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Inner Membrane (inner + membrane)
Kinds of Inner Membrane Terms modified by Inner Membrane Selected AbstractsTopology of the Mitochondrial Inner Membrane: Dynamics and Bioenergetic ImplicationsIUBMB LIFE, Issue 3-5 2001Carmen A. Mannella Abstract Electron tomography indicates that the mitochondrial inner membrane is not normally comprised of baffle-like folds as depicted in textbooks. In actuality, this membrane is pleomorphic, with narrow tubular regions connecting the internal compartments (cristae) to each other and to the membrane periphery. The membrane topologies observed in condensed (matrix contracted) and orthodox (matrix expanded) mitochondria cannot be interconverted by passive folding and unfolding. Instead, transitions between these morphological states likely involve membrane fusion and fission. Formation of tubular junctions in the inner membrane appears to be energetically favored, because they form spontaneously in yeast mitochondria following large-amplitude swelling and recontraction. However, aberrant, unattached, vesicular cristae are also observed in these mitochondria, suggesting that formation of cristae junctions depends on factors (such as the distribution of key proteins and/or lipids) that are disrupted during extreme swelling. Computer modeling studies using the "Virtual Cell" program suggest that the shape of the inner membrane can influence mitochondrial function. Simulations indicate that narrow cristae junctions restrict diffusion between intracristal and external compartments, causing depletion of ADP and decreased ATP output inside the cristae. [source] Fine structure of unusual spermatozoa and spermiogenesis of the mite Megisthanus floridanus Banks, 1904 (Acari: Gamasida: Antennophorina)ACTA ZOOLOGICA, Issue 4 2002Gerd Alberti Abstract The aflagellate spermatozoa of the gamasid mite Megisthanus floridanus are characterized by a large vacuole which contains a cytoplasmic column protruding into the vacuole from the region defined as the posterior part of the cell. The membrane of the column and the inner membrane of the posterior part of the cytoplasmic mantle (outer sheath) surrounding the vacuole bear numerous so-called cellular processes. However, most of the outer sheath is reduced and represented solely by a very thin membrane-like envelope. The posterior part of the cell bears extensive folds. The cell, or, more precisely, the column, shows a deep posterior invagination. This invagination contains extracellular material composed of thin filaments or strands. Peripheral folds emerging from the posterior rim of the cell form a thin-walled tube that contains the same material as the invagination. The elongated nucleus is attached to a peculiar acrosomal complex consisting of a flat acrosomal cisterna that parallels most of the cell membrane, an attachment cone, and a short acrosomal filament which is embedded in a narrow canal within the nucleus. The spermatozoa of M. floridanus represent a peculiar version of the vacuolated type of sperm known to be plesiomorphic within the anactinotrichid Acari. Some details of spermiogenesis are described and consequences for phylogenetic and systematic considerations are discussed. [source] Analysis of mitochondria by capillary electrophoresis: cardiolipin levels decrease in response to carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazoneEUROPEAN JOURNAL OF LIPID SCIENCE AND TECHNOLOGY, Issue 9 2010Wenfeng Zhao Abstract Cardiolipin is an important phospholipid present in the inner membrane of mitochondria. It plays a critical role in adenosine triphosphate (ATP) synthesis mediated by oxidative phosphorylation. Exposure of HepG2 cells to carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP) caused the inhibition of ATP synthesis and the depolarization of mitochondria. Capillary electrophoresis with laser-induced fluorescence (CE-LIF) analysis of fluorescent mitochondrion-selective probe 10-N-nonyl acridine orange (NAO) labeled mitochondria was employed to in situ estimate the cardiolipin levels under FCCP-induced de-energization of mitochondria. NAO, stoichiometriclly bound to cardiolipin at a 1:1 or 2:1 molar ratio (NAO/cardiolipin), emitted green and red fluorescence, respectively. Green fluorescence was chosen for cardiolipin content analysis because it was more intense than red fluorescence. A significant decrease in the cardiolipin content, up to 11% of the control, was evident when the ATP content and mitochondrial membrane potential (MMP) correspondingly decreased. These related findings suggested that CE-LIF may provide a sensitive strategy to determine cardiolipin content in response to exposure to chemical uncouplers. This reinforces the hypothesis that alterations in ATP synthesis and MMP have a close association with cardiolipin content, which correlated tightly with mitochondrial membrane assembly and activity. [source] Cold-Induced Recruitment of Brown Adipose Tissue ThermogenesisEXPERIMENTAL PHYSIOLOGY, Issue 1 2003Martin Klingenspor Non-shivering thermogenesis in brown adipose tissue is the main mechanism for thermoregulatory heat production in small mammals and newborns. During cold acclimation the sympathetic innervation triggers the recruitment of brown adipose tissue by hyperplasia, which involves the proliferation and differentiation of precursor cells, and by hypertrophy of mature brown adipocytes. Mitochondrial biogenesis and increased synthesis of the uncoupling protein 1 (UCP-1) are hallmarks of the thermogenic recruitment process. The severalfold increase of mitochondrial protein content during cold acclimation recruits a large capacity for oxidative phosphorylation. However, UCP-1 increases proton leakage across the inner membrane of brown adipocyte mitochondria and thereby dissipates proton motive force as heat instead of ATP synthesis. During recent years considerable progress has been achieved in the analysis of transcriptional mechanisms controlling Ucp1 gene expression. However, so far only little is known about the molecular basis of cold-induced mitochondrial biogenesis in brown adipose tissue. [source] Submitochondrial localization of 6- N -trimethyllysine dioxygenase , implications for carnitine biosynthesisFEBS JOURNAL, Issue 22 2007Naomi Van Vlies The first enzyme of carnitine biosynthesis is the mitochondrial 6- N -trimethyllysine dioxygenase, which converts 6- N -trimethyllysine to 3-hydroxy-6- N -trimethyllysine. Using progressive membrane solubilization with digitonin and protease protection experiments, we show that this enzyme is localized in the mitochondrial matrix. Latency experiments with intact mitochondria showed that 3-hydroxy-6- N -trimethyllysine formation is limited by 6- N -trimethyllysine transport across the mitochondrial inner membrane. Because the subsequent carnitine biosynthesis enzymes are cytosolic, after production, 3-hydroxy-6- N -trimethyllysine must be transported out of the mitochondria by a putative mitochondrial 6- N -trimethyllysine/3-hydroxy-6- N -trimethyllysine transporter system. This transport system represents an additional step in carnitine biosynthesis that could have considerable implications for the regulation of carnitine biosynthesis. [source] Complete reconstitution of an ATP-binding cassette transporter LolCDE complex from separately isolated subunitsFEBS JOURNAL, Issue 12 2007Kyoko Kanamaru The LolCDE complex of Escherichia coli belongs to the ATP-binding cassette transporter superfamily and mediates the detachment of lipoproteins from the inner membrane, thereby initiating lipoprotein sorting to the outer membrane. The complex is composed of one copy each of membrane subunits LolC and LolE, and two copies of ATPase subunit LolD. To establish the conditions for reconstituting the LolCDE complex from separately isolated subunits, the ATPase activities of LolD and LolCDE were examined under various conditions. We found that both LolD and LolCDE were inactivated on incubation at 30 °C in a detergent solution. ATP and phospholipids protected LolCDE, but not LolD. Furthermore, phospholipids reactivated LolCDE even after its near complete inactivation. LolD was also protected from inactivation when membrane subunits and phospholipids were present together, suggesting the phospholipid-dependent reassembly of LolCDE subunits. Indeed, the functional lipoprotein-releasing machinery was reconstituted into proteoliposomes with E. coli phospholipids and separately purified LolC, LolD and LolE. Preincubation with phospholipids at 30 °C was essential for the reconstitution of the functional machinery from subunits. Strikingly, the lipoprotein-releasing activity was also reconstituted from LolE and LolD without LolC, suggesting the intriguing possibility that the minimum lipoprotein-releasing machinery can be formed from LolD and LolE. We report here the complete reconstitution of a functional ATP-binding cassette transporter from separately purified subunits. [source] Uncoupling proteins 2 and 3 interact with members of the 14.3.3 familyFEBS JOURNAL, Issue 9 2000Benoit Pierrat Uncoupling proteins (UCPs) are members of the superfamily of the mitochondrial anion carrier proteins (MATP). Localized in the inner membrane of the organelle, they are postulated to be regulators of mitochondrial uncoupling. UCP2 and 3 may play an important role in the regulation of thermogenesis and, thus, on the resting metabolic rate in humans. To identify interacting proteins that may be involved in the regulation of the activity of UCPs, the yeast two-hybrid system was applied. Segments of hUCP2 containing the hydrophilic loops facing the intermembrane space, or combinations of these, were used to screen an adipocyte activation domain (AD) fusion library. The 14.3.3 protein isoforms ,, ,, , were identified as possible interacting partners of hUCP2. Screening of a human skeletal muscle AD fusion library, on the other hand, yielded several clones all of them encoding the , isoform of the 14.3.3 family. Mapping experiments further revealed that all these 14.3.3 proteins interact specifically with the C-terminal intermembrane space domain of both hUCP2 and hUCP3 whereas no interactions could be detected with the C-terminal part of hUCP1. Direct interaction between UCP3 and 14.3.3 , could be demonstrated after in vitro translation by coimmunoprecipitation. When coexpressed in a heterologous yeast system, 14.3.3 proteins potentiated the inhibitory effect of UCP3 overexpression on cell growth. These findings suggest that 14.3.3 proteins could be involved in the targeting of UCPs to the mitochondria. [source] Mitochondrial preprotein translocases as dynamic molecular machinesFEMS YEAST RESEARCH, Issue 6 2006Martin Van Der Laan Abstract Proteomic studies have demonstrated that yeast mitochondria contain roughly 1000 different proteins. Only eight of these proteins are encoded by the mitochondrial genome and are synthesized on mitochondrial ribosomes. The remaining 99% of mitochondrial precursors are encoded within the nuclear genome and after their synthesis on cytosolic ribosomes must be imported into the organelle. Targeting of these proteins to mitochondria and their import into one of the four mitochondrial subcompartments , outer membrane, intermembrane space (IMS), inner membrane and matrix , requires various membrane-embedded protein translocases, as well as numerous chaperones and cochaperones in the aqueous compartments. During the last years, several novel protein components involved in the import and assembly of mitochondrial proteins have been identified. The picture that emerges from these exciting new findings is that of highly dynamic import machineries, rather than of regulated, but static protein complexes. In this review, we will give an overview on the recent progress in our understanding of mitochondrial protein import. We will focus on the presequence translocase of the inner mitochondrial membrane, the TIM23 complex and the presequence translocase-associated motor, the PAM complex. These two molecular machineries mediate the multistep import of preproteins with cleavable N-terminal signal sequences into the matrix or inner membrane of mitochondria. [source] Current Views of the Structure of the Mammalian Mitochondrial RibosomeISRAEL JOURNAL OF CHEMISTRY, Issue 1 2010Emine Abstract Mammalian mitochondria synthesize polypeptides crucial for energy generation using ribosomes with a number of unique features. These ribosomes are very protein rich and have very truncated ribosomal RNAs. The bulk of the mammalian mitochondrial ribosome is composed of proteins, only about half of which are homologs of ribosomal proteins found in other translational systems. A number of distinctive features are found in these ribosomes. Among these is a gate-like structure that allows entrance of the primarily leaderless mRNAs that characterize this system. The exit tunnel of the large subunit is also quite unusual and includes a site in which the nascent peptide is visible to solvent prior to the normal exit site. Further, this region of the mitochondrial ribosome is dominated by ribosomal proteins rather than rRNA and is involved in the interaction of the ribosome with the inner membrane where all of the translation products are ultimately located. The proteins of the mitochondrial ribosome appear to play a number of important roles in the cell in addition to their function in protein biosynthesis, including roles in apoptosis and in cell cycle control. [source] Topology of the Mitochondrial Inner Membrane: Dynamics and Bioenergetic ImplicationsIUBMB LIFE, Issue 3-5 2001Carmen A. Mannella Abstract Electron tomography indicates that the mitochondrial inner membrane is not normally comprised of baffle-like folds as depicted in textbooks. In actuality, this membrane is pleomorphic, with narrow tubular regions connecting the internal compartments (cristae) to each other and to the membrane periphery. The membrane topologies observed in condensed (matrix contracted) and orthodox (matrix expanded) mitochondria cannot be interconverted by passive folding and unfolding. Instead, transitions between these morphological states likely involve membrane fusion and fission. Formation of tubular junctions in the inner membrane appears to be energetically favored, because they form spontaneously in yeast mitochondria following large-amplitude swelling and recontraction. However, aberrant, unattached, vesicular cristae are also observed in these mitochondria, suggesting that formation of cristae junctions depends on factors (such as the distribution of key proteins and/or lipids) that are disrupted during extreme swelling. Computer modeling studies using the "Virtual Cell" program suggest that the shape of the inner membrane can influence mitochondrial function. Simulations indicate that narrow cristae junctions restrict diffusion between intracristal and external compartments, causing depletion of ADP and decreased ATP output inside the cristae. [source] Protein Import Into MitochondriaIUBMB LIFE, Issue 3-5 2001Stefan A. Paschen Abstract Most mitochondrial proteins are encoded by the nuclear genome and thus have to be imported into mitochondria from the cytosol. Protein translocation across and into the mitochondrial membranes is a multistep process facilitated by the coordinated action of at least four specialized translocation systems in the outer and inner membranes of mitochondria. The outer membrane contains one general translocase, the TOM complex, whereas three distinct translocases are located in the inner membrane, which facilitates translocation of different classes of preproteins. The TIM23 complex mediates import of matrix-targeted preproteins with N -terminal presequences, whereas hydrophobic preproteins with internal targeting signals are inserted into the inner membrane via the TIM22 complex. The OXA translocase mediates the insertion of preproteins from the matrix space into the inner membrane. This review focuses on the structural organization and function of the import machinery of the model organisms of Saccharomyces cerevisiae and Neurospora crassa . In addition, the molecular basis of a new human mitochondrial disorder is discussed, the Mohr-Tranebjaerg syndrome. This is the first known disease, which is caused by an impaired mitochondrial protein import machinery leading to progressive neurodegeneration. [source] Effects of modification of membrane lipid composition on Bacillus subtilis sporulation and spore propertiesJOURNAL OF APPLIED MICROBIOLOGY, Issue 6 2009K.K. Griffiths Abstract Aims:, To determine effects of inner membrane lipid composition on Bacillus subtilis sporulation and spore properties. Methods and Results:, The absence of genes encoding lipid biosynthetic enzymes had no effect on B. subtilis sporulation, although the expected lipids were absent from spores' inner membrane. The rate of spore germination with nutrients was decreased c. 50% with mutants that lacked the major cardiolipin (CL) synthase and another enzyme for synthesis of a major phospholipid. Spores lacking the minor CL synthase or an enzyme essential for glycolipid synthesis exhibited 50,150% increases in rates of dodecylamine germination, while spores lacking enzymes for phosphatidylethanolamine (PE), phosphatidylserine (PS) and lysylphosphatidylglycerol (l-PG) synthesis exhibited a 30,50% decrease. Spore sensitivity to H2O2 and tert-butylhydroperoxide was increased 30,60% in the absence of the major CL synthase, but these spores' sensitivity to NaOCl or OxoneÔ was unaffected. Spores of lipid synthesis mutants were less resistant to wet heat, with spores lacking enzymes for PE, PS or l-PG synthesis exhibiting a two to threefold decrease and spores of other strains exhibiting a four to 10-fold decrease. The decrease in spore wet heat resistance correlated with an increase in core water content. Conclusions:, Changing the lipid composition of the B. subtilis inner membrane did not affect sporulation, although modest effects on spore germination and wet heat and oxidizing agent sensitivity were observed, especially when multiple lipids were absent. The increases in rates of dodecylamine germination were likely due to increased ability of this compound to interact with the spore's inner membrane in the absence of some CL and glycolipids. The effects on spore wet heat sensitivity are likely indirect, because they were correlated with changes in core water content. Significance and Impact of the Study:, The results of this study provide insight into roles of inner membrane lipids in spore properties. [source] Treatment with oxidizing agents damages the inner membrane of spores of Bacillus subtilis and sensitizes spores to subsequent stressJOURNAL OF APPLIED MICROBIOLOGY, Issue 4 2004D.E. Cortezzo Abstract Aims:, To determine if treatment of Bacillus subtilis spores with a variety of oxidizing agents causes damage to the spore's inner membrane. Methods and Results:, Spores of B. subtilis were killed 80,99% with wet heat or a variety of oxidizing agents, including betadine, chlorine dioxide, cumene hydroperoxide, hydrogen peroxide, OxoneTM, ozone, sodium hypochlorite and t-butylhydroperoxide, and the agents neutralized and/or removed. Survivors of spores pretreated with oxidizing agents exhibited increased sensitivity to killing by a normally minimal lethal heat treatment, while spores pretreated with wet heat did not. In addition, spores treated with wet heat or the oxidizing agents, except sodium hypochlorite, were more sensitive to high NaCl in plating media than were untreated spores. The core region of spores treated with at least two oxidizing agents was also penetrated much more readily by methylamine than was the core of untreated spores, and spores treated with oxidizing agents but not wet heat germinated faster with dodecylamine than did untreated spores. Spores of strains with very different levels of unsaturated fatty acids in their inner membrane exhibited essentially identical resistance to oxidizing agents. Conclusions:, Treatment of spores with oxidizing agents has been suggested to cause damage to the spore's inner membrane, a membrane whose integrity is essential for spore viability. The sensitization of spores to killing by heat and to high salt after pretreatment with oxidizing agents is consistent with and supports this suggestion. Presumably mild pretreatment with oxidizing agents causes some damage to the spore's inner membrane. While this damage may not be lethal under normal conditions, the damaged inner membrane may be less able to maintain its integrity, when dormant spores are exposed to high temperature or when germinated spores are faced with osmotic stress. Triggering of spore germination by dodecylamine likely involves action by this agent on the spore's inner membrane allowing release of the spore core's depot of dipicolinic acid. Presumably dodecylamine more readily alters the permeability of a damaged inner membrane and thus more readily triggers germination of spores pretreated with oxidizing agents. Damage to the inner spore membrane by oxidizing agents is also consistent with the more rapid penetration of methylamine into the core of treated spores, as the inner membrane is likely the crucial permeability barrier to methylamine entry into the spore core. As spores of strains with very different levels of unsaturated fatty acids in their inner membrane exhibited essentially identical resistance to oxidizing agents, it is not through oxidation of unsaturated fatty acids that oxidizing agents kill and/or damage spores. Perhaps these agents work by causing oxidative damage to key proteins in the spore's inner membrane. Significance and Impact of the Study:, The more rapid heat killing and germination with dodecylamine, the greater permeability of the spore core and the osmotic stress sensitivity in outgrowth of spores pretreated with oxidizing agents is consistent with such agents causing damage to the spore's inner membrane, even if this damage is not lethal under normal conditions. It may be possible to take advantage of this phenomenon to devise improved, less costly regimens for spore inactivation. [source] Mitochondria and aging: a role for the permeability transition?AGING CELL, Issue 1 2004M. Crompton Summary When mitochondria are subjected to oxidative stress and relatively high [Ca2+], they undergo a ,permeability transition' in which the inner membrane becomes freely permeable to low-molecular-weight solutes. This phenomenon reflects reversible deformation of the adenine nucleotide translocase, the loss of its native gating properties and the stabilization of the deformed state by cyclophilin-D. The permeability transition may be a factor in cell dysfunction associated with aging. This can manifest in a number of ways ranging, in the most severe, from impaired energy transduction and compromised viability to more subtle influences on the propagation of Ca2+ signals. This article critically examines data relevant to this issue. [source] Molecular characterization of mitocalcin, a novel mitochondrial Ca2+ -binding protein with EF-hand and coiled-coil domainsJOURNAL OF NEUROCHEMISTRY, Issue 1 2006Mitsutoshi Tominaga Abstract Here we have identified and characterized a novel mitochondrial Ca2+ -binding protein, mitocalcin. Western blot analysis demonstrated that mitocalcin was widely expressed in mouse tissues. The expression in brain was increased during post-natal to adult development. Further analyses were carried out in newly established neural cell lines. The protein was expressed specifically in neurons but not in glial cells. Double-labeling studies revealed that mitocalcin was colocalized with mitochondria in neurons differentiated from 2Y-3t cells. In addition, mitocalcin was enriched in the mitochondrial fraction purified from the cells. Immunohistochemical studies on mouse cerebellum revealed that the expression pattern of mitocalcin in glomeruli of the internal granular and molecular layers was well overlapped by the distribution pattern of mitochondria. Immunogold electron microscopy showed that mitocalcin was associated with mitochondrial inner membrane. Overexpression of mitocalcin in 2Y-3t cells resulted in neurite extension. Inhibition of the expression in 2Y-3t cells caused suppression of neurite outgrowth and then cell death. These findings suggest that mitocalcin may play roles in neuronal differentiation and function through the control of mitochondrial function. [source] Altered distribution of mitochondria impairs calcium homeostasis in rat hippocampal neurons in cultureJOURNAL OF NEUROCHEMISTRY, Issue 1 2003Guang Jian Wang Abstract The specificity of Ca2+ signals is conferred in part by limiting changes in cytosolic Ca2+ to subcellular domains. Mitochondria play a major role in regulating Ca2+ in neurons and may participate in its spatial localization. We examined the effects of changes in the distribution of mitochondria on NMDA-induced Ca2+ increases. Hippocampal cultures were treated with the microtubule-destabilizing agent vinblastine, which caused the mitochondria to aggregate and migrate towards one side of the neuron. This treatment did not appear to decrease the energy status of mitochondria, as indicated by a normal membrane potential and pH gradient across the inner membrane. Moreover, electron microscopy showed that vinblastine treatment altered the distribution but not the ultrastructure of mitochondria. NMDA (200 µm, 1 min) evoked a greater increase in cytosolic Ca2+ in vinblastine-treated cells than in untreated cells. This increase did not result from impaired Ca2+ efflux, enhanced Ca2+ influx, opening of the mitochondrial permeability transition pore or altered function of endoplasmic reticulum Ca2+ stores. Ca2+ uptake into mitochondria was reduced by 53% in vinblastine-treated cells, as reported by mitochondrially targeted aequorin. Thus, the distribution of mitochondria maintained by microtubules is critical for buffering Ca2+ influx. A subset of mitochondria close to a Ca2+ source may preferentially regulate Ca2+ microdomains, set the threshold for Ca2+ -induced toxicity and participate in local ATP production. [source] Hydrophobic peptides: novel regulators within bacterial membraneMOLECULAR MICROBIOLOGY, Issue 1 2009Eric Alix Summary Identification of short coding sequences is challenging, both experimentally and in silico, and functional natural peptides (< 50 amino acids) have to a large extent been overlooked in Gram-negative bacteria. Recent results have converged to highlight the role of hydrophobic peptides that form a novel class of active molecules in Escherichia coli and Salmonella enterica serovar Typhimurium. These peptides can play a regulatory role by interacting with protein partners at the inner membrane and by modulating protein partner activity or stability. Genome-wide analyses in both bacterial species have identified several conserved short open reading frames encoding a single transmembrane segment. We discuss the known and predicted membrane-associated peptides and the tools for their identification. Besides the identification of novel regulatory networks, characterization of peptides with a single transmembrane helix segment and proteins that interact with them provides a powerful opportunity to study interactions between alpha helices within biological membranes. In addition, some bioactive membrane peptides could provide a basis for engineering membrane protein antagonists. [source] Novel ultrastructures of Treponema primitia and their implications for motilityMOLECULAR MICROBIOLOGY, Issue 6 2008Gavin E. Murphy Summary Members of the bacterial phylum Spirochaetes are generally helical cells propelled by periplasmic flagella. The spirochete Treponema primitia is interesting because of its mutualistic role in the termite gut, where it is believed to cooperate with protozoa that break down cellulose and produce H2 as a by-product. Here we report the ultrastructure of T. primitia as obtained by electron cryotomography of intact, frozen-hydrated cells. Several previously unrecognized external structures were revealed, including bowl-like objects decorating the outer membrane, arcades of hook-shaped proteins winding along the exterior and tufts of fibrils extending from the cell tips. Inside the periplasm, cone-like structures were found at each pole. Instead of the single peptidoglycan layer typical of other Gram-negative bacteria, two distinct periplasmic layers were observed. These layers formed a central open space that contained two flagella situated adjacent to each other. In some areas, the inner membrane formed flattened invaginations that protruded into the cytoplasm. High-speed light microscopic images of swimming T. primitia cells showed that cell bodies remained rigid and moved in a helical rather than planar motion. Together, these findings support the ,rolling cylinder' model for T. primitia motility that posits rotation of the protoplasmic cylinder within the outer sheath. [source] Lipid trafficking to the outer membrane of Gram-negative bacteriaMOLECULAR MICROBIOLOGY, Issue 3 2006William T. Doerrler Summary The envelope of Gram-negative bacteria is composed of two distinct lipid membranes: an inner membrane and outer membrane. The outer membrane is an asymmetric bilayer with an inner leaflet of phospholipids and an outer leaflet of lipopolysaccharide. Most of the steps of lipid synthesis occur within the cytoplasmic compartment of the cell. Lipids must then be transported across the inner membrane and delivered to the outer membrane. These topological features combined with the ability to apply the tools of biochemistry and genetics make the Gram-negative envelope a fascinating model for the study of lipid trafficking. In addition, as lipopolysaccharide is essential for growth of most strains and is a potent inducer of the mammalian innate immune response via activation of Toll-like receptors, Gram-negative lipid transport is also a promising target for the development of novel antibacterial and anti-inflammatory compounds. This review focuses on recent developments in our understanding of lipid transport across the inner membrane and to the outer membrane of Gram-negative bacteria. [source] Flk prevents premature secretion of the anti-, factor FlgM into the periplasmMOLECULAR MICROBIOLOGY, Issue 3 2006Phillip Aldridge Summary The flk locus of Salmonella typhimurium was identified as a regulator of flagellar gene expression in strains defective in P- and l -ring formation. Flk acts as a regulator of flagellar gene expression by modulating the protein levels of the anti-,28 factor FlgM. Evidence is presented which suggests that Flk is a cytoplasmic-facing protein anchored to the inner membrane by a single, C-terminal transmembrane-spanning domain (TMS). The specific amino acid sequence of the TMS is not essential for Flk activity, but membrane anchoring is essential. Membrane fractionation and visualization of protein fusions of green fluorescent protein derivatives to Flk suggested that the Flk protein is present in the membrane as punctate spots in number that are much greater than the number of flagellar basal structures. The turnover of the anti-,28 factor FlgM was increased in flk mutant strains. Using FlgM,,-lactamase fusions we show the increased turnover of FlgM in flk null mutations is due to FlgM secretion into the periplasm where it is degraded. Our data suggest that Flk inhibits FlgM secretion by acting as a braking system for the flagellar-associated type III secretion system. A model is presented to explain a role for Flk in flagellar assembly and gene regulatory processes. [source] A subset of bacterial inner membrane proteins integrated by the twin-arginine translocaseMOLECULAR MICROBIOLOGY, Issue 5 2003Kostas Hatzixanthis Summary A group of bacterial exported proteins are synthesized with N-terminal signal peptides containing a SRRxFLK ,twin-arginine' amino acid motif. Proteins bearing twin-arginine signal peptides are targeted post-translationally to the twin-arginine translocation (Tat) system which transports folded substrates across the inner membrane. In Escherichia coli, most integral inner membrane proteins are assembled by a co-translational process directed by SRP/FtsY, the SecYEG translocase, and YidC. In this work we define a novel class of integral membrane proteins assembled by a Tat-dependent mechanism. We show that at least five E. coli Tat substrate proteins contain hydrophobic C-terminal transmembrane helices (or ,C-tails'). Fusions between the identified transmembrane C-tails and the exclusively Tat-dependent reporter proteins TorA and SufI render the resultant chimeras membrane-bound. Export-linked signal peptide processing and membrane integration of the chimeras is shown to be both Tat-dependent and YidC-independent. It is proposed that the mechanism of membrane integration of proteins by the Tat system is fundamentally distinct from that employed for other bacterial inner membrane proteins. [source] Imp/OstA is required for cell envelope biogenesis in Escherichia coliMOLECULAR MICROBIOLOGY, Issue 5 2002Martin Braun Summary In Gram-negative bacteria, all components of the outer membrane are synthesized in the cytoplasm or the cytoplasmic leaflet of the inner membrane and must thus traverse the inner membrane and the periplasm on the way to their final destination. In this study, we show Imp/OstA to have characteristics typical for proteins involved in envelope biogenesis. Imp is essential and forms a high-molecular-weight disulphide-bonded complex in the outer membrane. Upon depletion of Imp, lipids and outer membrane proteins appear in a novel membrane fraction with higher density than the outer membrane. We propose Imp to be part of a targeting/usher system for components of the outer membrane. [source] Topological analysis and role of the transmembrane domain in polar targeting of PilS, a Pseudomonas aeruginosa sensor kinaseMOLECULAR MICROBIOLOGY, Issue 4 2000Julie Ethier In Pseudomonas aeruginosa, synthesis of pilin, the major protein subunit of the pili, is regulated by a two-component signal transduction system in which PilS is the sensor kinase. PilS is an inner membrane protein found at the poles of the bacterial cell. It is composed of three domains: an N-terminal hydrophobic domain; a central cytoplasmic linker region; and the C-terminal transmitter region conserved among other sensor kinases. The signal that activates PilS and, consequently, pilin transcription remains unknown. The membrane topology of the hydrophobic domain was determined using the lacZ and phoA gene fusion approach. In this report, we describe a topological model for PilS in which the hydrophobic domain forms six transmembrane helices, whereas the N- and C-termini are cytoplasmic. This topology is very stable, and the cytoplasmic C-terminus cannot cross the inner membrane. We also show that two of the six transmembrane segments are sufficient for membrane anchoring and polar localization of PilS. [source] Tethering of CpxP to the inner membrane prevents spheroplast induction of the Cpx envelope stress responseMOLECULAR MICROBIOLOGY, Issue 5 2000Tracy L. Raivio The Cpx envelope stress response of Escherichia coli is controlled by a two-component regulatory system that senses misfolded proteins in extracytoplasmic compartments and responds by inducing the expression of envelope protein folding and degrading factors. We have proposed that in the absence of envelope stress the pathway is maintained in a downregulated state, in part through interactions between the periplasmic inhibitor molecule CpxP and the sensing domain of the histidine kinase CpxA. In this study, we show that depletion of the periplasmic contents of the cell by spheroplast formation does indeed lead to induction of the Cpx envelope stress response. Further, removal of CpxP is an important component of this induction because tethering an MBP,CpxP fusion protein to the spheroplast inner membranes prevents full activation by this treatment. Spheroplast formation has previously been demonstrated to induce the expression of a periplasmic protein of unknown function, Spy. Analysis of spy expression in response to spheroplast formation by Western blot analysis and by lacZ operon fusion in various cpx mutant backgrounds demonstrated that spy is a member of the Cpx regulon. Interestingly, although the only known spy homologue is cpxP, Spy does not appear to perform the same function as CpxP as it is not involved in inhibiting the Cpx envelope stress response. Rather, deletion of spy leads to activation of the ,E stress response. Because the ,E response is specifically affected by alterations in outer membrane protein biogenesis, we think it possible that Spy may be involved in this process. [source] Comparison of mitochondrial ascorbate peroxidase in the cultivated tomato, Lycopersicon esculentum, and its wild, salt-tolerant relative, L. pennellii, a role for matrix isoforms in protection against oxidative damagePLANT CELL & ENVIRONMENT, Issue 2 2004V. MITTOVA ABSTRACT Mitochondria require robust antioxidant defences to prevent lipid peroxidation and to protect tricarboxylic acid cycle enzymes from oxidative damage. Mitochondria from wild, salt-tolerant tomato, Lycopersicon pennellii (Lpa) did not exhibit lipid peroxidation in response to high salinity (100 mm NaCl), whereas those isolated from cultivated tomato, L. esculentum (Lem), accumulated malondialdehyde. The activity, intraorganellar distribution and salt response of mitochondrial ascorbate peroxidase (mAPX) differed dramatically in the two species. In Lem mitochondria, the majority (84%) of mAPX was associated with membranes, being located either on the inner membrane, facing the intermembrane space, or on the outer membrane. Total mAPX activity did not increase substantially in response to salt, although the proportion of matrix APX increased. In contrast, 61% of Lpa mAPX activity was soluble in the matrix, the remainder being bound to the matrix face of the inner membrane. Salt treatment increased the activity of all mAPX isoforms in Lpa, without altering their intramitochondrial distribution. The membrane-bound isoforms were detected in mitochondria of both species by western blotting and found to be induced by salt in Lpa. These observations suggest that matrix-associated APX isoforms could act in concert with other mitochondrial antioxidants to protect against salt-induced oxidative stress. [source] The ultrastructure of chilling stressPLANT CELL & ENVIRONMENT, Issue 4 2000H. A. Kratsch ABSTRACT Chilling injury to crop plants was first described 70 years ago and has been systematically investigated with electron microscopy since the late 1960s. Chloroplasts are the first and most severely impacted organelle. Thylakoids swell and distort, starch granules disappear, and a peripheral reticulum (vesicles arising from inner membrane of chloroplast envelope) appears. Chloroplast disintegration follows prolonged chilling. Mitochondria, nuclei and other organelles are less susceptible to chilling injury. Organellar development and ontogeny may also be disrupted. The inherent chilling sensitivity of a plant, as well as the ability of some species to acclimate to chilling, influence the timing and appearance of ultrastructural injury with the resulting outcome being mild, moderate, or severe. Other environmental factors that exacerbate injury are irradiance, chilling duration, and water status. The physiological basis for chloroplast swelling may be linked to chilling-stable starch-degrading enzymes that produce soluble sugars thus lowering stromal water potential at a time when chloroplast photosynthate export is reduced. Thylakoid dilation appears to be related to photo-oxidative conditions produced during chilling in the light. The peripheral reticulum is proposed to increase surface area of the transport-limiting membrane (chloroplast inner membrane) in response to the chilling-induced reduction in metabolite transport. Many of the ultrastructural symptoms appearing during moderate stress resemble those seen in programmed cell death. Future research directions are discussed. [source] Whole-seed development in Sicyos angulatus (Cucurbitaceae, Sicyeae) and a comparison with the development of water-impermeable seeds in five other familiesPLANT SPECIES BIOLOGY, Issue 3 2010XIAOXIA QU Abstract Sicyos is the only member of the Cucurbitaceae known to have water-impermeable seeds, that is, physical dormancy (PY), and development of seeds with PY has not been studied in detail in this family. Our primary aim was to describe seed development in Sicyos angulatus from pollination to seed maturity and to compare it with the development of water-impermeable seeds in other families reported in the literature. Full seed stage (highest moisture content [MC]) occurred 4,6 days after pollination (DAP) and physiological maturity occurred 20 DAP. Seeds became water impermeable 32 DAP, with 14.6% MC. The hilum was the site of water loss during the final stage of maturation drying. Excised embryos could germinate from 20 DAP to 36 DAP, whereas no intact seeds germinated during any stage of seed development. Mechanical scarification did not promote germination of seeds after the seed coat became water impermeable at 32,36 DAP. Based on a previous study of seed dormancy and germination in S. angulatus, we suggest that germination in these scarified seeds may have been prevented by resistance of inner membrane and fruit-seed coat to embryo expansion. The MC when seeds became impermeable (14.6%) is in the range of that reported for seeds of species in five other families with PY. Information on seed development in S. angulatus will be useful in planning the timing of management strategies for this pernicious annual weed. [source] Engineering antibody fragments to fold in the absence of disulfide bondsPROTEIN SCIENCE, Issue 2 2009Min Jeong Seo Abstract Disulfide bonds play a critical role in the stabilization of the immunoglobulin ,-sandwich sandwich. Under reducing conditions, such as those that prevail in the cytoplasm, disulfide bonds do not normally form and as a result most antibodies expressed in that compartment (intrabodies) accumulate in a misfolded and inactive state. We have developed a simple method for the quantitative isolation of antibody fragments that retain full activity under reducing conditions from large mutant libraries. In E. coli, inactivation of the cysteine oxidoreductase DsbA abolishes protein oxidation in the periplasm, which leads to the accumulation of scFvs and other disulfide-containing proteins in a reduced form. Libraries of mutant scFvs were tethered onto the inner membrane of dsbA cells and mutants that could bind fluorescently labeled antigen in the reducing periplasm were screened by Anchored Periplasmic Expression (APEx; Harvey et al., Proc Natl Acad Sci USA 2004;101:9193,9198.). Using this approach, we isolated scFv antibody variants that are fully active when expressed in the cytoplasm or when the four Cys residues that normally form disulfides are substituted by Ser residues. [source] Large-scale preparation of the homogeneous LolA,lipoprotein complex and efficient in vitro transfer of lipoproteins to the outer membrane in a LolB-dependent mannerPROTEIN SCIENCE, Issue 12 2007Shoji Watanabe Abstract An ATP-binding cassette transporter LolCDE complex of Escherichia coli releases lipoproteins destined to the outer membrane from the inner membrane as a complex with a periplasmic chaperone, LolA. Interaction of the LolA,lipoprotein complex with an outer membrane receptor, LolB, then causes localization of lipoproteins to the outer membrane. As far as examined, formation of the LolA,lipoprotein complex strictly depends on ATP hydrolysis by the LolCDE complex in the presence of LolA. It has been speculated, based on crystallographic and biochemical observations, that LolA undergoes an ATP-dependent conformational change upon lipoprotein binding. Thus, preparation of a large amount of the LolA,lipoprotein complex is difficult. Moreover, lipoproteins bound to LolA are heterogeneous. We report here that the coexpression of LolA and outer membrane-specific lipoprotein Pal from a very efficient plasmid causes the unusual accumulation of the LolA,Pal complex in the periplasm. The complex was purified to homogeneity and shown to be a functional intermediate of the lipoprotein localization pathway. In vitro incorporation of Pal into outer membranes revealed that a single molecule of LolB catalyzes the incorporation of more than 100 molecules of Pal into outer membranes. Moreover, the LolB-dependent incorporation of Pal was not affected by excess-free LolA, indicating that LolB specifically interacts with liganded LolA. Finally, the LolB depletion caused the accumulation of a significant amount of Pal in the periplasm, thereby establishing the conditions for preparation of the homogeneous LolA,lipoprotein complex. [source] Protein composition of Trypanosoma brucei mitochondrial membranesPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 24 2009Nathalie Acestor Abstract Mitochondria consist of four compartments, outer membrane, intermembrane space, inner membrane, and matrix; each harboring specific functions and structures. In this study, we used LC-MS/MS to characterize the protein composition of Trypanosoma brucei mitochondrial (mt) membranes, which were enriched by different biochemical fractionation techniques. The analyses identified 202 proteins that contain one or more transmembrane domain(s) and/or positive GRAVY scores. Of these, various criteria were used to assign 72 proteins to mt membranes with high confidence, and 106 with moderate-to-low confidence. The sub-cellular localization of a selected subset of 13 membrane assigned proteins was confirmed by tagging and immunofluorescence analysis. While most proteins assigned to mt membrane have putative roles in metabolic, energy generating, and transport processes, ,50% have no known function. These studies result in a comprehensive profile of the composition and sub-organellar location of proteins in the T. brucei mitochondrion thus, providing useful information on mt functions. [source] | ||||||||||||||||