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Membrane Localization (membrane + localization)
Selected AbstractsMembrane localization itself but not binding to IICBGlc is directly responsible for the inactivation of the global repressor Mlc in Escherichia coliMOLECULAR MICROBIOLOGY, Issue 3 2004Yuya Tanaka Summary Mlc is a global transcriptional repressor involved in the regulation of genes linked to glucose metabolism. The activity of Mlc is modulated through the interaction with a major glucose transporter, IICBGlc, in response to external glucose. To understand how IICBGlc,Mlc interaction controls the repressor activity of Mlc, we attempted to isolate Mlc mutants that retain the ability to repress target genes even in the presence of glucose. The Mlc mutants were tested for their ability to interact with IICBGlc. Mutants in which a single amino acid substitution occurs in the N-terminal portion were no longer able to bind to IICBGlc, suggesting that the N-terminal region of Mlc is primarily responsible for the interaction with IICBGlc. To examine whether the Mlc,IICBGlc interaction and/or the membrane localization of Mlc per se are essential for the inactivation of Mlc, the properties of several hybrid proteins in which either IIBGlc or Mlc is fused to membrane proteins were analysed. The cytoplasmic IIBGlc domain failed to inhibit the Mlc action although it retains the ability to bind Mlc in cells. However, it gained the ability to inhibit the Mlc activity when it was fused to a membrane protein LacY. In addition, we showed that Mlc is inactivated when fused to membrane proteins but not when fused to cytoplasmic proteins. We conclude that the IICBGlc,Mlc interaction is dispensable for the inactivation of Mlc, and that membrane localization is directly responsible for the inactivation of Mlc. [source] Distinctive E-cadherin and epidermal growth factor receptor expression in metastatic and nonmetastatic head and neck squamous cell carcinomaCANCER, Issue 1 2008Predictive, prognostic correlation Abstract BACKGROUND. The authors investigated whether coexpression and localization of E-cadherin (E-cad) and epidermal growth factor receptor (EGFR) had predictive and/or prognostic correlations with lymph node metastasis and/or survival in patients with squamous cell carcinoma of the head and neck (SCCHN). METHODS. Immunohistochemistry (IHC) of archival tissue was performed to measure expression of EGFR and E-cad in surgical specimens of SCCHN (n = 143) that included primary tumors (PTs) with positive lymph nodes (Tu+Met) and their paired lymph node metastases (LnMet), PTs with negative lymph nodes (Tu,Met), and benign tissue biopsies as normal controls. IHC staining was quantified as a weighted index and as the ratio of membrane to cytoplasmic staining. Correlative expression between EGFR and E-cad also was examined in SCCHN cell lines by immunoblotting and immunofluorescence analyses. RESULTS. Three distinct expression patterns of EGFR and E-cad were observed. Membrane localization of E-cad was significantly lower in the Tu+Met group than in the Tu,Met group (P = .01) and was associated inversely with lymph node status (P = .009). Wilcoxon analysis of the combined markers demonstrated that expression and/or membrane localization of EGFR and E-cad were correlated with disease-free survival and overall survival in patients with SCCHN. The study of SCCHN cell lines demonstrated that cells with positive but low EGFR expression and with negative E-cad expression were relatively resistant to the EGFR tyrosine kinase inhibitor erlotinib. CONCLUSIONS. The current study suggested that examining not only the expression but also the localization of EGFR and E-cad simultaneously may have clinical relevance in predicting lymph node metastasis, patient survival, and response to EGFR-targeted therapy in patients with SCCHN. Cancer 2008. © 2008 American Cancer Society. [source] Mechanism of activation of the double-stranded-RNA-dependent protein kinase, PKRFEBS JOURNAL, Issue 13 2001Role of dimerization, cellular localization in the stimulation of PKR phosphorylation of eukaryotic initiation factor-2 (eIF2) An important defense against viral infection involves inhibition of translation by PKR phosphorylation of the , subunit of eIF2. Binding of viral dsRNAs to two dsRNA-binding domains (dsRBDs) in PKR leads to relief of an inhibitory region and activation of eIF2 kinase activity. Interestingly, while deletion of the regulatory region of PKR significantly induces activity in vitro, the truncated kinase does not inhibit translation in vivo, suggesting that these sequences carry out additional functions required for PKR control. To delineate these functions and determine the order of events leading to activation of PKR, we fused truncated PKR to domains of known function and assayed the chimeras for in vivo activity. We found that fusion of a heterologous dimerization domain with the PKR catalytic domain enhanced autophosphorylation and eIF2 kinase function in vivo. The dsRBDs also mediate ribosome association and we proposed that such targeting increases the localized concentration of PKR, enhancing interaction between PKR molecules. We addressed this premise by linking the truncated PKR to RAS sequences mediating farnesylation and membrane localization and found that the fusion protein was functional in vivo. These results indicate that cellular localization along with oligomerization enhances interaction between PKR molecules. Alanine substitution for the phosphorylation site, threonine 446, impeded in vivo and in vitro activity of the PKR fusion proteins, while aspartate or glutamate substitutions partially restored the function of the truncated kinase. These results indicate that both dimerization and cellular localization play a role in transient protein,protein interactions and that trans -autophosphorylation is the final step in the mechanism of activation of PKR. [source] Localization of the mosaic transmembrane serine protease corin to heart myocytesFEBS JOURNAL, Issue 23 2000John D. Hooper Corin cDNA encodes an unusual mosaic type II transmembrane serine protease, which possesses, in addition to a trypsin-like serine protease domain, two frizzled domains, eight low-density lipoprotein (LDL) receptor domains, a scavenger receptor domain, as well as an intracellular cytoplasmic domain. In in vitro experiments, recombinant human corin has recently been shown to activate pro-atrial natriuretic peptide (ANP), a cardiac hormone essential for the regulation of blood pressure. Here we report the first characterization of corin protein expression in heart tissue. We generated antibodies to two different peptides derived from unique regions of the corin polypeptide, which detected immunoreactive corin protein of approximately 125,135 kDa in lysates from human heart tissues. Immunostaining of sections of human heart showed corin expression was specifically localized to the cross striations of cardiac myocytes, with a pattern of expression consistent with an integral membrane localization. Corin was not detected in sections of skeletal or smooth muscle. Corin has been suggested to be a candidate gene for the rare congenital heart disease, total anomalous pulmonary venous return (TAPVR) as the corin gene colocalizes to the TAPVR locus on human chromosome 4. However examination of corin protein expression in TAPVR heart tissue did not show evidence of abnormal corin expression. The demonstrated corin protein expression by heart myocytes supports its proposed role as the pro-ANP convertase, and thus a potentially critical mediator of major cardiovascular diseases including hypertension and congestive heart failure. [source] A role of the C-terminus of aquaporin 4 in its membrane expression in cultured astrocytesGENES TO CELLS, Issue 7 2002Ken-ichi Nakahama Background: Aquaporin 4 (AQP4) is a predominant water channel protein in mammalian brains, which is localized in the astrocyte plasma membrane. Membrane targeting of AQP4 is essential to perform its function. The mechanism(s) of membrane targeting is not clear in astrocytes. Results: We investigated the role of the C-terminus of AQP4 (short isoform) in its membrane targeting by an expression study of C-terminal mutants of AQP4 in cultured astrocytes. The deletion of 26 C-terminal residues of AQP4 (AQP4,276,301aa) results in the intracellular localization of the protein. However, smaller deletions than 21 C-terminal residues did not alter its plasma membrane localization. These results suggest that C-terminal residues between Val276 and Ile280 play an important role in the expression of AQP4 in the plasma membrane. However, the plasma membrane localization of the AQP4(A276AAAA280) mutant (alanine substitution of Val276 -Ile280 of AQP4) suggests that another signal for membrane targeting exists in the C-terminus of AQP4. The deletion or point mutations of the PDZ binding motif of the AQP4(A276AAAA280) mutant resulted in the intracellular localization of the proteins. These results suggest that the PDZ binding motif may also be involved in the membrane targeting of AQP4. Conclusions: We found that the C-terminal sequence of AQP4 contains two important signals for membrane expression of AQP4 in cultured astrocytes. One is a hydrophobic domain and the other is a PDZ binding motif that exists in the C-terminus. [source] Kir4.1 and AQP4 associate with Dp71- and utrophin-DAPs complexes in specific and defined microdomains of Müller retinal glial cell membraneGLIA, Issue 6 2008Patrice E. Fort Abstract The dystrophin-associated proteins (DAPs) complex consisting of dystroglycan, syntrophin, dystrobrevin, and sarcoglycans in muscle cells is associated either with dystrophin or its homolog utrophin. In rat retina, a similar complex was found associated with dystrophin-Dp71 that serves as an anchor for the inwardly rectifying potassium channel Kir4.1 and the aqueous pore, aquaporin-4 (AQP4). Here, using immunofluorescence imaging of isolated retinal Müller glial cells and co-immunoprecipitation experiments performed on an enriched Müller glial cells end-feet fraction, we investigated the effect of Dp71 deletion on the composition, anchoring, and membrane localization of the DAPs,Kir4.1 and/or ,AQP4 complex. Two distinct complexes were identified in the end-feet fraction associated either with Dp71 or with utrophin. Upon Dp71 deletion, the corresponding DAPs complex was disrupted and a compensating utrophin upregulation was observed, accompanied by diffuse overall staining of Kir4.1 along the Müller glial cells and redistribution of the K+ conductance. Dp71 deficiency was also associated with a marked reduction of AQP4 and ,-dystroglycan expression. Furthermore, it was observed that the Dp71,DAPs dependent complex could be, at least partially, associated with a specific membrane fraction. These results demonstrate that Dp71 has a central role in the molecular scaffold responsible for anchoring AQP4 and Kir4.1 in Müller cell end-feet membranes. They also show that despite its close relationship to the dystrophin proteins and its correlated upregulation, utrophin is only partially compensating for the absence of Dp71 in Müller glial cells. © 2008 Wiley-Liss, Inc. [source] Congenital insensitivity to pain: novel SCN9A missense and in-frame deletion mutations,HUMAN MUTATION, Issue 9 2010James J. Cox Abstract SCN9Aencodes the voltage-gated sodium channel Nav1.7, a protein highly expressed in pain-sensing neurons. Mutations in SCN9A cause three human pain disorders: bi-allelic loss of function mutations result in Channelopathy-associated Insensitivity to Pain (CIP), whereas activating mutations cause severe episodic pain in Paroxysmal Extreme Pain Disorder (PEPD) and Primary Erythermalgia (PE). To date, all mutations in SCN9A that cause a complete inability to experience pain are protein truncating and presumably lead to no protein being produced. Here, we describe the identification and functional characterization of two novel non-truncating mutations in families with CIP: a homozygously-inherited missense mutation found in a consanguineous Israeli Bedouin family (Nav1.7-R896Q) and a five amino acid in-frame deletion found in a sporadic compound heterozygote (Nav1.7-,R1370-L1374). Both of these mutations map to the pore region of the Nav1.7 sodium channel. Using transient transfection of PC12 cells we found a significant reduction in membrane localization of the mutant protein compared to the wild type. Furthermore, voltage clamp experiments of mutant-transfected HEK293 cells show a complete loss of function of the sodium channel, consistent with the absence of pain phenotype. In summary, this study has identified critical amino acids needed for the normal subcellular localization and function of Nav1.7. © 2010 Wiley-Liss, Inc. [source] Intracellular localization of the Epstein-Barr virus BFRF1 gene product in lymphoid cell lines and oral hairy leukoplakia lesionsJOURNAL OF MEDICAL VIROLOGY, Issue 1 2004Antonella Farina Abstract A novel protein encoded by the BFRF1 gene of the Epstein-Barr virus was identified recently [Farina et al. (2000) J Virol 74:3235,3244], which is antigenic "in vivo" and expressed early in the viral replicative cycle. In the present study, its subcellular localization was examined in greater detail comparing Epstein-Barr virus (EBV) induced producing and nonproducing cell lines by immunofluorescence: in 12-0-tetradecanoyl phorbol-13-acetate (TPA)-induced Raji and B95-8 cells, as well as in anti-IgG-stimulated Akata cells, the protein appeared to be localized over the cell nuclear membrane. A similar nuclear membrane localization was observed in epithelial cells of oral hairy leukoplakia, a pathological manifestation of permissive EBV infection. In contrast, upon transfection of BFRF1 in the EBV-negative Burkitt's lymphoma cell line DG75, the protein was localized predominantly over the plasma membrane. The membrane localization was abolished when DG75 cells were transfected with a C-terminal deletion mutant of BFRF1 lacking the transmembrane domain. Because induced Raji cells do not produce virus, the above observations indicate that the nuclear membrane localization is not associated with viral production, but requires the expression of EBV genes, and suggest that additional proteins, expressed early during viral lytic infection, might be necessary to target the protein to the nuclear membrane. Immunogold electron microscopy on ultrathin cryosections of induced B95-8 cells showed that BFRF1 on the nuclear membranes was concentrated over multilayered domains representing areas of active viral replication or at the sites of viral budding, suggesting that BFRF1 is involved in the process of viral assembly. J. Med. Virol. 72:102,111, 2004. © 2004 Wiley-Liss, Inc. [source] Poster Session BP07: Neurodegenerative DiseasesJOURNAL OF NEUROCHEMISTRY, Issue 2002F. Jayman Presynaptic terminals contain an abundant 140-amino acid phosphoprotein, dubbed ,-synuclein, which is accumulated in Lewy bodies typically observed in neurons in neurodegenerative diseases, such as Parkinson's disease. In this study, the role of ,-synuclein in regulating cycle, differentiation, and survival of neuronal cells was studied using a rat dopaminergic cell line ZN27D. To delineate specific effects of ,-synuclein the same cell line was engineered to express human ,-synuclein and a vector-transfected cell line RK27 was used as a second control. All three cell lines showed significant proliferation even in serum-free medium, and complete inhibition of cell division and differentiation could be achieved in the ZN27D cells only when both dibutyryl cAMP (dbcAMP) and retinoic acid were present. In contrast, the ,-synuclein expressing cells could be differentiated in the presence of only dbcAMP. Dose dependence of MPP+(1-methyl-4-phenylpyridinium iodide)-mediated caspase3 activation was studied in undifferentiated ZN27D cells. At 200 ,m MPP+ a significant cleavage of the caspase3 substrate PARP was observed and it was reversed in the presence of ,-synuclein. MPP+ also inhibited aminophospholipid translocase (APTL), a P-type ATPase that is responsible for inner plasma membrane localization of phophotidylserine in healthy cells. The role of ,-synuclein in regulating cell cycle, differentiation, APTL activity and cell death is being investigated further in the dopaminergic ZN27D cell line. [source] Oxytocin and Oxytocin Receptors in Cancer Cells and ProliferationJOURNAL OF NEUROENDOCRINOLOGY, Issue 4 2004P. Cassoni Abstract The hypothalamic nonapeptide oxytocin plays a crucial role in many reproductive and behavioural functions. However, in recent years, an additional new role for oxytocin has been identified in neoplastic pathology. In tumours, oxytocin acts as a growth regulator, through the activation of a specific G-coupled transmembrane receptor, the oxytocin receptor. In vitro, oxytocin inhibits proliferation of neoplastic cells of either epithelial (mammary and endometrial), nervous or bone origin, all expressing oxytocin receptor. Furthermore, an oxytocin growth-inhibiting effect was also tested and confirmed in vivo in mouse and rat mammary carcinomas. In neoplastic cells derived from two additional oxytocin target tissues, trophoblast and endothelium, oxytocin was found to promote cell proliferation, an effect opposite to that previously described in all other neoplastic oxytocin-responsive cells. The signal transduction pathways coupled to the biological effects of oxytocin are different in oxytocin growth-inhibited or growth-stimulated cells, and may depend on the membrane localization of the oxytocin receptor itself. The inhibitory effect of oxytocin is apparently mediated by activation of the cAMP-protein kinase A pathway, a nonconventional oxytocin signalling pathway, whereas the mitogenic effect is coupled to the increase of intracellular [Ca2+] and tyrosine phosphorylation, ,classical' oxytocin transducers. Moreover, the oxytocin receptor localization in lipid rafts enriched in caveolin-1 turns the inhibition of cell growth into a proliferative response, eliciting different epidermal growth factor receptor/mitogen-activated protein kinase activation patterns. This unexpected role of oxytocin (and oxytocin analogues) in regulating cell proliferation, as well as the widespread expression of oxytocin receptors in neoplastic tissues of different origin, opens up new perspectives on the biological role of the oxytocin,oxytocin receptor system in cancer. [source] Membrane localization itself but not binding to IICBGlc is directly responsible for the inactivation of the global repressor Mlc in Escherichia coliMOLECULAR MICROBIOLOGY, Issue 3 2004Yuya Tanaka Summary Mlc is a global transcriptional repressor involved in the regulation of genes linked to glucose metabolism. The activity of Mlc is modulated through the interaction with a major glucose transporter, IICBGlc, in response to external glucose. To understand how IICBGlc,Mlc interaction controls the repressor activity of Mlc, we attempted to isolate Mlc mutants that retain the ability to repress target genes even in the presence of glucose. The Mlc mutants were tested for their ability to interact with IICBGlc. Mutants in which a single amino acid substitution occurs in the N-terminal portion were no longer able to bind to IICBGlc, suggesting that the N-terminal region of Mlc is primarily responsible for the interaction with IICBGlc. To examine whether the Mlc,IICBGlc interaction and/or the membrane localization of Mlc per se are essential for the inactivation of Mlc, the properties of several hybrid proteins in which either IIBGlc or Mlc is fused to membrane proteins were analysed. The cytoplasmic IIBGlc domain failed to inhibit the Mlc action although it retains the ability to bind Mlc in cells. However, it gained the ability to inhibit the Mlc activity when it was fused to a membrane protein LacY. In addition, we showed that Mlc is inactivated when fused to membrane proteins but not when fused to cytoplasmic proteins. We conclude that the IICBGlc,Mlc interaction is dispensable for the inactivation of Mlc, and that membrane localization is directly responsible for the inactivation of Mlc. [source] Quantitation of the Effect of Hydroxylamine on Rhodopsin Palmitylation,PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 4 2008Wesley Jackson Rhodopsin (the photosensitive rod visual pigment) has been a model for photobiologic studies of the opsins as well as a structural model for G-protein-coupled receptors. The two palmitate groups attached to cysteines 322 and 323 are thought to serve as membrane anchors for the rhodopsin C-terminus, but the absence of the palmitates does not alter membrane localization. However, removal of the palmitates affects rhodopsin function. Therefore, it is important to quantitate the stability of rhodopsin palmitates to hydroxylamine, which is a widely utilized reagent in biochemical preparations of the apoprotein. We have developed a mass spectrometric method to quantitate the resulting opsin palmitylation. Our data show that both of the bovine rhodopsin palmitates are labile to hydroxylamine, with significant depalmitylation occurring at concentrations of ,100 mm, with an EC50 of 220 mm L,1. The palmitate at position 322 is the more stable to hydroxylamine. Samples prepared in the presence of >50 mm should therefore be considered to be at least partially depalmitylated and the results interpreted accordingly. [source] New Escherichia coli outer membrane proteins identified through prediction and experimental verificationPROTEIN SCIENCE, Issue 4 2006Paola Marani Abstract Many new Escherichia coli outer membrane proteins have recently been identified by proteomics techniques. However, poorly expressed proteins and proteins expressed only under certain conditions may escape detection when wild-type cells are grown under standard conditions. Here, we have taken a complementary approach where candidate outer membrane proteins have been identified by bioinformatics prediction, cloned and overexpressed, and finally localized by cell fractionation experiments. Out of eight predicted outer membrane proteins, we have confirmed the outer membrane localization for five,YftM, YaiO, YfaZ, CsgF, and YliI,and also provide preliminary data indicating that a sixth,YfaL,may be an outer membrane autotransporter. [source] Cellular efflux of auxin catalyzed by the Arabidopsis MDR/PGP transporter AtPGP1THE PLANT JOURNAL, Issue 2 2005Markus Geisler Summary Directional transport of the phytohormone auxin is required for the establishment and maintenance of plant polarity, but the underlying molecular mechanisms have not been fully elucidated. Plant homologs of human multiple drug resistance/P-glycoproteins (MDR/PGPs) have been implicated in auxin transport, as defects in MDR1 (AtPGP19) and AtPGP1 result in reductions of growth and auxin transport in Arabidopsis (atpgp1, atpgp19), maize (brachytic2) and sorghum (dwarf3). Here we examine the localization, activity, substrate specificity and inhibitor sensitivity of AtPGP1. AtPGP1 exhibits non-polar plasma membrane localization at the shoot and root apices, as well as polar localization above the root apex. Protoplasts from Arabidopsis pgp1 leaf mesophyll cells exhibit reduced efflux of natural and synthetic auxins with reduced sensitivity to auxin efflux inhibitors. Expression of AtPGP1 in yeast and in the standard mammalian expression system used to analyze human MDR-type proteins results in enhanced efflux of indole-3-acetic acid (IAA) and the synthetic auxin 1-naphthalene acetic acid (1-NAA), but not the inactive auxin 2-NAA. AtPGP1-mediated efflux is sensitive to auxin efflux and ABC transporter inhibitors. As is seen in planta, AtPGP1 also appears to mediate some efflux of IAA oxidative breakdown products associated with apical sites of high auxin accumulation. However, unlike what is seen in planta, some additional transport of the benzoic acid is observed in yeast and mammalian cells expressing AtPGP1, suggesting that other factors present in plant tissues confer enhanced auxin specificity to PGP-mediated transport. [source] Distinctive E-cadherin and epidermal growth factor receptor expression in metastatic and nonmetastatic head and neck squamous cell carcinomaCANCER, Issue 1 2008Predictive, prognostic correlation Abstract BACKGROUND. The authors investigated whether coexpression and localization of E-cadherin (E-cad) and epidermal growth factor receptor (EGFR) had predictive and/or prognostic correlations with lymph node metastasis and/or survival in patients with squamous cell carcinoma of the head and neck (SCCHN). METHODS. Immunohistochemistry (IHC) of archival tissue was performed to measure expression of EGFR and E-cad in surgical specimens of SCCHN (n = 143) that included primary tumors (PTs) with positive lymph nodes (Tu+Met) and their paired lymph node metastases (LnMet), PTs with negative lymph nodes (Tu,Met), and benign tissue biopsies as normal controls. IHC staining was quantified as a weighted index and as the ratio of membrane to cytoplasmic staining. Correlative expression between EGFR and E-cad also was examined in SCCHN cell lines by immunoblotting and immunofluorescence analyses. RESULTS. Three distinct expression patterns of EGFR and E-cad were observed. Membrane localization of E-cad was significantly lower in the Tu+Met group than in the Tu,Met group (P = .01) and was associated inversely with lymph node status (P = .009). Wilcoxon analysis of the combined markers demonstrated that expression and/or membrane localization of EGFR and E-cad were correlated with disease-free survival and overall survival in patients with SCCHN. The study of SCCHN cell lines demonstrated that cells with positive but low EGFR expression and with negative E-cad expression were relatively resistant to the EGFR tyrosine kinase inhibitor erlotinib. CONCLUSIONS. The current study suggested that examining not only the expression but also the localization of EGFR and E-cad simultaneously may have clinical relevance in predicting lymph node metastasis, patient survival, and response to EGFR-targeted therapy in patients with SCCHN. Cancer 2008. © 2008 American Cancer Society. [source] | |||||||||||||||||||||||||