Home About us Contact
|
Home About us Contact | ||
|
|
||
Membrane Association (membrane + association)
Selected AbstractsCalcium modulates endopeptidase 24.15 (EC 3.4.24.15) membrane association, secondary structure and substrate specificityFEBS JOURNAL, Issue 12 2005Vitor Oliveira The metalloendopeptidase 24.15 (EP24.15) is ubiquitously present in the extracellular environment as a secreted protein. Outside the cell, this enzyme degrades several neuropeptides containing from 5 to 17 amino acids (e.g. gonadotropin releasing hormone, bradykinin, opioids and neurotensin). The constitutive secretion of EP24.15 from glioma C6 cells was demonstrated to be stimulated linearly by reduced concentrations of extracellular calcium. In the present report we demonstrate that extracellular calcium concentration has no effect on the total amount of the extracellular (cell associated + medium) enzyme. Indeed, immuno-cytochemical analyses by confocal and electron microscopy suggested that the absence of calcium favors the enzyme shedding from the plasma membrane into the medium. Two putative calcium-binding sites on EP24.15 (D93 and D159) were altered by site-directed mutagenesis to investigate their possible contribution to binding of the enzyme at the cell surface. These mutated recombinant proteins behave similarly to the wild-type enzyme regarding enzymatic activity, secondary structure, calcium sensitivity and immunoreactivity. However, immunocytochemical analyses by confocal microscopy consistently show a reduced ability of the D93A mutant to associate with the plasma membrane of glioma C6 cells when compared with the wild-type enzyme. These data and the model of the enzyme's structure as determined by X-ray diffraction suggest that D93 is located at the enzyme surface and is consistent with membrane association of EP24.15. Moreover, calcium was also observed to induce a major change in the EP24.15 cleavage site on distinctive fluorogenic substrates. These data suggest that calcium may be an important modulator of ep24.15 cell function. [source] Membrane binding of SRP pathway components in the halophilic archaea Haloferax volcaniiFEBS JOURNAL, Issue 7 2004Tovit Lichi Across evolution, the signal recognition particle pathway targets extra-cytoplasmic proteins to membranous translocation sites. Whereas the pathway has been extensively studied in Eukarya and Bacteria, little is known of this system in Archaea. In the following, membrane association of FtsY, the prokaryal signal recognition particle receptor, and SRP54, a central component of the signal recognition particle, was addressed in the halophilic archaea Haloferax volcanii. Purified H. volcanii FtsY, the FtsY C-terminal GTP-binding domain (NG domain) or SRP54, were combined separately or in different combinations with H. volcanii inverted membrane vesicles and examined by gradient floatation to differentiate between soluble and membrane-bound protein. Such studies revealed that both FtsY and the FtsY NG domain bound to H. volcanii vesicles in a manner unaffected by proteolytic pretreatment of the membranes, implying that in Archaea, FtsY association is mediated through the membrane lipids. Indeed, membrane association of FtsY was also detected in intact H. volcanii cells. The contribution of the NG domain to FtsY binding in halophilic archaea may be considerable, given the low number of basic charges found at the start of the N-terminal acidic domain of haloarchaeal FtsY proteins (the region of the protein thought to mediate FtsY,membrane association in Bacteria). Moreover, FtsY, but not the NG domain, was shown to mediate membrane association of H. volcanii SRP54, a protein that did not otherwise interact with the membrane. [source] Membrane potential and endocytic activity control disintegration of cell,cell adhesion and cell fusion in vinculin-injected MDBK cellsJOURNAL OF CELLULAR PHYSIOLOGY, Issue 3 2004Riitta Palovuori Cell fusion occurs during fertilization and in the formation of organs such as muscles, placenta, and bones. We have developed an experimental model for epithelial cell fusion which permits analysis of the processes during junction disintegration and formation of polykaryons (Palovuori and Eskelinen [2000] Eur. J. Cell. Biol. 79: 961,974). In the present work, we analyzed the process in detail. Cell fusion was achieved by microinjecting into the cytoplasm of kidney epithelial Madin-Darby bovine kidney (MDBK) cells TAMRA-tagged vinculin, which incorporated into lateral membranes, focal adhesions and nucleus, and, prior fusion, induced internalization of actin, cadherin and plakoglobin to small clusters in cytoplasm. Injected vinculin was still visible at lateral membranes after removal of junctional proteins indicating that it was tightly associated and perturbed the cell,cell contact sites resulting in membrane fragmentation. Injection of active Rac together with vinculin induced accumulation of cadherin to the membranes, but did not affect vinculin,membrane association. However, it hampered cell fusion probably by supporting adherens junctions. In order to stop endocytosis, we lowered intracellular pH of vinculin-injected cells to 5.5 with the aid of nigericin in KCl buffer. In acidified cells, injected vinculin delineated lateral membranes as thick layers, cadherin remained in situ, and cell fusion was completely inhibited. Since this treatment also leads to cell depolarization, we checked the vinculin incorporation in a KCl solution containing nigericin at neutral pH. In these circumstances, both endogenous and injected vinculin delineated lateral membranes as very thin discontinuous layers, but still fusion was hampered most likely due to perturbation in the initial vinculin,membrane association. We suggest that vinculin might function as a sensor of the environment triggering cell fusion during development in circumstances where membrane potential and local and transient pH gradients play a role. © 2004 Wiley-Liss, Inc. [source] RGS7 Is Palmitoylated and Exists as Biochemically Distinct FormsJOURNAL OF NEUROCHEMISTRY, Issue 5 2000Jeremy J. Rose Abstract:Regulator of G protein signaling (RGS) proteins are GTPase-activating proteins that modulate neurotransmitter and G protein signaling. RGS7 and its binding partners G, and G,5 are enriched in brain, but biochemical mechanisms governing RGS7/G,/G,5 interactions and membrane association are poorly defined. We report that RGS7 exists as one cytosolic and three biochemically distinct membrane-bound fractions (salt-extractable, detergent-extractable, and detergent-insensitive) in brain. To define factors that determine RGS7 membrane attachment, we examined the biochemical properties of recombinant RGS7 and G,5 synthesized in Spodoptera frugiperda insect cells. We have found that membrane-bound but not cytosolic RGS7 is covalently modified by the fatty acid palmitate. G,5 is not palmitoylated. Both unmodified (cytosolic) and palmitoylated (membrane-derived) forms of RGS7, when complexed with G,5, are equally effective stimulators of G,o GTPase activity, suggesting that palmitoylation does not prevent RGS7/G,o interactions. The isolated core RGS domain of RGS7 selectively binds activated G,i/o in brain extracts and is an effective stimulator of both G,o and G,i1 GTPase activities in vitro. In contrast, the RGS7/G,5 complex selectively interacts with G,o only, suggesting that features outside the RGS domain and/or G,5 association dictate RGS7-G, interactions. These findings define previously unrecognized biochemical properties of RGS7, including the first demonstration that RGS7 is palmitoylated. [source] Up-Regulation of Cell Surface Insulin Receptor by Protein Kinase C-, in Adrenal Chromaffin CellsJOURNAL OF NEUROCHEMISTRY, Issue 2 2000Involvement of Transcriptional, Translational Events Our previous study showed that treatment of cultured bovine adrenal chromaffin cells with phorbol 12, 13-dibutyrate (PDBu) or 12- O -tetradecanoylphorbol 13-acetate (TPA) caused a rapid (<15 min) and persistent (>15 h) translocation of both conventional (c) protein kinase C-, (PKC-,) and novel PKC-, (but not atypical PKC-,) from cytosol to membranes, whereas thymeleatoxin (TMX) increased the similar but selective membrane association of only cPKC-,. In the present study, chronic (,12 h) treatment of chromaffin cells with PDBu raised cell surface 125I-insulin binding without altering the KD value ; it developed in a concentration (EC50 = 1.9 nM)-and time (t1/2 = 14.6 h)-dependent manner, reaching its maximum 115% increase at 48 h. Either TPA (30 nM) or TMX (EC50 = 6.4 nM) also increased 125I-insulin binding by 97 or 88%, whereas the biologically inactive 4,-TPA had no effect. The increasing effect of PDBu (30 nM for 24 h) on 125I-insulin binding was significantly blocked, even when H7, an inhibitor of PKC, was added at 8 h after the initiation of PDBu treatment. Concurrent treatment with brefeldin A, an inhibitor of vesicular transport from the trans -Golgi network, cycloheximide, an inhibitor of protein synthesis, or 5,6-dichlorobenzimidazole riboside, an inhibitor of RNA synthesis, abolished the PDBu-induced increment of 125I-insulin binding. Western blot analysis, using antibody against the ,-subunit of the insulin receptor, showed that treatment with PDBu (30 nM) or TMX (EC50 = 2.3 nM) increased levels of insulin receptor precursor (~190 kDa ; t1/2 = 7.1 h) and insulin receptor ,-subunit (t1/2 = 15.4 h), causing their almost maximum 52 and 59% rises, respectively, at 24 h. Northern blot analysis revealed that PDBu or TMX increased levels of insulin receptor mRNAs by ~35% as soon as 3 h, producing its monophasic peak ~76% increases at 24 h. All of these increasing effects of PDBu and TMX on 125I-insulin binding and insulin receptor ,-subunit and insulin receptor mRNA levels were entirely prevented by simultaneous treatment with Gö6976, a selective inhibitor of cPKC. These results suggest that long-term activation of cPKC-, up-regulates the density of the cell surface insulin receptor via transcriptional/translational events. [source] New insights into the cellular organization of the RNA processing and degradation machinery of Escherichia coliMOLECULAR MICROBIOLOGY, Issue 4 2008Aziz Taghbalout Summary Ribonuclease E (RNase E) is a component of the Escherichia coli RNA degradosome, a multiprotein complex that also includes RNA helicase B (RhlB), polynucleotide phosphorylase (PNPase) and enolase. The degradosome plays a key role in RNA processing and degradation. The degradosomal proteins are organized as a cytoskeletal-like structure within the cell that has been thought to be associated with the cytoplasmic membrane. The article by Khemici et al. in the current issue of Molecular Microbiology reports that RNase E can directly interact with membrane phospholipids in vitro. The RNase E,membrane interaction is likely to play an important role in the membrane association of the degradosome system. These findings shed light on important but largely unexplored aspects of cellular structure and function, including the organization of the RNA processing machinery of the cell and of bacterial cytoskeletal elements in general. [source] Clenbuterol increases muscle fiber size and GATA-2 protein in rat skeletal muscle in uteroMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 5 2008Diane Downie Abstract Certain ,2 -adrenoceptor agonists, such as clenbuterol, are known to elicit a muscle-specific anabolism or hypertrophy in both normal and catabolic muscle in a wide variety of species. However, the underlying mechanism(s) of the ,2 -agonist-induced anabolism remains unclear. This study aimed to determine the effects of clenbuterol administration in utero on skeletal muscle and to examine the underlying molecular mechanisms. Pregnant rats were fed clenbuterol (2 mg/kg diet) from Day 4 of gestation (4 dg) until weanling and fetal samples were taken from 13.5, 15.5, 17.5, and 19.5 dg and from 1d neonatal pups. Muscles were analyzed for total DNA, RNA and protein and sections examined morphologically for changes in muscle development. Western and immunohistochemical analyses were performed to identify changes in known myogenic signaling proteins. Clenbuterol increased the size of both fast and slow fibers in utero which was associated with a decreased DNA:protein ratio (28%) and an increased RNA:DNA ratio (36%). Additionally, drug treatment in utero induced a decrease in the fast:slow fiber ratio (38%). These myogenic changes were correlated with an increase in the GATA-2 hypertrophic transcription factor at both 17.5 dg (by 250%) and 19.5 dg (by 40%) in fetuses from clenbuterol treated dams. In addition, drug treatment resulted in increased membrane association of PKC-µ at 17.5 dg (325%) and increased PKC-, cytosolic abundance (40%) and PKC-, membrane abundance at 19.5 dg (250%). These results are the first demonstration that ,2 -agonists such as clenbuterol may act through upregulating the GATA-2 transcription factor and implicate certain PKC isoforms in the drug-induced regulation of skeletal muscle development. Mol. Reprod. Dev. 75: 785,794, 2008. © 2007 Wiley-Liss, Inc. [source] A mutant form of PTEN linked to autismPROTEIN SCIENCE, Issue 10 2010Roberta E. Redfern Abstract The tumor suppressor, phosphatase, and tensin homologue deleted on chromosome 10 (PTEN), is a phosphoinositide (PI) phosphatase specific for the 3-position of the inositol ring. PTEN has been implicated in autism for a subset of patients with macrocephaly. Various studies identified patients in this subclass with one normal and one mutated PTEN gene. We characterize the binding, structural properties, activity, and subcellular localization of one of these autism-related mutants, H93R PTEN. Even though this mutation is located at the phosphatase active site, we find that it affects the functions of neighboring domains. H93R PTEN binding to phosphatidylserine-bearing model membranes is 5.6-fold enhanced in comparison to wild-type PTEN. In contrast, we find that binding to phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) model membranes is 2.5-fold decreased for the mutant PTEN in comparison to wild-type PTEN. The structural change previously found for wild-type PTEN upon interaction with PI(4,5)P2, is absent for H93R PTEN. Consistent with the increased binding to phosphatidylserine, we find enhanced plasma membrane association of PTEN-GFP in U87MG cells. However, this enhanced plasma membrane association does not translate into increased PI(3,4,5)P3 turnover, since in vivo studies show a reduced activity of the H93R PTEN-GFP mutant. Because the interaction of PI(4,5)P2 with PTEN's N-terminal domain is diminished by this mutation, we hypothesize that the interaction of PTEN's N-terminal domain with the phosphatase domain is impacted by the H93R mutation, preventing PI(4,5)P2 from inducing the conformational change that activates phosphatase activity. [source] Effect of Rds abundance on cone outer segment morphogenesis, photoreceptor gene expression, and outer limiting membrane integrityTHE JOURNAL OF COMPARATIVE NEUROLOGY, Issue 6 2007Rafal Farjo Abstract We examined the molecular, structural, and functional consequences on cone photoreceptors of the neural retinal leucine zipper knockout (Nrl,/,) mice when only one allele of retinal degeneration slow (Rds) is present (Rds+/,/Nrl,/,). Quantitative RT-PCR and immunoblot analysis were used to assess the expression levels of several phototransduction genes; electroretinography was used to assess quantitatively the retinal responsiveness to light; and immunohistochemistry and ultrastructural analysis were used to examine retinal protein distribution and morphology, respectively. In Rds/Nrl double-null mice, S-cones form dysmorphic outer segments that lack lamellae and fail to associate properly with the cone matrix sheath and the outer limiting membrane. In Rds+/,/Nrl,/, mice, cones form oversized and disorganized outer segment lamellae; although outer limiting membrane associations are maintained, normal interactions with cone matrix sheaths are not, and photoreceptor rosette formation is observed. These retinas produce significantly higher photopic a-wave and b-wave amplitudes than do those of Rds,/,/Nrl,/, mice, and the levels of several cone phototransduction genes are significantly increased coincidently with the presence of Rds and partial lamellae formation. Thus, as in rod photoreceptors, expression of only one Rds allele is unable to support normal outer segment morphogenesis in cones. However, cone lamellae assembly, albeit disorganized, concomitantly permits outer limiting membrane association, and this appears to be linked to photoreceptor rosette formation in the rodless (cone-only) Nrl,/, retina. In addition, photoreceptor gene expression alterations occur in parallel with changes in Rds levels. J. Comp. Neurol. 504:619,630, 2007. © 2007 Wiley-Liss, Inc. [source] The Arabidopsis thaliana TIR-NB-LRR R-protein, RPP1A; protein localization and constitutive activation of defence by truncated alleles in tobacco and ArabidopsisTHE PLANT JOURNAL, Issue 6 2006L. Michael Weaver Summary Specific recognition of Hyaloperonospora parasitica isolate Cala2 by Arabidopsis thaliana Ws-0 is mediated by the resistance gene RPP1A. Transient expression of different truncations of RPP1A in tobacco leaves revealed that its TIR-NB-ARC portion is sufficient to induce an elicitor-independent cell death. In stable transgenic lines of Arabidopsis, overexpression of the RPP1A TIR-NB-ARC domains (E12) using the 35S promoter leads to broad-spectrum resistance to virulent strains of H. parasitica and Pseudomonas syringae DC3000. The TIR-NB-ARC-mediated constitutive immunity is due to activation of the salicylic acid-dependent resistance pathway and is relieved by either a mutation in EDS1 or the presence of the salicylate hydroxylase gene, NahG. Growth of 35S::E12 plants is reduced, a phenotype observed in many constitutively resistant mutants. RPP1A carries a hydrophobic peptide at its N-terminus that directs the RPP1A protein into membranes, though it may not be the sole determinant mediating membrane association of RPP1A. Two-phase partitioning and sucrose density gradient sedimentation established that RPP1A resides in the endoplasmic reticulum and/or Golgi apparatus. [source] Crystallographic characterization of the radixin FERM domain bound to the cytoplasmic tail of the adhesion protein ICAM-2ACTA CRYSTALLOGRAPHICA SECTION D, Issue 6 2001Keisuke Hamada Radixin is a member of the ERM proteins, which cross-link plasma membranes and actin filaments. The FERM domains located at the N-terminal regions of ERM proteins are responsible for membrane association through direct interactions with the cytoplasmic domains of integral membrane proteins. Here, crystals of the complex between the radixin FERM domain and the full-length cytoplasmic tail (28-residue peptide) of intercellular adhesion molecule 2, ICAM-2, have been obtained. The crystals were found to belong to space group P3121 or P3221, with unit-cell parameters a = b = 100.44,(9), c = 99.49,(6),Å, and contain one complex in the crystallographic asymmetric unit. An intensity data set was collected to a resolution of 2.60,Å. [source] Crystallographic characterization of the radixin FERM domain bound to the cytoplasmic tails of adhesion molecules CD43 and PSGL-1ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 1 2007Yumiko Takai Radixin is a member of the ERM proteins that cross-link plasma membranes and actin filaments. The FERM domains located in the N-terminal regions of ERM proteins are responsible for membrane association through direct interaction with the cytoplasmic tails of integral membrane proteins. Here, crystals of the radixin FERM domain bound to the cytoplasmic peptides of two adhesion molecules, CD43 and PSGL-1, have been obtained. Crystals of the radixin FERM domain bound to CD43 belong to space group P4322, with unit-cell parameters a = b = 68.72, c = 201.39,Å, and contain one complex in the crystallographic asymmetric unit. Crystals of the radixin FERM domain bound to PSGL-1 belong to space group P212121, with unit-cell parameters a = 80.74, b = 85.73, c = 117.75,Å, and contain two complexes in the crystallographic asymmetric unit. Intensity data sets were collected to a resolution of 2.9,Å for the FERM,CD43 complex and 2.8,Å for the FERM,PSGL-1 complex. [source] The pattern-recognition molecule Nod1 is localized at the plasma membrane at sites of bacterial interactionCELLULAR MICROBIOLOGY, Issue 2 2008Thomas A. Kufer Summary The pattern-recognition molecule Nod1 is a critical sensor for bacterial derived diaminopimelic acid-containing peptidoglycan fragments which induces innate immune responses in epithelial cells. Here we report the subcellular localization of this protein in human epithelial cells. Nod1 is localized in the cytosol and at the plasma membrane in human cells. This membrane association is dependent on the integrity of the protein, on its signalling capacity and on an intact actin cytoskeleton. Signalling-inactive mutants of Nod1 or disruption of the actin cytoskeleton interferes with this localization pattern and impacts on downstream NF-,B activation. Moreover, the invasive bacterium Shigella flexneri was used as a model for physiological activation of Nod1. Imaging revealed that Nod1 is recruited to the site of bacterial entry, where it colocalizes with NEMO. Our data provide evidence that membrane association is linked to Nod1 function and, in view of recent findings on Nod2, that this may be a common feature of NLR family members. [source] Effect of Rds abundance on cone outer segment morphogenesis, photoreceptor gene expression, and outer limiting membrane integrityTHE JOURNAL OF COMPARATIVE NEUROLOGY, Issue 6 2007Rafal Farjo Abstract We examined the molecular, structural, and functional consequences on cone photoreceptors of the neural retinal leucine zipper knockout (Nrl,/,) mice when only one allele of retinal degeneration slow (Rds) is present (Rds+/,/Nrl,/,). Quantitative RT-PCR and immunoblot analysis were used to assess the expression levels of several phototransduction genes; electroretinography was used to assess quantitatively the retinal responsiveness to light; and immunohistochemistry and ultrastructural analysis were used to examine retinal protein distribution and morphology, respectively. In Rds/Nrl double-null mice, S-cones form dysmorphic outer segments that lack lamellae and fail to associate properly with the cone matrix sheath and the outer limiting membrane. In Rds+/,/Nrl,/, mice, cones form oversized and disorganized outer segment lamellae; although outer limiting membrane associations are maintained, normal interactions with cone matrix sheaths are not, and photoreceptor rosette formation is observed. These retinas produce significantly higher photopic a-wave and b-wave amplitudes than do those of Rds,/,/Nrl,/, mice, and the levels of several cone phototransduction genes are significantly increased coincidently with the presence of Rds and partial lamellae formation. Thus, as in rod photoreceptors, expression of only one Rds allele is unable to support normal outer segment morphogenesis in cones. However, cone lamellae assembly, albeit disorganized, concomitantly permits outer limiting membrane association, and this appears to be linked to photoreceptor rosette formation in the rodless (cone-only) Nrl,/, retina. In addition, photoreceptor gene expression alterations occur in parallel with changes in Rds levels. J. Comp. Neurol. 504:619,630, 2007. © 2007 Wiley-Liss, Inc. [source] | |||||||||||||