Rab4 GTP/GDP modulates amiloride-sensitive sodium channel (ENaC) function in colonic epithelia

The sodium-selective amiloride-sensitive epithelial sodium channel (ENaC) mediates electrogenic sodium re-absorption in tight epithelia. ENaC expression at the plasma membrane requires regulated transport, processing, and macromolecular assembly of subunit proteins in a defined and highly compartmentalized manner. Ras-related Rab GTPases monitor these processes in a highly regulated sequence of events. In order to evaluate the role of Rab proteins in ENaC function, Rab4 wild-type (WT), the GTPase-deficient mutant Rab4Q67L, and the dominant negative GDP-locked mutant Rab4S22N were over-expressed in the colon cancer cell line, HT-29 and amiloride-sensitive currents were recorded. Rab4 over-expression inhibited amiloride-sensitive currents. The effect was reversed by introducing Rab4-neutralizing antibody and Rab4 specific SiRNA. The GDP-locked Rab4 mutant inhibited, while GTPase-deficient mutant moderately stimulated amiloride-sensitive currents. Active status of Rab4 was confirmed by GTP overlay assay, while its expression was verified by Western blotting. Immunoprecipitation and pull-down assay suggest protein-protein interaction between Rab4 and ENaC. In addition, the functional modulation coincides with concomitant changes in ENaC expression at the cell surface and in intracellular pool. We propose that Rab4 is a critical element that regulates ENaC function by mechanisms that include GTP-GDP status, recycling, and expression level. Our observations imply that channel expression in apical membranes of epithelial cell system incorporates RabGTPase as an essential determinant of channel function and adds an exciting paradigm to ENaC therapeutics.     

Small GTPase protein Rac-1 is activated with maturation and regulates cell morphology and function in chondrocytes

During maturation, chondrocytes undergo changes in morphology, matrix production, and gene expression; however, it remains unclear whether these are interrelated. In this study, we examined whether Rho GTPases were involved in these regulatory interplays. Levels of active Rho GTPases were assayed in immature and mature primary chondrocytes. We found that activation of Rac-1 and Cdc42 increased with maturation, whereas RhoA levels remained unchanged. GFP-tagged Rho GTPases tracked cellular localization. Rac-1 was enriched at the cell membrane where it co-localized with cortical actin, while RhoA and Cdc42 were cytoplasmic. To test the roles of Rac-1 in chondrocyte maturation, we force-expressed constitutively active or dominant negative forms of Rac-1 and assessed phenotypic consequences in primary chondrocytes. Activated Rac-1 expression induced chondrocyte enlargement and increased matrix metalloproteinase expression, which are characteristic of mature chondrocytes. Conversely, Rac-1 inactivation diminished adhesion, decreased alkaline phosphatase activity, and stimulated functions typical of immature chondrocytes. Exposure to a pro-maturation factor, Wnt3A, induced a flattened and enlarged morphology accompanied by peripheral Rac-1 re-arrangement. Wnt3A stimulated Tiam1 expression and Rac-1 activation, while DN-Rac-1 inhibited Wnt3A-induced cell spreading. Our data provide strong evidence that Rac-1 coordinates changes in chondrocyte phenotype and function and stimulates the maturation process essential for skeletal development.     

GTP is required to stabilize and display transamidation activity of transglutaminase 2

Transglutaminase 2 (TGase 2) is a bifunctional enzyme that catalyzes calcium-dependent transamidation and GTP binding/hydrolysis. The transamidation activity is proposed to be associated with several neurodegenerative disorders such as Alzheimer's and Hungtinton's disease. However, the regulation mechanism by which TGase 2 causes neurodegeneration is unknown. In this study, we show that two activities of TGase 2 have a differential stability; transamidation activity is less stable than GTP hydrolytic activity, and that GTP was required to stabilize and to display transamidation activity. Moreover, GTP binding-defective mutant of TGase 2 did not show any transamidation activity in transfection experiments. These results indicate that GTP binding is crucial for transamidation activity of TGase 2, suggesting that protein cross-linking by TGase 2 might be associated with G-protein coupled receptor signaling system. Thus, our data could contribute to understand the regulation of TGase 2 activity and TGase 2-associated pathogenesis.     

RhoGDI-3 regulates RhoG and targets this protein to the Golgi complex through its unique N-terminal domain

Guanine nucleotide dissociation inhibitors (GDIs) regulate both GDP/GTP and membrane association/dissociation cycles of Rho/Rac and Rab proteins.RhoGDI-3 is distinguishable from other rhoGDI proteins by its partial association with a detergent-resistant subcellular fraction. Here, we investigate the activity of this unusual rhoGDI using confocal laser scanning microscopy, immuno-isolation, and rhoGDI-3 mutants. We establish that the noncytosolic fraction of rhoGDI-3 is associated with the Golgi apparatus. The domain involved in this association is the unique N-terminal segment of rhoGDI-3 predicted to form an amphipathic α helix. This peptide is indispensable for Golgi association of rhoGDI-3 and sufficient to address a green fluorescent protein to the Golgi apparatus. Site-directed mutations, decreasing the hydrophobic surface of the helix, localize rhoGDI-3 into the cytoplasm. We establish that rhoGDI-3 is able to inhibit activation of the RhoG protein and to target this protein to the Golgi apparatus. Furthermore, we demonstrate the importance of the rhoGDI-3 N-terminal segment for both Golgi targeting and stability of the cytoplasmic RhoG/rhoGDI-3 complex. RhoGDI-3 is the first example of a GDI directly involved in the delivery of a Rho protein to a specific subcellular compartment.     

Molecular dynamics analysis of the effects of GTP,GDP and benzimidazole derivative on structural dynamics of a cell division protein FtsZ from Mycobacterium tuberculosis

Abstract

The prevailing multi-drug resistance in Mycobacterium tuberculosis continues to remain one of the main challenges to combat tuberculosis. Hence, it becomes imperative to focus on novel drug targets. Filamenting temperature-sensitive mutant Z (FtsZ) is an essential cell division protein, a eukaryotic tubulin homologue and a promising drug target. During cytokinesis, FtsZ polymerises in the presence of GTP to form Z-ring and recruits other proteins at this site that eventually lead to the formation of daughter cells. Benzimidazoles were experimentally shown to inhibit Mtb-FtsZ, with one of the benzimidazole derivatives, M1, being reported to have the minimum inhibitory concentration (MIC) value of 3.13 µg/mL. In the present study, mechanism of destabilisation of FtsZ in the presence of M1 was computationally investigated in the presence of its substrate GTP/GDP employing molecular dynamics (MD) simulation analysis, principal component analysis (PCA), molecular mechanics combined with the generalised Born and surface area continuum salvation (MM-GBSA) and density functional theory (DFT). From the analyses, it is proposed that binding of M1 in the inter-domain cleft induces structural changes in the GTP-binding region that affect GTP binding, thus switching the preference of this protein towards depolymerised state and eventually inhibiting the cell division. Hence, this study provides mechanistic insights into the design of novel benzimidazole inhibitors against Mtb-FtsZ.

Communicated by Ramaswamy H. Sarma     

Aberrant alternative splicing of RHOA is associated with loss of its expression and activity in diffuse-type gastric carcinoma cells

RhoA is a member of Rho family small GTPases that regulates diverse cellular functions. Recent large-scale sequencing studies have identified recurrent somatic mutations of RHOA in diffuse-type gastric carcinoma (DGC), indicating that RHOA is a driver of DGC. In this study, we investigated the possible abnormalities of RHOA in a panel of gastric carcinoma (GC) cell lines. Pulldown assay and immunoblot analysis showed that the activity and expression of RhoA were detectable in all GC cell lines tested, except for two DGC cell lines, HSC-59 and GSU. RHOA coding region sequencing revealed that aberrant alternative splicing of RHOA occurred in these cell lines. Quantitative real-time PCR analysis showed that the expression of wild-type RHOA was nearly undetectable, whereas splicing variants were almost exclusively expressed in HSC-59 and GSU cell lines. However, the expression levels of RHOA splicing variants were very low and the corresponding proteins were not detected by immunoblotting. Moreover, the splicing isoforms of RhoA protein were neither efficiently expressed nor activated even if ectopically expressed in cells. These results indicate that aberrant alternative splicing of RHOA results in the loss of its activity and expression in DGC cells.     

The hinge region of Escherichia coli ribosomal protein L7/L12 is required for factor binding and GTP hydrolysis

A variant form of Escherichia coli ribosomal protein L7/L12 that lacked residues 42 to 52 (L7/L12 Δ42–52) in the hinge region was shown previously to be completely inactive in supporting polyphenylalanine synthesis although it bound to L7/L12 deficient core particles with the normal stoichiometry of four copies per particle (Oleinikov AV, Perroud B, Wang B, Traut RR (1993) J Biol Chem, 268, 917–922). The result suggested that the hinge confers flexibility that is required for activity because the resulting bent conformation allows the distal C-terminal domain to occupy a location on the body of the large ribosomal subunit proximal to the base of the L7/L12 stalk where elongation factors bind. Factor binding to the hinge-truncated variant was tested. As an alternative strategy to deleting residues from the hinge, seven amino acid residues within the putative hinge region were replaced by seven consecutive proline residues in an attempt to confer increased rigidity that might reduce or eliminate the bending of the molecule inferred to be functionally important. This variant, L7/L12: (Pro)₇, remained fully active in protein synthesis. Whereas the binding of both factors in ribosomes containing L7/L12:Δ42–52 was decreased by about 50%, there was no loss of factor binding in ribosomes containing L7/L12:(Pro)₇, as predicted from the retention of protein synthesis activity. The factor:ribosome complexes that contained L7/L12:Δ42–52 had the same low level of GTP hydrolysis as the core particles completely lacking L7/L12 and EF-G did not support translocation measured by the reaction of phe-tRNA bounds in hr Asite with puromycin. It is concluded that the hinge region is required for the functionally productive binding of elongation factors, and the defect in protein synthesis reported previously is due to this defect. The variant produced by the introduction of the putative rigid Pro₇ sequence retains sufficient flexibility for full activity.     

G protein activation is prerequisite for functional coupling between Galpha/Gbetagamma and tubulin/microtubules

Heterotrimeric G proteins participate in signal transduction by transferring signals from cell surface receptors to intracellular effector molecules. Interestingly, recent results suggest that G proteins also interact with microtubules and participate in cell division and differentiation. It has been shown earlier that both alpha and betagamma subunits of G proteins modulate microtubule assembly in vitro. Since G protein activation and subsequent dissociation of alpha and betagamma subunits are necessary for G proteins to participate in signaling processes, here we asked if similar activation is required for modulation of microtubule assembly by G proteins. We reconstituted Galphabetagamma heterotrimer from myristoylated-Galpha and prenylated-Gbetagamma, and found that the heterotrimer blocks Gi1alpha activation of tubulin GTPase and inhibits the ability of Gbeta1gamma2 to promote in vitro microtubule assembly. Results suggest that G protein activation is required for functional coupling between Galpha/Gbetagamma and tubulin/microtubules, and supports the notion that regulation of microtubules is an integral component of G protein mediated signaling.     

Identification of a bipartite focal adhesion localization signal in RhoU/Wrch-1, a Rho family GTPase that regulates cell adhesion and migration

Background information. Rho GTPases are important regulators of cytoskeleton dynamics and cell adhesion. RhoU/Wrch‐1 is a Rho GTPase which shares sequence similarities with Rac1 and Cdc42 (cell division cycle 42), but has also extended N‐ and C‐terminal domains. The N‐terminal extension promotes binding to SH3 (Src homology 3)‐domain‐containing adaptors, whereas the C‐terminal extension mediates membrane targeting through palmitoylation of its non‐conventional CAAX box. RhoU/Wrch‐1 possesses transforming activity, which is negatively regulated by its N‐terminal extension and depends on palmitoylation. Results. In the present study, we have shown that RhoU is localized to podosomes in osteoclasts and c‐Src‐expressing cells, and to focal adhesions of HeLa cells and fibroblasts. The N‐terminal extension and the palmitoylation site were dispensable, whereas the C‐terminal extension and effector binding loop were critical for RhoU targeting to focal adhesions. Moreover, the number of focal adhesions was reduced and their distribution changed upon expression of activated RhoU. Conversely, RhoU silencing increased the number of focal adhesions. As RhoU was only transiently associated with adhesion structures, this suggests that RhoU may modify adhesion turnover and cell migration rate. Indeed, we found that migration distances were increased in cells expressing activated RhoU and decreased when RhoU was knocked‐down. Conclusions. Our data indicate that RhoU localizes to adhesion structures, regulates their number and distribution and increases cell motility. It also suggests that the RhoU effector binding and C‐terminal domains are critical for these functions.     

Actin polymerization in the equatorial and postacrosomal regions of guinea pig spermatozoa during the acrosome reaction is regulated by G proteins

The acrosome reaction (AR) is an exocytotic process of spermatozoa, and an absolute requirement for fertilization. During AR, actin polymerization is necessary in the equatorial and postacrosomal regions of guinea pig sperm for spermatozoa incorporation deep into the egg cytoplasm, but not for plasma membrane (PM) fusion nor the early steps of egg activation. To identify the mechanisms involved in this sperm actin polymerization, we searched for the protein members, known to be involved in a highly conserved model, that may apply to any cellular process in which de novo actin polymerization occurs from G protein activation. WASP, Arp 2/3, profilins I and II, and Cdc42, RhoA and RhoB GTPases were localized by indirect immunofluorescence (IIF) in guinea pig spermatozoa and their presence corroborated by Western blotting. WASP and profilin II were translocated to the postacrosomal region (Arp2/3 already were there) in long-term capacitated and acrosome-reacted spermatozoa, at the same time as actin polymerization occurred. These events were inhibited by GDP-beta-S and promoted by lysophosphatidic acid (LPA) and GTP-gamma-S, a small GTPase inhibitor and two activators, respectively. By immunoprecipitation, Cdc42-WASp association was identified in capacitated but not in noncapacitated gametes. Polymerized actin in the postacrosomal region is apparently anchored both to the postacrosomal perinuclear theca region and the overlying PM. Results suggest that GTPases are involved in sperm actin polymerization, in the postacrosomal region and the mechanism for polymerization might fit a previously proposed model (Mullins, 2000: Curr Opin Cell Biol 12:91-96).     

Myogenic differentiation is dependent on both the kinase function and the N-terminal sequence of mammalian target of rapamycin

The mammalian target of rapamycin (mTOR) is a serine/threonine protein kinase known to control initiation of translation through two downstream pathways: eukaryotic initiation factor 4E-binding protein 1 (4E-BP1)/eukaryotic initiation factor 4E and ribosomal p70 S6 kinase (S6K1). We previously showed in C2C12 murine myoblasts that rapamycin arrests cells in G(1) phase and completely inhibits terminal myogenesis. To elucidate the pathways that regulate myogenesis, we established stable C2C12 cell lines that express rapamycin-resistant mTOR mutants (mTORrr; S2035I) that have N-terminal deletions (Delta10 or Delta91) or are full-length kinase-dead mTORrr proteins. Additional clones expressing a constitutively active S6K1 were also studied. Our results show that Delta10mTORrr signals 4E-BP1 and permits rapamycin-treated myoblasts to differentiate, confirming the mTOR dependence of the inhibition of myogenesis by rapamycin. C2C12 cells expressing either Delta91mTORrr or kinase-dead mTORrr(D2338A) could not phosphorylate 4E-BP1 in the presence of rapamycin and could not abrogate the inhibition of myogenesis. Taken together, our results indicate that both the kinase function of mTOR and the N terminus (residues 11-91, containing part of the first HEAT domain) are essential for myogenic differentiation. In contrast, constitutive activation of S6K1 does not abrogate rapamycin inhibition of either proliferation or myogenic differentiation.     

Monocyte adhesion and spreading on human endothelial cells is dependent on Rho-regulated receptor clustering.

The GTPase Rho is known to mediate the assembly of integrin-containing focal adhesions and actin stress fibers. Here, we investigate the role of Rho in regulating the distribution of the monocyte-binding receptors E-selectin, ICAM-1, and VCAM-1 in human endothelial cells. Inhibition of Rho activity with C3 transferase or N19RhoA, a dominant negative RhoA mutant, reduced the adhesion of monocytes to activated endothelial cells and inhibited their spreading. Similar effects were observed after pretreatment of endothelial cells with cytochalasin D. In contrast, dominant negative Rac and Cdc42 proteins did not affect monocyte adhesion or spreading. C3 transferase and cytochalasin D did not alter the expression levels of monocyte-binding receptors on endothelial cells, but did inhibit clustering of E-selectin, ICAM-1, and VCAM-1 on the cell surface induced by monocyte adhesion or cross-linking antibodies. Similarly, N19RhoA inhibited receptor clustering. Monocyte adhesion and receptor cross-linking induced stress fiber assembly, and inhibitors of myosin light chain kinase prevented this response but did not affect receptor clustering. Finally, receptor clusters colocalized with ezrin/moesin/ radixin proteins. These results suggest that Rho is required in endothelial cells for the assembly of stable adhesions with monocytes via the clustering of monocyte-binding receptors and their association with the actin cytoskeleton, independent of stress fiber formation.     

The nuclear localization of 3'-phosphoinositide-dependent kinase-1 is dependent on its association with the protein tyrosine phosphatase SHP-1

3'-Phosphoinositide-dependent protein kinase-1 (PDK1), the direct upstream kinase of Akt, can localize to the nucleus during specific signalling events. The mechanism used for its import into the nucleus, however, remains unresolved as it lacks a canonical nuclear localization signal (NLS). Expression of activated Src kinase in C6 glioblastoma cells promotes the association of tyrosylphosphorylated PDK1 with the NLS-containing tyrosine phosphatase SHP-1 as well as the nuclear localization of both proteins. A constitutive nucleo-cytoplasmic SHP-1:PDK1 shuttling complex is supported by several lines of evidence including (i) the distribution of both proteins to similar subcellular compartments following manipulation of the nuclear pore complex, (ii) the nuclear retention of SHP-1 upon overexpression of a PDK1 protein bearing a disrupted nuclear export signal (NES), and (iii) the exclusion of PDK1 from the nucleus upon overexpression of SHP-1 lacking the NLS or following siRNA-mediated knock-down of SHP-1. The latter case results in a perinuclear distribution of PDK1 that corresponds with the distribution of PIP3 (phosphatidylinositol 3,4,5-triphosphate), while a PDK1 protein bearing a mutated PH domain that abrogates PIP3-binding is excluded from the nucleus. Our data suggest that the SHP-1:PDK1 complex is recruited to the nuclear membrane by binding to perinuclear PIP3, whereupon SHP-1 (and its NLS) facilitates active import. Export from the nucleus relies on PDK1 (and its NES). The intact complex contributes to Src kinase-induced, Akt-sensitive podial formation in C6 cells.     

Interaction of CacyBP/SIP with NPM1 and its influence on NPM1 localization and function in oxidative stress

Calcyclin (S100A6) binding protein/Siah‐1 interacting protein (CacyBP/SIP) is mainly a cytoplasmic protein; however, some literature data suggested its presence in the nucleus. In this work we examined more precisely the nuclear localization and function of CacyBP/SIP. By applying mass spectrometry, we have identified several nuclear proteins, among them is nucleophosmin (NPM1), that may interact with CacyBP/SIP. Subsequent assays revealed that CacyBP/SIP forms complexes with NPM1 in the cell and that the interaction between these two proteins is direct. Interestingly, although CacyBP/SIP exhibits phosphatase activity, we have found that its overexpression favors phosphorylation of NPM1 on S125. In turn, the RNA immunoprecipitation assay indicated that the altered CacyBP/SIP level has an impact on the amount of 28S and 18S rRNA bound to NPM1. The overexpression of CacyBP/SIP resulted in a significant increase in the binding of 28S and 18S rRNA to NPM1, whereas silencing of CacyBP/SIP expression decreased 28S rRNA binding and had no effect on the binding of 18S rRNA. Further studies have shown that under oxidative stress, CacyBP/SIP overexpression alters NPM1 distribution in cell nuclei. In addition, staining for a nucleolar marker, fibrillarin, revealed that CacyBP/SIP is indispensable for maintaining the nucleolar structure. These results are in agreement with data obtained by western blot analysis, which show that upon oxidative stress the NPM1 level decreases but that CacyBP/SIP overexpression counteracts the effect of stress. Altogether, our results show for the first time that CacyBP/SIP binds to and affects the properties of a nuclear protein, NPM1, and that it is indispensable for preserving the structure of nucleoli under oxidative stress.     

Antinociceptive effects of spinally administered nociceptin/orphanin FQ and its N-terminal fragments on capsaicin-induced nociception

Nociceptin/orphanin FQ (N/OFQ), the endogenous ligand for the N/OFQ peptide (NOP) receptors, has been shown to be metabolized into some fragments. We examined to determine whether intrathecal (i.t.) N/OFQ (1-13), (1-11) and (1-7) have antinociceptive activity in the pain-related behavior after intraplantar injection of capsaicin. The i.t. administration of N/OFQ (0.3-1.2 nmol) produced an appreciable and dose-dependent inhibition of capsaicin-induced paw-licking/biting response. The N-terminal fragments of N/OFQ, (1-13) and (1-11), were antinociceptive with a potency lower than N/OFQ. Calculated ID₅₀ values (nmol, i.t.) were 0.83 for N/OFQ, 2.5 for N/OFQ (1-13) and 4.75 for N/OFQ (1-11), respectively. The time-course effect revealed that the antinociceptive effects of these N-terminal fragments lasted longer than those of N/OFQ. Removal of amino acids down to N/OFQ (1-7) led to be less potent than N/OFQ and its fragments, (1-13) and (1-11). Antinociception induced by N/OFQ or N/OFQ (1-13) was reversed significantly by i.t. co-injection of [Nphe¹]N/OFQ (1-13)NH₂, a peptidergic antagonist for NOP receptors, whereas i.t. injection of the antagonist did not interfere with the action of N/OFQ (1-11) and (1-7). Pretreatment with the opioid receptor antagonist naloxone hydrochloride did not affect the antinociception induced by N/OFQ and its N-terminal fragments. These results suggest that N-terminal fragments of N/OFQ are active metabolites and may modulate the antinociceptive effect of N/OFQ in the spinal cord. The results also indicate that N/OFQ (1-13) still possess antinociceptive activity through NOP receptors.     

Effects of NaCl and GTP on the inhibition of platelet adenylate cyclase by 1-O-octadecyl-2-O-acetyl-sn-glyceryl-3-phosphorylcholine (synthetic platelet-activating factor)

We have investigated the effects of NaCl and GTP on the inhibition of platelet adenylate cyclase by 1-O-octadecyl-2-O-acetyl-sn-glyceryl-3-phosphorylcholine (1-octadecyl-2-acetyl-G-3-PC), using particulate fractions from human and rabbit platelets that had been frozen and thawed in the presence of ethylene glycol bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetate to prevent Ca²⁺-dependent proteolysis. When 10 μM GTP was present, 100 mM NaCl stimulated the activity of the rabbit enzyme 5.6-fold and that of the human enzyme 2.2-fold. Under these conditions, maximum inhibitions of 90% and 64% were obtained on addition of 100 nM 1-octadecyl-2-acetyl-G-3-PC to rabbit and human preparations, respectively. These inhibitions resulted partly from an NaCl-independent inhibition of basal enzyme activity and partly from reversal of the stimulatory effect of NaCl. The relative abilities of the chlorides of different monovalent cations to enhance inhibition of rabbit platelet adenylate cyclase were: NaCl >LiCl >KCl >choline chloride. NaCl also increased the concentrations of 1-octadecyl-2-acetyl-G-3-PC required for half-maximal inhibition of adenylate cyclase but this action of NaCl did not correlate with its stimulatory effect on enzyme activity. After particulate fractions from platelets of either species were washed, 10 μM GTP inhibited basal adenylate cyclase activity in the absence of NaCl but stimulated the enzyme in the presence of NaCl. Inhibition of adenylate cyclase by 1-octadecyl-2-acetyl-G-3-PC was then either enhanced by GTP (rabbit material) or completely dependent on added GTP (human material). Stimulation of the activity of the washed human preparations by NaCl required GTP, but concentrations lower than required for potentiation of the inhibitory effect of 1-octadecyl-2-acetyl-G-3-PC by NaCl were effective.     

The N-terminal region of acyl-CoA synthetase 3 is essential for both the localization on lipid droplets and the function in fatty acid uptake

Cytosolic lipid droplets (LDs) are storage organelles for neutral lipids derived from endogenous metabolism. Acyl-CoA synthetase family proteins are essential enzymes in this biosynthetic pathway, contributing activated fatty acids. Fluorescence microscopy showed that ACSL3 is localized to the endoplasmic reticulum (ER) and LDs, with the distribution dependent on the cell type and the supply of fatty acids. The N-terminus of ACSL3 was necessary and sufficient for targeting reporter proteins correctly, as demonstrated by subcellular fractionation and confocal microscopy. The N-terminal region of ACSL3 was also found to be functionally required for the enzyme activity. Selective permeabilization and in silico analysis suggest that ACSL3 assumes a hairpin membrane topology, with the N-terminal hydrophobic amino acids forming an amphipathic helix restricted to the cytosolic leaflet of the ER membrane. ACSL3 was effectively translocated from the ER to nascent LDs when neutral lipid synthesis was stimulated by the external addition of fatty acids. Cellular fatty acid uptake was increased by overexpression and reduced by RNA interference of ACSL3. In conclusion, the structural organization of ACSL3 allows the fast and efficient movement from the ER to emerging LDs. ACSL3 not only esterifies fatty acids with CoA but is also involved in the cellular uptake of fatty acids, presumably indirectly by metabolic trapping. The unique localization of the acyl-CoA synthetase ACSL3 on LDs suggests a function in the local synthesis of lipids.