of the integrin transmembrane domain required for bidirectional signal transmission across the cell membrane

Studying the tight activity regulation of platelet-specific integrin α_IIbβ₃ is foundational and paramount to our understanding of integrin structure and activation. α_IIbβ₃ is essential for the aggregation and adhesion function of platelets in hemostasis and thrombosis. Structural and mutagenesis studies have previously revealed the critical role of α_IIbβ₃ transmembrane (TM) association in maintaining the inactive state. Gain-of-function TM mutations were identified and shown to destabilize the TM association leading to integrin activation. Studies using isolated TM peptides have suggested an altered membrane embedding of the β₃ TM α-helix coupled with α_IIbβ₃ activation. However, controversies remain as to whether and how the TM α-helices change their topologies in the context of full-length integrin in native cell membrane. In this study, we utilized proline scanning mutagenesis and cysteine scanning accessibility assays to analyze the structure and function correlation of the α_IIbβ₃ TM domain. Our identification of loss-of-function proline mutations in the TM domain suggests the requirement of a continuous TM α-helical structure in transmitting activation signals bidirectionally across the cell membrane, characterized by the inside-out activation for ligand binding and the outside-in signaling for cell spreading. Similar results were found for α_Lβ₂ and α₅β₁ TM domains, suggesting a generalizable mechanism. We also detected a topology change of β₃ TM α-helix within the cell membrane, but only under conditions of cell adhesion and the absence of α_IIb association. Our data demonstrate the importance of studying the structure and function of the integrin TM domain in the native cell membrane.     

Cysteine Palmitoylation of the γ Subunit Has a Dominant Role in Modulating Activity of the Epithelial Sodium Channel

The epithelial sodium channel (ENaC) is composed of three homologous subunits (α, β, and γ) with cytoplasmic N and C termini. Our previous work revealed that two cytoplasmic Cys residues in the β subunit, βCys-43 and βCys-557, are Cys-palmitoylated. ENaCs with mutant βC43A/C557A exhibit normal surface expression but enhanced Na⁺ self-inhibition and reduced channel open probability. Although the α subunit is not palmitoylated, we now show that the two cytoplasmic Cys residues in the γ subunit are palmitoylated. ENaCs with mutant γC33A, γC41A, or γC33A/C41A exhibit reduced activity compared with wild type channels but normal surface expression and normal levels of α and γ subunit-activating cleavage. These mutant channels have significantly enhanced Na⁺ self-inhibition and reduced open probability compared with wild type ENaCs. Channel activity was enhanced by co-expression with the palmitoyltransferase DHHC2 that also co-immunoprecipitates with ENaCs. Secondary structure prediction of the N terminus of the γ subunit places γCys-33 within an α-helix and γCys-44 on a coil before the first transmembrane domain within a short tract that includes a well conserved His-Gly motif, where mutations have been associated with altered channel gating. Our current and previous results suggest that palmitoylation of the β and γ subunits of ENaCs enhances interactions of their respective cytoplasmic domains with the plasma membrane and stabilizes the open state of the channel. Comparison of activities of channels lacking palmitoylation sites in individual or multiple subunits revealed that γ subunit palmitoylation has a dominant role over β subunit palmitoylation in modulating ENaC gating.     

Down-regulating protein kinase C alpha: Functional cooperation between the proteasome and the endocytic system

Ubiquitination, proteasome, caveolae and endosomes have been implicated in controlling protein kinase Cα (PKCα) down-regulation. However, the molecular mechanism remained obscure. Here we show that endosomes and proteasome cooperate in phorbol ester 12-O-tetradecanoyl phorbol acetate (TPA)-induced down-regulation of PKCα. We show that following TPA treatment and translocation to the plasma membrane, PKCα undergoes multimonoubiquitination prior to its degradation by the proteasome. However, to reach the proteasome, PKCα must travel through the endocytic system from early to late endosomes. This route requires functional endosomes, whereby endosomal alkalinization, or ablation, abrogates completely PKCα degradation maintaining the enzyme at the plasma membrane. This route also depends on synaptotagmin (Syt) II and the Rab7 GTPase, whereby Syt II knock-down or expression of the GDP-locked Rab7 inactive mutant prevents PKCα degradation. We further show that proteasome plays a dual role, where an active proteasome is required for deubiquitination of PKCα, a step crucial to prevent PKCα targeting to the endocytic recycling compartment. Finally, we show that the association with rafts-localized cell surface proteins that internalize in a clathrin-independent fashion is necessary to allow the trafficking of PKCα from the plasma membrane to the proteasome, its ultimate degradation station.     

Cys Palmitoylation of the β Subunit Modulates Gating of the Epithelial Sodium Channel

The epithelial Na⁺ channel (ENaC) is comprised of three homologous subunits (α, β, and γ) that have a similar topology with two transmembrane domains, a large extracellular region, and cytoplasmic N and C termini. Although ENaC activity is regulated by a number of factors, palmitoylation of its cytoplasmic Cys residues has not been previously described. Fatty acid-exchange chemistry was used to determine whether channel subunits were Cys-palmitoylated. We observed that only the β and γ subunits were modified by Cys palmitoylation. Analyses of ENaCs with mutant β subunits revealed that Cys-43 and Cys-557 were palmitoylated. Xenopus oocytes expressing ENaC with a β C43A,C557A mutant had significantly reduced amiloride-sensitive whole cell currents, enhanced Na⁺ self-inhibition, and reduced single channel P_o when compared with wild-type ENaC, while membrane trafficking and levels of surface expression were unchanged. Computer modeling of cytoplasmic domains indicated that β Cys-43 is in proximity to the first transmembrane α helix, whereas β Cys-557 is within an amphipathic α-helix contiguous with the second transmembrane domain. We propose that β subunit palmitoylation modulates channel gating by facilitating interactions between cytoplasmic domains and the plasma membrane.     

Curcumin Prevents Palmitoylation of Integrin β4 in Breast Cancer Cells

Curcumin has been shown to mitigate cancer phenotypes such as invasive migration, proliferation, and survival by disrupting numerous signaling pathways. Our previous studies showed that curcumin inhibits integrin β4 (ITG β4)-dependent migration by blocking interaction of this integrin with growth factor receptors in lipid rafts. In the current study, we investigated the possibility that curcumin inhibits ITG β4 palmitoylation, a post-translational modification required for its lipid raft localization and signaling activity. We found that the levels of ITG β4 palmitoylation correlated with the invasive potential of breast cancer cells, and that curcumin effectively reduced the levels of ITG β4 palmitoylation in invasive breast cancer cells. Through studies of ITG β4 palmitoylation kinetics, we concluded curcumin suppressed palmitoylation independent of growth factor-induced phosphorylation of key ITG β4 Ser and Tyr residues. Rather, curcumin blocked autoacylation of the palmitoyl acyltransferase DHHC3 that is responsible for ITG β4 palmitoylation. Moreover, these data reveal that curcumin is able to prevent the palmitoylation of a subset of proteins, but not indiscriminately bind to and block all cysteines from modifications. Our studies reveal a novel paradigm for curcumin to account for much of its biological activity, and specifically, how it is able to suppress the signaling function of ITG β4 in breast cancer cells.     

Hypoxic preconditioning promotes the translocation of protein kinase C ? binding with caveolin-3 at cell membrane not mitochondrial in rat heart

Protein kinase C has been shown to play a central role in the cardioprotection of ischemic preconditioning. However, the mechanism underlying PKC-mediated cardioprotection is not completely understood. Given that caveolae are critical for PKC signaling, we sought to determine whether hypoxic preconditioning promotes translocation and association of PKC isoforms with caveolin-3. A cellular model of hypoxic preconditioning from adult rat cardiac myocytes (ARCM) or H9c2 cells was employed to examine PKC isoforms by molecular, biochemical and cellular imaging analysis. Hypoxia was induced by incubating the cells in an airtight chamber in which O₂ was replaced by N₂ with glucose-free Tyrode''s solution. Cells were subjected to hypoxic preconditioning with 10 minutes of hypoxia followed by 30 minutes of reoxygenation. Western blot data indicated that the band intensity for PKCϵ, PKCδ or PKCα, but not PKCβ and PKCζ was enhanced significantly by hypoxic preconditioning from the caveolin-enriched plasma membrane interactions. Immunoprecipitation experiments from the caveolin-enriched membrane fractions of ARCM showed that the level of PKCϵ, PKCδ and PKCα in the anti-caveolin-3 immunoprecipitates was also increased by hypoxic preconditioning. Further, our FRET analysis in H9c2 cells suggested that there is a minimum FRET signal for caveolin-3 and PKCϵ along cell peripherals, but hypoxic preconditioning enhanced the FRET signal, indicating a potential interaction between caveolin-3 and PKCϵ. And also treatment of the cells with hypoxic preconditioning led to a smaller amount of translocation of PKCϵ to the mitochondria than that to the membrane. We demonstrate that hypoxic preconditioning promotes rapid association of PKCϵ, PKCδ and PKCα with the caveolin-enriched plasma membrane microdomain of cardiac myocytes, and PKCϵ via direct molecular interaction with caveolin-3. This regulatory mechanism may play an important role in cardioprotection.     

Prothrombin Activation by Platelet-associated Prothrombinase Proceeds through the Prethrombin-2 Pathway via a Concerted Mechanism

The protease α-thrombin is a key enzyme of the coagulation process as it is at the cross-roads of both the pro- and anti-coagulant pathways. The main source of α-thrombin in vivo is the activation of prothrombin by the prothrombinase complex assembled on either an activated cell membrane or cell fragment, the most relevant of which is the activated platelet surface. When prothrombinase is assembled on synthetic phospholipid vesicles, prothrombin activation proceeds with an initial cleavage at Arg-320 yielding the catalytically active, yet effectively anticoagulant intermediate meizothrombin, which is released from the enzyme complex ∼30–40% of the time. Prothrombinase assembled on the surface of activated platelets has been shown to proceed through the inactive intermediate prethrombin-2 via an initial cleavage at Arg-271 followed by cleavage at Arg-320. The current work tests whether or not platelet-associated prothrombinase proceeds via a concerted mechanism through a study of prothrombinase assembly and function on collagen-adhered, thrombin-activated, washed human platelets in a flow chamber. Prothrombinase assembly was demonstrated through visualization of bound factor Xa by confocal microscopy using a fluorophore-labeled anti-factor Xa antibody, which demonstrated the presence of distinct platelet subpopulations capable of binding factor Xa. When prothrombin activation was monitored at a typical venous shear rate over preassembled platelet-associated prothrombinase neither potential intermediate, meizothrombin or prethrombin-2, was observed in the effluent. Collectively, these findings suggest that platelet-associated prothrombinase activates prothrombin via an efficient concerted mechanism in which neither intermediate is released.     

Differential Ligand Binding Affinities of Human Estrogen Receptor-α Isoforms

Rapid non-genomic effects of 17β-estradiol are elicited by the activation of different estrogen receptor-α isoforms. Presence of surface binding sites for estrogen have been identified in cells transfected with full-length estrogen receptor-α66 (ER66) and the truncated isoforms, estrogen receptor-α46 (ER46) and estrogen receptor-α36 (ER36). However, the binding affinities of the membrane estrogen receptors (mERs) remain unknown due to the difficulty of developing of stable mER-transfected cell lines with sufficient mER density, which has largely hampered biochemical binding studies. The present study utilized cell-free expression systems to determine the binding affinities of 17β-estradiol to mERs, and the relationship among palmitoylation, membrane insertion and binding affinities. Saturation binding assays of human mERs revealed that [³H]-17β-estradiol bound ER66 and ER46 with K_d values of 68.81 and 60.72 pM, respectively, whereas ER36 displayed no specific binding within the tested concentration range. Inhibition of palmitoylation or removal of the nanolipoprotein particles, used as membrane substitute, reduced the binding affinities of ER66 and ER46 to 17β-estradiol. Moreover, ER66 and ER46 bound differentially with some estrogen receptor agonists and antagonists, and phytoestrogens. In particular, the classical estrogen receptor antagonist, ICI 182,780, had a higher affinity for ER66 than ER46. In summary, the present study defines the binding affinities for human estrogen receptor-α isoforms, and demonstrates that ER66 and ER46 show characteristics of mERs. The present data also indicates that palmitoylation and membrane insertion of mERs are important for proper receptor conformation allowing 17β-estradiol binding. The differential binding of ER66 and ER46 with certain compounds substantiates the prospect of developing mER-selective drugs.     

Visualization of a functional Galpha q-green fluorescent protein fusion in living cells. Association with the plasma membrane is disrupted by mutational activation and by elimination of palmitoylation sites, but not be activation mediated by receptors or AlF4-

To investigate how G protein alpha subunit localization is regulated under basal and activated conditions, we inserted green fluorescent protein (GFP) into an internal loop of Galpha(q). alpha(q)-GFP stimulates phospholipase C in response to activated receptors and inhibits betagamma-dependent activation of basal G protein-gated inwardly rectifying K(+) currents as effectively as alpha(q) does. Association of alpha(q)-GFP with the plasma membrane is reduced by mutational activation and eliminated by mutation of the alpha(q) palmitoylation sites, suggesting that alpha(q) must be in the inactive, palmitoylated state to be targeted to this location. We tested the effects of activation by receptors and by AlF(4)(-) on the localization of alpha(q)-GFP in cells expressing both alpha(q)-GFP and a protein kinase Cgamma-red fluorescent protein fusion that translocates to the plasma membrane in response to activation of G(q). In cells that clearly exhibit protein kinase Cgamma-red fluorescent protein translocation responses, relocalization of alpha(q)-GFP is not observed. Thus, under conditions associated with palmitate turnover and betagamma dissociation, alpha(q)-GFP remains associated with the plasma membrane. These results suggest that upon reaching the plasma membrane alpha(q) receives an anchoring signal in addition to palmitoylation and association with betagamma, or that during activation, one or both of these factors continues to retain alpha(q) in this location.     

The cysteine-rich domain of synaptosomal-associated protein of 23 kDa (SNAP-23) regulates its membrane association and regulated exocytosis from mast cells

Synaptosomal-associated protein of 23 kDa (SNAP-23) plays an important role during regulated exocytosis of various inflammatory mediators, stored in secretory granules, from mast cells in response to physiological triggers. It is however synthesized as a soluble protein, and the mechanisms by which free SNAP-23 gets peripherally associated with membrane for the regulation of exocytosis, are poorly defined. SNAP-23 contains a hydrophobic domain with five closely spaced cysteines which get palmitoylated, and we show that SNAP-23 cysteine mutants show differential membrane association when transfected in rat basophilic leukemia (RBL) mast cells. SNAP-23 Cys⁻ mutant, devoid of all five cysteines, and SNAP-23 P119A (proline to alanine) mutant, that likely interferes with palmitoylation of SNAP-23 by palmitoyl transferases are completely cytosolic. Mutating specific cysteines (Cys; C) to leucine or phenylalanine (L or F; retains hydrophobicity but lacks palmitoylation) partially decreases the membrane association of SNAP-23 which is further hampered by alanine (A; has lesser hydrophobicity, and lacks palmitoylation) mutation at C79, C80 or C83 position. Cloning a transmembrane domain MDR3_1–145 from multidrug resistance protein into SNAP-23 Cys⁻ mutant is able to partially restore its membrane association. Regulated exocytosis studies using co-transfected human growth hormone (hGH) secretion reporter plasmid revealed that overexpression of SNAP-23 Cys⁻ and P119A mutants significantly inhibits the overall extent of exocytosis from RBL mast cells, whereas expression of SNAP-23 Cys⁻-MDR3_1–145 fusion protein is able to restore exocytosis. These results establish that the cysteine-rich domain of SNAP-23 regulates its membrane association and thereby also regulates exocytosis from mast cells.     

Palmitoylation of brain capillary proteins

Palmitoylation is a reversible posttranslational modification which is involved in the regulation of several membrane proteins such as β₂-adrenergic receptor, p21^ras and trimeric G-protein α-subunits. This covalent modification could be involved in the regulation of the numerous membrane proteins present in the blood-brain barrier capillaries. The palmitoylation activity present in brain capillaries was characterized using [³H]palmitate labeling followed by chloroform methanol precipitation. Palmitate solubilizing agents such as detergents and bovine serum albumin (BSA), were used for optimizing activity. Some palmitoylated substrates were identified using [³H]palmitate labeling followed by immunoprecipitation with specific antibodies. Two optimal palmitate solubilization conditions were found, one involves cell permeabilization (Triton X-100) and the other represents a more physiological condition where membrane integrity is conserved (BSA). Sensitivity to the cysteine modifier N-ethylmaleimide and to hydrolysis, using hydroxylamine or alkaline methanolysis, indicated that palmitic acid was bound to the proteins by a thioester bond. Maximal palmitate incorporation was reached after 30 or 60 min of incubation in the presence of Triton or BSA, respectively. Depalmitoylation was observed in the presence of BSA, but not with detergents. The palmitoylation reaction was optimal at pH 8 or 9 in the presence of Triton or BSA, respectively, but palmitoylated substrates were detectable over a wide range of pH values. In the presence of Triton X-100, the addition of ATP, CoA and Mg²⁺ to the incubation medium increased palmitoylation by up to 80-fold. Two palmitoylated substrates were identified, a 42 kDa G-protein α subunit and p21^ras. The study shows that the utilization of palmitate solubilizing agents is essential to measure in vitro palmitoylation in brain capillaries. Several palmitoylated proteins are present in the blood-brain barrier including five major substrates of 12, 21, 35, 42 and 55 kDa. It is suggested that palmitoylation could play a crucial role in the regulation of brain capillary function, since the two substrates identified in this study are known to be involved in signal transduction, vesicular transport and cell differentiation.     

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.     

Wortmannin-Sensitive Phosphorylation, Translocation, and Activation of PLCγ1, but Not PLCγ2, in Antigen-stimulated RBL-2H3 Mast Cells

In RBL-2H3 tumor mast cells, cross-linking the high affinity IgE receptor (FcεRI) with antigen activates cytosolic tyrosine kinases and stimulates Ins(1,4,5)P₃ production. Using immune complex phospholipase assays, we show that FcεRI cross-linking activates both PLCγ1 and PLCγ2. Activation is accompanied by the increased phosphorylation of both PLCγ isoforms on serine and tyrosine in antigen-treated cells. We also show that the two PLCγ isoforms have distinct subcellular localizations. PLCγ1 is primarily cytosolic in resting RBL-2H3 cells, with low levels of plasma membrane association. After antigen stimulation, PLCγ1 translocates to the plasma membrane where it associates preferentially with membrane ruffles. In contrast, PLCγ2 is concentrated in a perinuclear region near the Golgi and adjacent to the plasma membrane in resting cells and does not redistribute appreciably after FcεRI cross-linking. The activation of PLCγ1, but not of PLCγ2, is blocked by wortmannin, a PI 3-kinase inhibitor previously shown to block antigen-stimulated ruffling and to inhibit Ins(1,4,5)P₃ synthesis. In addition, wortmannin strongly inhibits the antigen-stimulated phosphorylation of both serine and tyrosine residues on PLCγ1 with little inhibition of PLCγ2 phosphorylation. Wortmannin also blocks the antigen-stimulated translocation of PLCγ1 to the plasma membrane. Our results implicate PI 3-kinase in the phosphorylation, translocation, and activation of PLCγ1. Although less abundant than PLCγ2, activated PLCγ1 may be responsible for the bulk of antigen-stimulated Ins(1,4,5)P₃ production in RBL-2H3 cells.     

Cortical Granule Exocytosis Is Mediated by Alpha-SNAP and N-Ethilmaleimide Sensitive Factor in Mouse Oocytes

Cortical granule exocytosis (CGE), also known as cortical reaction, is a calcium- regulated secretion that represents a membrane fusion process during meiotic cell division of oocytes. The molecular mechanism of membrane fusion during CGE is still poorly understood and is thought to be mediated by the SNARE pathway; nevertheless, it is unkown if SNAP (acronym for soluble NSF attachment protein) and NSF (acronym for N-ethilmaleimide sensitive factor), two key proteins in the SNARE pathway, mediate CGE in any oocyte model. In this paper, we documented the gene expression of α-SNAP, γ-SNAP and NSF in mouse oocytes. Western blot analysis showed that the expression of these proteins maintains a similar level during oocyte maturation and early activation. Their localization was mainly observed at the cortical region of metaphase II oocytes, which is enriched in cortical granules. To evaluate the function of these proteins in CGE we set up a functional assay based on the quantification of cortical granules metaphase II oocytes activated parthenogenetically with strontium. Endogenous α-SNAP and NSF proteins were perturbed by microinjection of recombinant proteins or antibodies prior to CGE activation. The microinjection of wild type α-SNAP and the negative mutant of α-SNAP L294A in metaphase II oocytes inhibited CGE stimulated by strontium. NEM, an irreversibly inhibitor of NSF, and the microinjection of the negative mutant NSF D1EQ inhibited cortical reaction. The microinjection of anti-α-SNAP and anti-NSF antibodies was able to abolish CGE in activated metaphase II oocytes. The microinjection of anti-γ SNAP antibody had no effect on CGE. Our findings indicate, for the first time in any oocyte model, that α-SNAP, γ-SNAP, and NSF are expressed in mouse oocytes. We demonstrate that α-SNAP and NSF have an active role in CGE and propose a working model.     

The membrane scaffold CD82 regulates cell adhesion by altering α4 integrin stability and molecular density

Hematopoietic stem/progenitor cell (HSPC) interactions with the bone marrow microenvironment are important for maintaining HSPC self-renewal and differentiation. In recent work, we identified the tetraspanin protein, CD82, as a regulator of HPSC adhesion and homing to the bone marrow, although the mechanism by which CD82 mediated adhesion was unclear. In the present study, we determine that CD82 expression alters cell–matrix adhesion, as well as integrin surface expression. By combining the superresolution microscopy imaging technique, direct stochastic optical reconstruction microscopy, with protein clustering algorithms, we identify a critical role for CD82 in regulating the membrane organization of α4 integrin subunits. Our data demonstrate that CD82 overexpression increases the molecular density of α4 within membrane clusters, thereby increasing cellular adhesion. Furthermore, we find that the tight packing of α4 into membrane clusters depend on CD82 palmitoylation and the presence of α4 integrin ligands. In combination, these results provide unique quantifiable evidence of CD82’s contribution to the spatial arrangement of integrins within the plasma membrane and suggest that regulation of integrin density by tetraspanins is a critical component of cell adhesion.