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1.
Cell microparticles (MPs) released in the extracellular milieu can embark plasma membrane and intracellular components which are specific of their cellular origin, and transfer them to target cells. The MP-mediated, cell-to-cell transfer of three human membrane glycoproteins of different degrees of complexity was investigated in the present study, using a CHO cell model system. We first tested the delivery of CAR and CD46, two monospanins which act as adenovirus receptors, to target CHO cells. CHO cells lack CAR and CD46, high affinity receptors for human adenovirus serotype 5 (HAdV5), and serotype 35 (HAdV35), respectively. We found that MPs derived from CHO cells (MP-donor cells) constitutively expressing CAR (MP-CAR) or CD46 (MP-CD46) were able to transfer CAR and CD46 to target CHO cells, and conferred selective permissiveness to HAdV5 and HAdV35. In addition, target CHO cells incubated with MP-CD46 acquired the CD46-associated function in complement regulation. We also explored the MP-mediated delivery of a dodecaspanin membrane glycoprotein, the CFTR to target CHO cells. CFTR functions as a chloride channel in human cells and is implicated in the genetic disease cystic fibrosis. Target CHO cells incubated with MPs produced by CHO cells constitutively expressing GFP-tagged CFTR (MP-GFP-CFTR) were found to gain a new cellular function, the chloride channel activity associated to CFTR. Time-course analysis of the appearance of GFP-CFTR in target cells suggested that MPs could achieve the delivery of CFTR to target cells via two mechanisms: the transfer of mature, membrane-inserted CFTR glycoprotein, and the transfer of CFTR-encoding mRNA. These results confirmed that cell-derived MPs represent a new class of promising therapeutic vehicles for the delivery of bioactive macromolecules, proteins or mRNAs, the latter exerting the desired therapeutic effect in target cells via de novo synthesis of their encoded proteins.  相似文献   

2.
Dok1 is believed to be a mainly cytoplasmic adaptor protein which down-regulates mitogen-activated protein kinase activation, inhibits cell proliferation and transformation, and promotes cell spreading and cell migration. Here we show that Dok1 shuttles between the nucleus and cytoplasm. Treatment of cells with leptomycin B (LMB), a specific inhibitor of the nuclear export signal (NES)-dependent receptor CRM1, causes nuclear accumulation of Dok1. We have identified a functional NES (348LLKAKLTDPKED359) that plays a major role in the cytoplasmic localization of Dok1. Src-induced tyrosine phosphorylation prevented the LMB-mediated nuclear accumulation of Dok1. Dok1 cytoplasmic localization is also dependent on IKKbeta. Serum starvation or maintaining cells in suspension favor Dok1 nuclear localization, while serum stimulation, exposure to growth factor, or cell adhesion to a substrate induce cytoplasmic localization. Functionally, nuclear NES-mutant Dok1 had impaired ability to inhibit cell proliferation and to promote cell spreading and cell motility. Taken together, our results provide the first evidence that Dok1 transits through the nucleus and is actively exported into the cytoplasm by the CRM1 nuclear export system. Nuclear export modulated by external stimuli and phosphorylation may be a mechanism by which Dok1 is maintained in the cytoplasm and membrane, thus regulating its signaling functions.  相似文献   

3.
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.  相似文献   

4.
After synaptic vesicles fuse with the plasma membrane and release their contents, vesicle membrane proteins recycle by endocytosis and are targeted to newly formed synaptic vesicles. The membrane traffic of an epitope-tagged form of VAMP-2 (VAMP-TAg) was observed in transfected cells to identify sequence requirements for recycling of a synaptic vesicle membrane protein. In the neuroendocrine PC12 cell line VAMP-TAg is found not only in synaptic vesicles, but also in endosomes and on the plasma membrane. Endocytosis of VAMP-TAg is a rapid and saturable process. At high expression levels VAMP-TAg accumulates at the cell surface. Rapid endocytosis of VAMP-TAg also occurs in transfected CHO cells and is therefore independent of other synaptic proteins. The majority of the measured endocytosis is not directly into synaptic vesicles since mutations in VAMP-TAg that enhance synaptic vesicle targeting did not affect endocytosis. Nonetheless, mutations that inhibited synaptic vesicle targeting, in particular replacement of methionine-46 by alanine, inhibited endocytosis by 85% in PC12 cells and by 35% in CHO cells. These results demonstrate that the synaptic vesicle targeting signal is also used for endocytosis and can be recognized in cells lacking synaptic vesicles.  相似文献   

5.
Initiation and termination of signaling of the type I angiotensin receptor (AT(1)-R) can lead to dynamic changes in its localization in plasma membrane microdomains. Several markers were recently developed to investigate membrane microdomains. Here, we used several YFP-labeled fusion constructs (i.e. raft or non-raft plasma membrane markers) to analyze the agonist-induced changes in compartmentalization of AT(1)-R, including internalization or lateral movement between plasma membrane compartments in response to stimulation using bioluminescence resonance energy transfer measurements. Our data demonstrate that angiotensin II (AngII) stimulus changes the microdomain localization of wild type or mutated (DRY → AAY or TSTS → AAAA) AT(1)-Rs co-expressed with the fluorescent probes in HEK293 cells. The comparison of the trafficking of AT(1)-R upon AngII stimulus with those of [Sar(1),Ile(8)]AngII or [Sar(1),Ile(4),Ile(8)]AngII stimulus revealed different types of changes, depending on the nature of the ligand. The observed changes in receptor compartmentalization of the AT(1)-R are strikingly different from those of 5HT-2C and EGF receptors, which demonstrate the usefulness of the bioluminescence resonance energy transfer-based measurements in the investigation of receptor trafficking in the plasma membrane in living cell experiments.  相似文献   

6.
It has been possible for several years to study the dynamics of fluorescently labeled proteins by single-molecule microscopy, but until now this technology has been applied only to individual cells in culture. In this study, it was extended to stem cells and living vertebrate organisms. As a molecule of interest we used yellow fluorescent protein fused to the human H-Ras membrane anchor, which has been shown to serve as a model for proteins anchored in the plasma membrane. We used a wide-field fluorescence microscopy setup to visualize individual molecules in a zebrafish cell line (ZF4) and in primary embryonic stem cells. A total-internal-reflection microscopy setup was used for imaging in living organisms, in particular in epidermal cells in the skin of 2-day-old zebrafish embryos. Our results demonstrate the occurrence of membrane microdomains in which the diffusion of membrane proteins in a living organism is confined. This membrane organization differed significantly from that observed in cultured cells, illustrating the relevance of performing single-molecule microscopy in living organisms.  相似文献   

7.
Previously, we presented evidence that the vesicles routinely exfoliated from the surface of T27A tumor cells arise from vesicle-forming regions of the plasma membrane and possess a set of lateral microdomains distinct from those of the plasma membrane as a whole. We also showed that docosahexaenoic acid (DHA, or 22:6n-3), a fatty acyl chain known to alter microdomain structure in model membranes, also alters the structure and composition of exfoliated vesicles, implying a DHA-induced change in microdomain structure on the cell surface. In this report we show that enrichment of the cells with DHA reverses some of the characteristic differences in composition between the parent plasma membrane and shed microdomain vesicles, but does not alter their phospholipid class composition. In untreated cells, DHA-containing species were found to be a much greater proportion of the total phosphatidylethanolamine (PE) pool than the total phosphatidylcholine (PC) pool in both the plasma membrane and the shed vesicles. After DHA treatment, the proportion of DHA-containing species in the PE and PC pools of the plasma membrane were elevated, and unlike in untreated cells, their proportions were equal in the two pools. In the vesicles shed from DHA-loaded cells, the proportion of DHA-containing species of PE was the same as in the plasma membrane. However, the proportion of DHA-containing species of PC in the vesicles (0.089) was much lower than that found in the plasma membrane (0.194), and was relatively devoid of species with 16-carbon acyl components. These data suggested that DHA-containing species of PC, particularly those having a 16-carbon chain in the sn-1 position, were preferentially retained in the plasma membrane. The data can be interpreted as indicating that DHA induces a restructuring of lateral microdomains on the surface of living cells similar to that predicted by its behavior in model membranes.  相似文献   

8.
Abstract: This report presents the first evidence that a member of the L1 family of nervous system cell-adhesion molecules is covalently modified by thioesterification with palmitate, and identifies a highly conserved cysteine in the predicted membrane-spanning domain as the site of modification. Neurofascin is constitutively palmitoylated at cysteine-1213 at close to a 1:1 molar stoichiometry. Kinetics of palmitate incorporation into neurofascin expressed in resting neuroblastoma cells indicate that the palmitate modification has the same turnover rate as the polypeptide chain and does not affect the protein stability of neurofascin. Palmitoylation of neurofascin expressed in dorsal root ganglion neurons is not required for delivery of neurofascin to the plasma membrane or targeting to axons. Palmitoylation also has no effect on ankyrin-binding activity of neurofascin, on the oligomeric state of neurofascin in solution, or on cell-adhesion activity of neurofascin expressed in neuroblastoma cells. A significant difference between native and C1213L neurofascin is that these proteins were localized in distinct fractions within a low-density membrane population enriched in signaling molecules. These results indicate a palmitate-dependent targeting of neurofascin to a specialized membrane microdomain.  相似文献   

9.
Previous results indicate that apoE (apolipoprotein E) may be associated with the nucleus in specific cell types, particularly under stress conditions such as serum starvation. In addition, nuclear apoE localization in ovarian cancer was recently shown to be correlated with patient survival. In order to better understand the factors associated with apoE nuclear localization, we examined intracellular apoE trafficking using live-cell imaging of CHO (Chinese-hamster ovary) cells that constitutively expressed apoE-GFP (green fluorescent protein). In addition, we used biotinylated apoE (in a lipid-free state and as a lipidated discoidal complex) to track the uptake and potential nuclear targeting of exogenous apoE. Our results indicate that a small proportion of apoE-GFP is detected in the nucleus of living apoE-GFP-expressing CHO cells and that the level of apoE-GFP in the nucleus is increased with serum starvation. Exposure of control CHO cells to exogenous apoE-GFP did not result in nuclear apoE-GFP localization in the recipient cells. Similarly, biotinylated apoE did not reach the nucleus of control CHO cells or SK-N-SH neurons. In contrast, when biotinylated apoE was delivered to recipient cells as a lipidated apoE disc, apoE was detected in the nucleus, suggesting that the lipoprotein complex alters the intracellular degradation or trafficking of apoE. Biotinylated apoE discs containing each of the three common human apoE isoforms (E2, E3 and E4) were also tested for nuclear trafficking. All three apoE isoforms were equally detected in the nucleus. These studies provide new evidence that apoE may be targeted to the nucleus and shed light on factors that regulate this process.  相似文献   

10.
Junctional microdomains, paradigm for membrane protein segregation in functional assemblies, in eye lens fiber cell membranes are constituted of lens-specific aquaporin-0 tetramers (AQP0(4)) and connexin (Cx) hexamers, termed connexons. Both proteins have double function to assure nutrition and mediate adhesion of lens cells. Here we use high-speed atomic force microscopy to examine microdomain protein dynamics at the single-molecule level. We found that the adhesion function of head-to-head associated AQP0(4) and Cx is cooperative. This finding provides first experimental evidence for the mechanistic importance for junctional microdomain formation. From the observation of lateral association-dissociation events of AQP0(4), we determine that the enthalpic energy gain of a single AQP0(4)-AQP0(4) interaction in the membrane plane is -2.7 k(B)T, sufficient to drive formation of microdomains. Connexon association is stronger as dynamics are rarely observed, explaining their rim localization in junctional microdomains.  相似文献   

11.
The formin mDia2 mediates the formation of lamellipodia and filopodia during cell locomotion. The subcellular localization of activated mDia2 depends on interactions with actin filaments and the plasma membrane. We investigated the poorly understood mechanism of plasma membrane targeting of mDia2 and found that the entire N-terminal region of mDia2 preceding the actin-polymerizing formin homology domains 1 and 2 (FH1-FH2) module was potently targeted to the membrane. This localization was enhanced by Rif, but not by other tested small GTPases, and depended on a positively charged N-terminal basic domain (BD). The BD bound acidic phospholipids in vitro, suggesting that in vivo it may associate with the plasma membrane through electrostatic interactions. Unexpectedly, a fragment consisting of the GTPase-binding region and the diaphanous inhibitory domain (G-DID), thought to mediate the interaction with GTPases, was not targeted to the plasma membrane even in the presence of constitutively active Rif. Addition of the BD or dimerization/coiled coil domains to G-DID rescued plasma membrane targeting in cells. Direct binding of Rif to mDia2 N terminus required the presence of both G and DID. These results suggest that the entire N terminus of mDia2 serves as a coincidence detection module, directing mDia2 to the plasma membrane through interactions with phospholipids and activated Rif.  相似文献   

12.
Paranodin/contactin-associated protein (caspr) is a transmembrane glycoprotein of the neurexin superfamily that is highly enriched in the paranodal regions of myelinated axons. We have investigated the role of its association with F3/contactin, a glycosylphosphatidyl inositol (GPI)-anchored neuronal adhesion molecule of the Ig superfamily. Paranodin was not expressed at the cell surface when transfected alone in CHO or neuroblastoma cells. Cotransfection with F3 resulted in plasma membrane delivery of paranodin, as analyzed by confocal microscopy and cell surface biotinylation. The region that mediates association with paranodin was mapped to the Ig domains of F3 by coimmunoprecipitation experiments. The association of paranodin with F3 allowed its recruitment to Triton X-100-insoluble microdomains. The GPI anchor of F3 was necessary, but not sufficient for surface expression of paranodin. F3-Ig, a form of F3 deleted of the fibronectin type III (FNIII) repeats, although GPI-linked and expressed at the cell surface, was not recovered in the microdomain fraction and was unable to promote cell surface targeting of paranodin. Thus, a cooperative effect between the GPI anchor, the FNIII repeats, and the Ig regions of F3 is required for recruitment of paranodin into lipid rafts and its sorting to the plasma membrane.  相似文献   

13.
Membrane- associated guanylate kinase proteins (MAGUKs) are important determinants of localization and organization of ion channels into specific plasma membrane domains. However, their exact role in channel function and cardiac excitability is not known. We examined the effect of synapse-associated protein 97 (SAP97), a MAGUK abundantly expressed in the heart, on the function and localization of Kv1.5 subunits in cardiac myocytes. Recombinant SAP97 or Kv1.5 subunits tagged with green fluorescent protein (GFP) were overexpressed in rat neonatal cardiac myocytes and in Chinese hamster ovary (CHO) cells from adenoviral or plasmidic vectors. Immunocytochemistry, fluorescence recovery after photobleaching, and patch-clamp techniques were used to study the effects of SAP97 on the localization, mobility, and function of Kv1.5 subunits. Adenovirus-mediated SAP97 overexpression in cardiac myocytes resulted in the clustering of endogenous Kv1.5 subunits at myocyte-myocyte contacts and an increase in both the maintained component of the outward K(+) current, I(Kur) (5.64 +/- 0.57 pA/pF in SAP97 myocytes vs. 3.23 +/- 0.43 pA/pF in controls) and the number of 4-aminopyridine-sensitive potassium channels in cell-attached membrane patches. In live myocytes, GFP-Kv1.5 subunits were mobile and organized in clusters at the basal plasma membrane, whereas SAP97 overexpression reduced their mobility. In CHO cells, Kv1.5 channels were diffusely distributed throughout the cell body and freely mobile. When coexpressed with SAP97, Kv subunits were organized in plaquelike clusters and poorly mobile. In conclusion, SAP97 regulates the K(+) current in cardiac myocytes by retaining and immobilizing Kv1.5 subunits in the plasma membrane. This new regulatory mechanism may contribute to the targeting of Kv channels in cardiac myocytes.  相似文献   

14.
The C2 domain is a Ca(2+)-dependent membrane-targeting module found in many cellular proteins involved in signal transduction or membrane trafficking. C2 domains are unique among membrane targeting domains in that they show a wide range of lipid selectivity for the major components of cell membranes, including phosphatidylserine and phosphatidylcholine. To understand how C2 domains show diverse lipid selectivity and how this functional diversity affects their subcellular targeting behaviors, we measured the binding of the C2 domains of group IVa cytosolic phospholipase A(2) (cPLA(2)) and protein kinase C-alpha (PKC-alpha) to vesicles that model cell membranes they are targeted to, and we monitored their subcellular targeting in living cells. The surface plasmon resonance analysis indicates that the PKC-alpha C2 domain strongly prefers the cytoplasmic plasma membrane mimic to the nuclear membrane mimic due to high phosphatidylserine content in the former and that Asn(189) plays a key role in this specificity. In contrast, the cPLA(2) C2 domain has specificity for the nuclear membrane mimic over the cytoplasmic plasma membrane mimic due to high phosphatidylcholine content in the former and aromatic and hydrophobic residues in the calcium binding loops of the cPLA(2) C2 domain are important for its lipid specificity. The subcellular localization of enhanced green fluorescent protein-tagged C2 domains and mutants transfected into HEK293 cells showed that the subcellular localization of the C2 domains is consistent with their lipid specificity and could be tailored by altering their in vitro lipid specificity. The relative cell membrane translocation rate of selected C2 domains was also consistent with their relative affinity for model membranes. Together, these results suggest that biophysical principles that govern the in vitro membrane binding of C2 domains can account for most of their subcellular targeting properties.  相似文献   

15.
Synaptophysin, an integral membrane protein of small synaptic vesicles, was expressed by transfection in fibroblastic CHO-K1 cells. The properties and localization of synaptophysin were compared between transfected CHO-K1 cells and native neuroendocrine PC12 cells. Both cell types similarly glycosylate synaptophysin and sort it into indistinguishable microvesicles. These become labeled by endocytic markers and are primarily concentrated below the plasmalemma and at the area of the Golgi complex and the centrosomes. A small pool of synaptophysin is transiently found on the plasma membrane. In CHO-K1 cells synaptophysin co-localizes with transferrin that has been internalized by receptor-mediated endocytosis. These findings suggest that synaptophysin in transfected CHO-K1 cells and neuroendocrine PC12 cells is directed into a pathway of recycling microvesicles which, in CHO cells, is shown to coincide with that of the transferrin receptor. They further indicate that fibroblasts have the ability to sort a synaptic vesicle membrane protein. Our results suggest a pathway for the evolution of small synaptic vesicles from a constitutively recycling organelle which is normally present in all cells.  相似文献   

16.
Ward BM  Moss B 《Journal of virology》2000,74(8):3771-3780
The vaccinia virus B5R type I integral membrane protein accumulates in the Golgi network, from where it becomes incorporated into the envelope of extracellular virions. Our objective was to determine the domains of B5R responsible for Golgi membrane targeting in the absence of other viral components. Fusion of an enhanced green fluorescent protein to the C terminus of B5R allowed imaging of the chimeric protein without altering intracellular trafficking and Golgi network localization in transfected cells. Deletion or swapping of B5R domains with corresponding regions of the vesicular stomatitis virus G protein, which is targeted to the plasma membrane, indicated that (i) the N-terminal extracellular domain of B5R had no specific role in Golgi apparatus localization, (ii) the transmembrane domain of B5R was sufficient for exiting the endoplasmic reticulum, and (iii) removal of the cytoplasmic tail impaired Golgi network localization and increased the accumulation of B5R in the plasma membrane. Further experiments demonstrated that the cytoplasmic tail mediated internalization of B5R from the plasma membrane, suggesting a retrieval mechanism. Mutagenesis revealed residues required for Golgi membrane localization and efficient plasma membrane retrieval of the B5R protein: a tyrosine at residue 310 and two adjacent leucines at residues 315 and 316.  相似文献   

17.
GPR30, or G protein-coupled estrogen receptor, is a G protein-coupled receptor reported to bind 17β-estradiol (E2), couple to the G proteins Gs and Gi/o, and mediate non-genomic estrogenic responses. However, controversies exist regarding the receptor pharmacological profile, effector coupling, and subcellular localization. We addressed the role of the type I PDZ motif at the receptor C terminus in receptor trafficking and coupling to cAMP production in HEK293 cells and CHO cells ectopically expressing the receptor and in Madin-Darby canine kidney cells expressing the native receptor. GPR30 was localized both intracellularly and in the plasma membrane and subject to limited basal endocytosis. E2 and G-1, reported GPR30 agonists, neither stimulated nor inhibited cAMP production through GPR30, nor did they influence receptor localization. Instead, GPR30 constitutively inhibited cAMP production stimulated by a heterologous agonist independently of Gi/o. Moreover, siRNA knockdown of native GPR30 increased cAMP production. Deletion of the receptor PDZ motif interfered with inhibition of cAMP production and increased basal receptor endocytosis. GPR30 interacted with membrane-associated guanylate kinases, including SAP97 and PSD-95, and protein kinase A-anchoring protein (AKAP) 5 in the plasma membrane in a PDZ-dependent manner. Knockdown of AKAP5 or St-Ht31 treatment, to disrupt AKAP interaction with the PKA RIIβ regulatory subunit, decreased inhibition of cAMP production, and St-Ht31 increased basal receptor endocytosis. Therefore, GPR30 forms a plasma membrane complex with a membrane-associated guanylate kinase and AKAP5, which constitutively attenuates cAMP production in response to heterologous agonists independently of Gi/o and retains receptors in the plasma membrane.  相似文献   

18.
Enveloped viruses often use membrane lipid rafts to assemble and bud, augment infection and spread efficiently. However, the molecular bases and functional consequences of the partitioning of viral glycoproteins into microdomains remain intriguing questions in virus biology. Here, we measured Foerster resonance energy transfer by fluorescence lifetime imaging microscopy (FLIM‐FRET) to study the role of distinct membrane proximal regions of the human immunodeficiency virus glycoprotein gp41 for lipid raft partitioning in living Chinese hamster ovary cells (CHO‐K1). Gp41 was labelled with a fluorescent protein at the exoplasmic face of the membrane, preventing any interference of the fluorophore with the proposed role of the transmembrane and cytoplasmic domains in lateral organization of gp41. Raft localization was deduced from interaction with an established raft marker, a fluorescently tagged glycophosphatidylinositol anchor and the cholesterol recognition amino acid consensus (CRAC) was identified as the crucial lateral sorting determinant in CHO‐K1 cells. Interestingly, the raft association of gp41 indicates a substantial cell‐to‐cell heterogeneity of the plasma membrane microdomains. In complementary fluorescence polarization microscopy, a distinct CRAC requirement was found for the oligomerization of the gp41 variants. Our data provide further insight into the molecular basis and biological implications of the cholesterol dependent lateral sorting of viral glycoproteins for virus assembly at cellular membranes.  相似文献   

19.
The endocytosis of transferrin receptor (TfR) has served as a model to study the receptor-targeted cargo delivery system for cancer therapy for many years. To accurately evaluate and optically measure this TfR targeting delivery in vitro, a CHO cell line with enhanced green fluorescent protein (EGFP)-tagged human TfR was established. A chimera of the hTfR and EGFP was engineered by fusing EGFP to the amino terminus of hTfR. Data were provided to demonstrate that hTfR-EGFP chimera was predominantly localized on the plasma membrane with some intracellular fluorescent structures on CHO cells and the EGFP moiety did not affect the endocytosis property of hTfR. Receptor internalization occurred similarly to that of HepG2 cells expressing wild-type hTfR. The internalization percentage of this chimeric receptor was about 81±3% of wild type. Time-dependent co-localization of hTfR-EGFP and PE-conjugated anti-hTfR mAb in living cells demonstrated the trafficking of mAb-receptor complexes through the endosomes followed by segregation of part of the mAb and receptor at the late stages of endocytosis. The CHO-hTfR cells preferentially took up anti-hTfR mAb conjugated nanoparticles. This CHO-hTfR cell line makes it feasible for accurate evaluation and visualization of intracellular trafficking of therapeutic agents conjugated with transferrin or Abs targeting the hTfRs.  相似文献   

20.
Bacterial toxins require localization to specific intracellular compartments following injection into host cells. In this study, we examined the membrane targeting of a broad family of bacterial proteins, the patatin-like phospholipases. The best characterized member of this family is ExoU, an effector of the Pseudomonas aeruginosa type III secretion system. Upon injection into host cells, ExoU localizes to the plasma membrane, where it uses its phospholipase A2 activity to lyse infected cells. The targeting mechanism of ExoU is poorly characterized, but it was recently found to bind to the phospholipid phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), a marker for the plasma membrane of eukaryotic cells. We confirmed that the membrane localization domain (MLD) of ExoU had a direct affinity for PI(4,5)P2, and we determined that this binding was required for ExoU localization. Previously uncharacterized ExoU homologs from Pseudomonas fluorescens and Photorhabdus asymbiotica also localized to the plasma membrane and required PI(4,5)P2 for this localization. A conserved arginine within the MLD was critical for interaction of each protein with PI(4,5)P2 and for localization. Furthermore, we determined the crystal structure of the full-length P. fluorescens ExoU and found that it was similar to that of P. aeruginosa ExoU. Each MLD contains a four-helical bundle, with the conserved arginine exposed at its cap to allow for interaction with the negatively charged PI(4,5)P2. Overall, these findings provide a structural explanation for the targeting of patatin-like phospholipases to the plasma membrane and define the MLD of ExoU as a member of a new class of PI(4,5)P2 binding domains.  相似文献   

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