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Membrane Insertion (membrane + insertion)
Selected AbstractsPhylogenetic and structural analyses of the oxa1 family of protein translocasesFEMS MICROBIOLOGY LETTERS, Issue 2 2001Ming-Ren Yen Abstract Mitochondrial Oxa1p homologs have been shown to function in protein export and membrane insertion in bacteria, mitochondria and chloroplasts, but their mode of action, organismal distribution and evolutionary origins are poorly understood. All sequenced homologs of Oxa1p were retrieved from the databases and multiply aligned. All organisms with a fully sequenced genome possess at least one Oxa1p homolog showing that the family is truly ubiquitous. Most prokaryotes possess just one Oxa1p homolog, but several Gram-positive bacteria and one archaeon possess two, and eukaryotes may have as many as six. Although these proteins vary in length over a 5-fold range, they exhibit a common hydrophobic core region of about 200 residues. Multiple sequence alignments reveal conserved residues and provide the basis for structural and phylogenetic analyses that serve to characterize the Oxa1 family. [source] Glucagon induces the plasma membrane insertion of functional aquaporin-8 water channels in isolated rat hepatocytesHEPATOLOGY, Issue 6 2003Sergio A. Gradilone Although glucagon is known to stimulate the cyclic adenosine monophosphate (cAMP)-mediated hepatocyte bile secretion, the precise mechanisms accounting for this choleretic effect are unknown. We recently reported that hepatocytes express the water channel aquaporin-8 (AQP8), which is located primarily in intracellular vesicles, and its relocalization to plasma membranes can be induced with dibutyryl cAMP. In this study, we tested the hypothesis that glucagon induces the trafficking of AQP8 to the hepatocyte plasma membrane and thus increases membrane water permeability. Immunoblotting analysis in subcellular fractions from isolated rat hepatocytes indicated that glucagon caused a significant, dose-dependent increase in the amount of AQP8 in plasma membranes (e.g., 102% with 1 ,mol/L glucagon) and a simultaneous decrease in intracellular membranes (e.g., 38% with 1 ,mol/L glucagon). Confocal immunofluorescence microscopy in cultured hepatocytes confirmed the glucagon-induced redistribution of AQP8 from intracellular vesicles to plasma membrane. Polarized hepatocyte couplets showed that this redistribution was specifically to the canalicular domain. Glucagon also significantly increased hepatocyte membrane water permeability by about 70%, which was inhibited by the water channel blocker dimethyl sulfoxide (DMSO). The inhibitors of protein kinase A, H-89, and PKI, as well as the microtubule blocker colchicine, prevented the glucagon effect on both AQP8 redistribution to hepatocyte surface and cell membrane water permeability. In conclusion, our data suggest that glucagon induces the protein kinase A and microtubule-dependent translocation of AQP8 water channels to the hepatocyte canalicular plasma membrane, which in turn leads to an increase in membrane water permeability. These findings provide evidence supporting the molecular mechanisms of glucagon-induced hepatocyte bile secretion. [source] Protein- and energy-mediated targeting of chloroplast outer envelope membrane proteinsTHE PLANT JOURNAL, Issue 6 2005Nancy R. Hofmann Summary While the import of nuclear-encoded chloroplast proteins is relatively well studied, the targeting of proteins to the outer membrane of the chloroplast envelope is not. The insertion of most outer membrane proteins (OMP) is generally considered to occur without the utilization of energy or proteinaceous components. Recently, however, proteins have been shown to be involved in the integration of outer envelope protein 14 (OEP14), whose outer membrane insertion was previously thought to be spontaneous. Here we investigate the insertion of two proteins from Physcomitrella patens, PpOEP64-1 and PpOEP64-2 (formerly known as PpToc64-1 and PpToc64-2), into the outer membrane of chloroplasts. The association of PpOEP64-1 with chloroplasts was not affected by chloroplast pre-treatments. Its insertion into the membrane was affected, however, demonstrating the importance of measuring insertion specifically in these types of assays. We found that the insertion of PpOEP64-1, PpOEP64-2 and two other OMPs, OEP14 and digalactosyldiacylglycerol synthase 1 (DGD1), was reduced by either nucleotide depletion or proteolysis of the chloroplasts. Integration was also inhibited in the presence of an excess of an imported precursor protein. In addition, OEP14 competed with the insertion of the OEP64s and DGD1. These data demonstrate that the targeting of several OMPs involves proteins present in chloroplasts and requires nucleotides. Together with previous reports, our data suggest that OMPs in general do not insert spontaneously. [source] Crystallization and preliminary crystallographic analysis of fragaceatoxin C, a pore-forming toxin from the sea anemone Actinia fragaceaACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 4 2009A. E. Mechaly Sea anemones produce water-soluble toxins that have the ability to interact with cell membranes and form pores within them. The mechanism of pore formation is based on an initial binding step followed by oligomerization and membrane insertion. Although the final structure of the pore remains unclear, biochemical studies indicate that it consists of a tetramer with a functional radius of ,1.1,nm. Since four monomers seem to be insufficient to build a pore of this size, the currently accepted model suggests that lipids might also participate in its structure. In this work, the crystallization and preliminary crystallographic analysis of two crystal forms of fragaceatoxin C (FraC), a newly characterized actinoporin from Actinia fragacea, are described. The crystals diffracted up to 1.8,Å resolution and the preliminary molecular-replacement solution supports an oligomeric structure of about 120,Å in diameter. [source] Genetic analysis of G protein-coupled receptor expression in Escherichia coli: Inhibitory role of DnaJ on the membrane integration of the human central cannabinoid receptorBIOTECHNOLOGY & BIOENGINEERING, Issue 2 2009Georgios Skretas Abstract The overexpression of G protein-coupled receptors (GPCRs) and of many other heterologous membrane proteins in simple microbial hosts, such as the bacterium Escherichia coli, often results in protein mistargeting, aggregation into inclusion bodies or cytoplasmic degradation. Furthermore, membrane protein production is very frequently accompanied by severe cell toxicity. In this work, we have employed a genetic strategy to isolate E. coli mutants that produce markedly increased amounts of the human central cannabinoid receptor (CB1), a pharmacologically significant GPCR that expresses very poorly in wild-type E. coli. By utilizing a CB1 fusion with the green fluorescent protein (GFP) and fluorescence-activated cell sorting (FACS), we screened an E. coli transposon library and identified an insertion in dnaJ that resulted in a large increase in CB1-GFP fluorescence and a dramatic enhancement in bacterial production of membrane-integrated CB1. Furthermore, the dnaJ::Tn5 inactivation suppressed the severe cytotoxicity associated with CB1 production. This revealed an unexpected inhibitory role of the chaperone/ co-chaperone DnaJ in the protein folding or membrane insertion of bacterially produced CB1. Our strategy can be easily adapted to identify expression bottlenecks for different GPCRs or any other integral membrane protein, provide useful and unanticipated mechanistic insights, and assist in the construction of genetically engineered E. coli strains for efficient heterologous membrane protein production. Biotechnol. Bioeng. 2009;102: 357,367. © 2008 Wiley Periodicals, Inc. [source] Hypertonic upregulation of betaine transport in renal cells is blocked by a proteasome inhibitorCELL BIOCHEMISTRY AND FUNCTION, Issue 5 2005Philip E. Lammers Abstract The renal betaine transporter (BGT1) protects cells in the hypertonic medulla by mediating uptake and accumulation of the osmolyte betaine. Transcription plays an essential role in upregulating BGT1 transport in MDCK cells subjected to hypertonic stress. During hypertonic stress, the abundance of the transcription factor TonEBP increases and it shifts from the cytoplasm to the nucleus where it activates transcription of the BGT1 gene. Little is known about post-transcriptional regulation of BGT1 protein. In the presence of the proteasome inhibitor MG-132, which blocked nuclear translocation of TonEBP, the hypertonic upregulation of BGT1 protein and transport was prevented and cell viability in hypertonic medium was impaired over 24,h. Urea also prevented the hypertonic upregulation of BGT1 protein and transport, but did not interfere with TonEBP translocation and cell viability. Shorter treatments of hypertonic cells with MG-132 avoided viability problems and produced dose-dependent inhibition of translocation and transport. When stably transfected MDCK cells that over-expressed BGT1 were treated for 6,h with hypertonic medium containing 3,µM MG-132, there was 43% inhibition of nuclear translocation, 83% inhibition of BGT1 transport, and no change in viability. While other proteasome functions may be involved, these data are consistent with a critical role for nuclear translocation of TonEBP in upregulation and membrane insertion of BGT1 protein. Copyright © 2005 John Wiley & Sons, Ltd. [source] Conformation and Interaction of a d,l -Alternating Peptide with a Bilayer Membrane: X-ray Reflectivity, CD, and FTIR Spectroscopy,CHEMPHYSCHEM, Issue 16 2007Andrea Küsel Dr. Abstract Peptides with alternating amino acid configuration provide helical secondary structures that are especially known from the membrane channel and pore-forming gramicidin A. In analogy to this natural d,l- alternating pentadecapeptide, the potential of d,l- alternating peptides for membrane insertion is investigated using the model dodecamer peptide H -(Phe- Tyr)5 -Trp- Trp - OH. This aromatic peptide is introduced as a novel pore-forming synthetic analogue of gramicidin A. It forms a well-organized homodimer similar to one of the gramicidin A transmembrane motifs. X-ray reflectivity measurements are performed on solid-supported peptide,lipid complexes to obtain information about the influence of the artificial dodecamer peptide on the bilayer parameters. In addition, Fourier-transform infrared (FTIR) and circular dichroism (CD) spectroscopic studies determine the conformational state of H -(Phe- Tyr)5 -Trp- Trp - OH within the model membrane. Site-specific iodine labeling assists in determining the topology of the membrane-embedded peptide by pinpointing the position of the iodine label within the bilayers. [source] Functional imaging: New views on lens structure and functionCLINICAL AND EXPERIMENTAL PHARMACOLOGY AND PHYSIOLOGY, Issue 12 2004Paul J Donaldson SUMMARY 1.,We have developed an experimental imaging approach that allows the distribution of lens membrane proteins to be mapped with subcellular resolution over large distances as a function of fibre cell differentiation. 2.,Using this approach in the rat lens, we have localized precisely histological sites of connexin 46 cleavage, quantitatively mapped changes in gap junction distribution and fibre cell morphology and correlated these changes to differences in intercellular dye transfer. 3.,Profiling of glucose transporter isoform expression showed that lens epithelial cells express GLUT1, whereas deeper cortical fibre cells express the higher-affinity GLUT3 isoform. Near the lens periphery, GLUT3 was located in the cytoplasm of fibre cells, but it underwent a differentiation-dependent membrane insertion. 4.,Similarly, the putative adhesion protein membrane protein 20 is inserted into fibre cell membranes at the stage when the cells lose their nuclei. This redistribution is strikingly rapid in terms of fibre cell differentiation and correlates with a barrier to extracellular diffusion. 5.,Our imaging-orientated approach has facilitated new insights into the relationships between fibre cell differentiation and lens function. Taken together, our results indicate that a number of strategies are used by the lens during the course of normal differentiation to change the subcellular distribution, gross spatial location and functional properties of key membrane transport proteins. [source] | ||||||||||||||||