Heterogeneity of photosystem II as it occurs in domain specific regions of the thylakoid membrane of spinach (spinacia oleracia L.)

Thylakoid membrane fractions were prepared from specific regions of thylakoid membranes of spinach (Spinacia oleracea). These fractions, which include grana (83), stroma (T3), grana core (8S), margins (Ma) and purified stroma (Y100) were prepared using a non-detergent method including a mild sonication and aqueous two-phase partitioning. The significance of PSlla and PSII~ centres have been described extensively in the literature. Previous work has characterized two types of PSII centres which are proposed to exist in different regions of the thylakoid membrane. a-centres are suggested to aggregate in stacked regions of grana whereas ~-centres are located in unstacked regions of stroma lamellae. The goal of this study is to characterize photosystem II from the isolated membrane vesicles representing different regions of the higher plant thylakoid membrane. The low temperature absorption spectra have been deconvoluted via Gaussian decomposition to estimate the relative sub-components that contribute to each fractions signature absorption spectrum. The relative sizes of the functional PSII antenna and the fluorescence induction kinetics were measured and used to determine the relative contributions of PSlla and PSII~ to each fraction. Picosecond chlorophyll fluorescence decay kinetics were collected for each fraction to characterize and gain insight into excitation energy transfer and primary electron transport in PSlla and PSII~ centres. The results presented here clearly illustrate the widely held notions of PSII/PS·I and PSlIa/PSII~ spatial separation. This study suggests that chlorophyll fluorescence decay lifetimes of PSII~ centres are shorter than those of PSlIa centres and, at FM, the longer lived of the two PSII components renders a larger yield in PSlIa-rich fractions, but smaller in PSIlr3-rich fractions.

Transport d’iode par le transporteur de sodium/acide monocarboxylique SMCT1

Le transporteur de Na+/ acide monocarboxylique sensible à l’ibuprofène (SMCT1) est exprimé dans la membrane apicale de plusieurs épithélia. Son rôle physiologique dans la glande thyroïde reste cependant obscur mais on présume qu’il pourrait agir comme un transporteur apical d’iode nécessaire pour la synthèse des hormones thyroïdiennes. Récemment, on a montré que SMCT1 possède un courant de fuite anionique sensible à [Na+]e qui permettrait de transporter l’iode de façon électrogénique. Cependant, un efflux d’iode sensible à l’ibuprofène, mais indépendant de la [Na+]e a été aussi observé sur des cultures primaires des thyrocytes porcins, suggérant un autre mécanisme de transport d’iode par SMCT1. Ce travail vise à comprendre les caractéristiques de ce genre de transport en utilisant comme modèle d’expression les ovocytes de Xenopus laevis. Les résultats obtenus des essais de captation d’iode radioactif montrent que SMCT1 présente un transport d’iode sensible à l’ibuprofène de l’ordre de 30nmol/ovocyte/h. Si ce transport est non saturable en iode (0-100 mM), il nécessite du Na+ dans la solution externe. En effet, le remplacement du Na+ extracellulaire par le NMDG inhibe complètement le transport. En outre, on s’est intéressé à exclure la possibilité de différents artefacts. En ayant trouvé que la grande majorité de l’iode radioactif se trouve dans la partie soluble de l’ovocyte, on exclut une liaison non spécifique de l’iode à la membrane cellulaire. Cependant, une bonne proportion de l’iode transporté pourrait être liée à des protéines à l’intérieur de l`ovocyte. En effet, on observe une réduction du transport d’iode dans les ovocytes exprimant SMCT1 de 81,6 ± 2 % en présence de 2 % BSA dans la solution extracellulaire. Également, on écarte la possibilité que le transport d’iode soit le résultat de la surexpression de protéines de transport endogènes dont les canaux chlore. Le transport d’iode semble spécifique à l’expression de SMCT1 et de manière intéressante à l’expression d’un autre transporteur de monocarboxylates, MCT1. L’analyse de l’ensemble des essais, y compris le fait que l’amplitude du transport observé est 20 fois plus grande que celle du courant de fuite nous mène à proposer que SMCT1 puisse transporter l’iode de façon électroneutre. Cependant, le mécanisme par lequel ceci est accompli n’est pas évident à identifier. L’utilisation d’un autre modèle cellulaire serait surement utile pour répondre à cette question.

The role of the TGN in the transport of herpes simplex virus type I capsids

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.

EfeO-cupredoxins: major new members of the cupredoxin superfamily with roles in bacterial iron transport

The EfeUOB system of Escherichia coli is a tripartite, low pH, ferrous iron transporter. It resembles the high-affinity iron transporter (Ftr1p-Fet3p) of yeast in that EfeU is homologous to Ftr1p, an integral-membrane iron-permease. However, EfeUOB lacks an equivalent of the Fet3p component—the multicopper oxidase with three cupredoxin-like domains. EfeO and EfeB are periplasmic but their precise roles are unclear. EfeO consists primarily of a C-terminal peptidase-M75 domain with a conserved ‘HxxE’ motif potentially involved in metal binding. The smaller N-terminal domain (EfeO-N) is predicted to be cupredoxin (Cup) like, suggesting a previously unrecognised similarity between EfeO and Fet3p. Our structural modelling of the E. coli EfeO Cup domain identifies two potential metal-binding sites. Site I is predicted to bind Cu2+ using three conserved residues (C41 and 103, and E66) and M101. Of these, only one (C103) is conserved in classical cupredoxins where it also acts as a Cu ligand. Site II most probably binds Fe3+ and consists of four well conserved surface Glu residues. Phylogenetic analysis indicates that the EfeO-Cup domains form a novel Cup family, designated the ‘EfeO-Cup’ family. Structural modelling of two other representative EfeO-Cup domains indicates that different subfamilies employ distinct ligand sets at their proposed metal-binding sites. The ~100 efeO homologues in the bacterial sequence databases are all associated with various iron-transport related genes indicating a common role for EfeO-Cup proteins in iron transport, supporting a new copper-iron connection in biology.

Characterization of a gamma-aminobutyric acid transport system of rhizobium leguminosarum bv. viciae 3841

Spontaneous mutants of Rhizobium leguminosarum bv. viciae 3841 were isolated that grow faster than the wild type on gamma-aminobutyric acid (GABA) as the sole carbon and nitrogen source. These strains (RU1736 and RU1816) have frameshift mutations (gtsR101 and gtsR102, respectively) in a GntR-type regulator (GtsR) that result in a high rate of constitutive GABA transport. Tn5 mutagenesis and quantitative reverse transcription-PCR showed that GstR regulates expression of a large operon (pRL100242 to pRL100252) on the Sym plasmid that is required for GABA uptake. An ABC transport system, GtsABCD (for GABA transport system) (pRL100248-51), of the spermidine/putrescine family is part of this operon. GtsA is a periplasmic binding protein, GtsB and GtsC are integral membrane proteins, and GtsD is an ATP-binding subunit. Expression of gtsABCD from a lacZ promoter confirmed that it alone is responsible for high rates of GABA transport, enabling rapid growth of strain 3841 on GABA. Gts transports open-chain compounds with four or five carbon atoms with carboxyl and amino groups at, or close to, opposite termini. However, aromatic compounds with similar spacing between carboxyl and amino groups are excellent inhibitors of GABA uptake so they may also be transported. In addition to the ABC transporter, the operon contains two putative mono-oxygenases, a putative hydrolase, a putative aldehyde dehydrogenase, and a succinate semialdehyde dehydrogenase. This suggests the operon may be involved in the transport and breakdown of a more complex precursor to GABA. Gts is not expressed in pea bacteroids, and gtsB mutants are unaltered in their symbiotic phenotype, suggesting that Bra is the only GABA transport system available for amino acid cycling.

Feo - Transport of ferrous iron into bacteria

Bacteria commonly utilise a unique type of transporter, called Feo, to specifically acquire the ferrous (Fe2+) form of iron from their environment. Enterobacterial Feo systems are composed of three proteins: FeoA, a small, soluble SH3-domain protein probably located in the cytosol; FeoB, a large protein with a cytosolic N-terminal G-protein domain and a C-terminal integral inner-membrane domain containing two 'Gate' motifs which likely functions as the Fe2+ permease; and FeoC, a small protein apparently functioning as an [Fe-S]-dependent transcriptional repressor. We provide a review of the current literature combined with a bioinformatic assessment of bacterial Feo systems showing how they exhibit common features, as well as differences in organisation and composition which probably reflect variations in mechanisms employed and function.

Proteomic analysis of plasma membrane vesicles isolated from the rat renal cortex

Polarized epithelial cells are responsible for the vectorial transport of solutes and have a key role in maintaining body fluid and electrolyte homeostasis. Such cells contain structurally and functionally distinct plasma membrane domains. Brush border and basolateral membranes of renal and intestinal epithelial cells can be separated using a number of different separation techniques, which allow their different transport functions and receptor expressions to be studied. In this communication, we report a proteomic analysis of these two membrane segments, apical and basolateral, obtained from the rat renal cortex isolated by two different methods: differential centrifugation and free-flow electrophoresis. The study was aimed at assessing the nature of the major proteins isolated by these two separation techniques. Two analytical strategies were used: separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) at the protein level or by cation-exchange high-performance liquid chromatography (HPLC) after proteolysis (i.e., at the peptide level). Proteolytic peptides derived from the proteins present in gel pieces or from HPLC fractions after proteolysis were sequenced by on-line liquid chromatography-tandem mass spectrometry (LC-MS/MS). Several hundred proteins were identified in each membrane section. In addition to proteins known to be located at the apical and basolateral membranes, several novel proteins were also identified. In particular, a number of proteins with putative roles in signal transduction were identified in both membranes. To our knowledge, this is the first reported study to try and characterize the membrane proteome of polarized epithelial cells and to provide a data set of the most abundant proteins present in renal proximal tubule cell membranes.

Proteomic analysis of plasma membrane vesicles isolated from the rat renal cortex

Polarized epithelial cells are responsible for the vectorial transport of solutes and have a key role in maintaining body fluid and electrolyte homeostasis. Such cells contain structurally and functionally distinct plasma membrane domains. Brush border and basolateral membranes of renal and intestinal epithelial cells can be separated using a number of different separation techniques, which allow their different transport functions and receptor expressions to be studied. In this communication, we report a proteomic analysis of these two membrane segments, apical and basolateral, obtained from the rat renal cortex isolated by two different methods: differential centrifugation and free-flow electrophoresis. The study was aimed at assessing the nature of the major proteins isolated by these two separation techniques. Two analytical strategies were used: separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) at the protein level or by cation-exchange high-performance liquid chromatography (HPLC) after proteolysis (i.e., at the peptide level). Proteolytic peptides derived from the proteins present in gel pieces or from HPLC fractions after proteolysis were sequenced by on-line liquid chromatography-tandem mass spectrometry (LC-MS/MS). Several hundred proteins were identified in each membrane section. In addition to proteins known to be located at the apical and basolateral membranes, several novel proteins were also identified. In particular, a number of proteins with putative roles in signal transduction were identified in both membranes. To our knowledge, this is the first reported study to try and characterize the membrane proteome of polarized epithelial cells and to provide a data set of the most abundant proteins present in renal proximal tubule cell membranes.

Feo-transport of ferrous iron into bacteria

Bacteria commonly utilise a unique type of transporter, called Feo, to specifically acquire the ferrous (Fe2+) form of iron from their environment. Enterobacterial Feo systems are composed of three proteins: FeoA, a small, soluble SH3-domain protein probably located in the cytosol; FeoB, a large protein with a cytosolic N-terminal G-protein domain and a C-terminal integral inner-membrane domain containing two 'Gate' motifs which likely functions as the Fe2+ permease; and FeoC, a small protein apparently functioning as an [Fe-S]-dependent transcriptional repressor. We provide a review of the current literature combined with a bioinformatic assessment of bacterial Feo systems showing how they exhibit common features, as well as differences in organisation and composition which probably reflect variations in mechanisms employed and function.

A novel transport mechanism for MOMP in Chlamydophila pneumoniae and its putative role in immune-therapy

Major outer membrane proteins (MOMPs) of Gram negative bacteria are one of the most intensively studied membrane proteins. MOMPs are essential for maintaining the structural integrity of bacterial outer membranes and in adaptation of parasites to their hosts. There is evidence to suggest a role for purified MOMP from Chlamydophila pneumoniae and corresponding MOMP-derived peptides in immune-modulation, leading to a reduced atherosclerotic phenotype in apoE−/− mice via a characteristic dampening of MHC class II activity. The work reported herein tests this hypothesis by employing a combination of homology modelling and docking to examine the detailed molecular interactions that may be responsible. A three-dimensional homology model of the C. pneumoniae MOMP was constructed based on the 14 transmembrane β-barrel crystal structure of the fatty acid transporter from Escherichia coli, which provides a plausible transport mechanism for MOMP. Ligand docking experiments were used to provide details of the possible molecular interactions driving the binding of MOMP-derived peptides to MHC class II alleles known to be strongly associated with inflammation. The docking experiments were corroborated by predictions from conventional immuno-informatic algorithms. This work supports further the use of MOMP in C. pneumoniae as a possible vaccine target and the role of MOMP-derived peptides as vaccine candidates for immune-therapy in chronic inflammation that can result in cardiovascular events.

TNF-alpha mediated transport of NF-kappaB to the nucleus is independent of the cytoskeleton-based transport system in non-neuronal cells

In unstimulated cells, proteins of the nuclear factor kappaB (NF-kappaB) transcription factor family are sequestered in the cytoplasm through interactions with IkappaB inhibitor proteins. Tumor necrosis factor alpha (TNF-alpha) activates the degradation of IkappaB-alpha and the nuclear import of cytoplasmic NF-kappaB. Nuclear localization of numerous cellular proteins is mediated by the ability of the cytoskeleton, usually microtubules, to direct their perinuclear accumulation. In a former study we have shown that activated NF-kappaB rapidly moves from distal processes in neurons towards the nucleus. The fast transport rate suggests the involvement of motor proteins in the transport of NF-kappaB. Here we address the question how NF-kappaB arrives at the nuclear membrane before import in non-neuronal cells, i.e., by diffusion alone or with the help of active transport mechanisms. Using confocal microscopy imaging and analysis of nuclear protein extracts, we show that NF-kappaB movement through the cytoplasm to the nucleus is independent of the cytoskeleton, in the three cell lines investigated here. Additionally we demonstrate that NF-kappaB p65 is not associated with the dynein/dynactin molecular motor complex. We propose that cells utilize two distinct mechanisms of NF-kappaB transport: (1) signaling via diffusion over short distances in non-neuronal cells and (2) transport via motor proteins that move along the cytoskeleton in neuronal processes where the distances between sites of NF-kappaB activation and nucleus can be vast.

Biochemical characterization of serine transport in Leishmania (Leishmania) amazonensis

In addition to its role as a protein component in Leishmania, serine is also a precursor for the synthesis of both phosphatidylserine, which is a membrane molecule involved in parasite invasion and inactivation of macrophages, and sphingolipids, which are necessary for Leishmania to differentiate into its infective forms. We have characterized serine uptake in both promastigote and amastigote forms of Leishmania (Leishmania) amazonensis. In promastigotes, kinetic data show a single, saturable transport system, with a Km of 0.253 +/- 0.01 mM and a maximum velocity of 0.246 +/- 0.04 nmol/min per 107 cells. Serine transport increased linearly with temperature in the range from 20 degrees C to 45 degrees C, allowing the calculation of an activation energy of 7.09 kJ/mol. Alanine, cysteine, glycine, threonine, valine and ethanolamine competed with the substrate at a ten-fold excess concentration. Serine uptake was dependent on pH, with an optimum activity at pH 7.5. The characterization of the serine transport process in amastigotes revealed a transport system with a similar Km, energy of activation and pH response to that found in promastigotes, suggesting that the same transport system is active in both insect vector and mammalian host Leishmania stages. This could constitute an evolutionary mechanism that guarantees the provision of such an essential molecule during host change events, such as differentiation into amastigotes and macrophage invasion, as well as to ensure that the parasite maintains the infection in the mammalian host. (C) 2008 Elsevier B.V. All rights reserved.

Active Transport of Glutamate in Leishmania (Leishmania) amazonensis

Leishmania spp. are the causative agents of leishmaniasis, a complex of diseases with a broad spectrum of clinical manifestations. Leishmania (Leishmania) amazonensis is a main etiological agent of diffuse cutaneous leishmaniasis. Leishmania spp., as other trypanosomatids, possess a metabolism based significantly on the consumption of amino acids. However, the transport of amino acids in these organisms remains poorly understood with few exceptions. Glutamate transport is an important biological process in many organisms. In the present work, the transport of glutamate is characterized. This process is performed by a single kinetic system (K-m=0.59 +/- 0.04 mM, V-max=0.123 +/- 0.003 nmol/min per 20 x 10(6) cells) showing an energy of activation of 52.38 +/- 4.7 kJ/mol and was shown to be partially inhibited by analogues, such as glutamine, aspartate, alpha-ketoglutarate and oxaloacetate, methionine, and alanine. The transport activity was sensitive to the extracellular concentration of H+ but not to Na+ or K+. However, unlike other amino acid transporters presently characterized, the treatment with specific ionophores confirmed the participation of a K+, and not H+ membrane gradient in the transport process.

(65)Zn(2+) transport by isolated gill epithelial cells of the American lobster, Homarus americanus

Gills are the first site of impact by metal ions in contaminated waters. Work on whole gill cells and metal uptake has not been reported before in crustaceans. In this study, gill filaments of the American lobster, Homarus americanus, were dissociated in physiological saline and separated into several cell types on a 30, 40, 50, and 80% sucrose gradient. Cells from each sucrose solution were separately resuspended in physiological saline and incubated in (65)Zn(2+) in order to assess the nature of metal uptake by each cell type. Characteristics of zinc accumulation by each kind of cell were investigated in the presence and absence of 10 mM calcium, variable NaCl concentrations and pH values, and 100 mu M verapamil, nifedipine, and the calcium ionophore A23187. (65)Zn(2+) influxes were hyperbolic functions of zinc concentration (1-1,000 mu M) and followed Michaelis-Menten kinetics. Calcium reduced both apparent zinc binding affinity (K (m)) and maximal transport velocity (J (max)) for 30% sucrose cells, but doubled the apparent maximal transport velocity for 80% sucrose cells. Results suggest that calcium, sodium, and protons enter gill epithelial cells by an endogenous broad-specificity cation channel and trans-stimulate metal uptake by a plasma membrane carrier system. Differences in zinc transport observed between gill epithelial cell types appear related to apparent affinity differences of the transporters in each kind of cell. Low affinity cells from 30% sucrose were inhibited by calcium, while high affinity cells from 80% sucrose were stimulated. (65)Zn(2+) transport was also studied by isolated, intact, gill filament tips. These intact gill fragments generally displayed the same transport properties as did cells from 80% sucrose and provided support for metal uptake processes being an apical phenomenon. A working model for zinc transport by lobster gill cells is presented.

Transepithelial transport of glucose and mRNA of glucose transporters in the small intestine of rats with iron-deficiency anemia

Objective: To evaluate the transepithelial transport of sodium, glucose, potassium, and water and the mRNA level of the sodium-glucose cotransporter (SGLT1) and the facilitated sugar transporter (GLUT2) in the small intestine of iron-deficient rats. Methods: After 6 wk of receiving diets with low or normal iron content, rats (Wistar-EPM) were subjected to two experiments: 1) evaluation of the transepithelial transport of sodium, glucose, potassium, and water by an ""in vivo"" experimental model of intestinal perfusion and 2) determination of relative SGLT1 and GLUT2 mRNA levels in the proximal, intermediate, and distal portions of the small intestine by the northern blotting technique. Results: Hemoglobin and hepatic iron levels were statistically lower in the anemic rats. The mean transepithelial transports of sodium (-33.0 mu Eq . min(-1) . cm(-1)), glucose (426.0 mu M . min(-1) . cm(-1)), and water (0.4 mu L . min(-1) . cm(-1)) in the small intestine of the anemic rats were significantly lower than in the control group (349.1 mu Eq . min(-1) cm(-1), 842.6 mu M . min(-1) . cm(-1), and 4.3 mu l . min(-1) cm(-1), respectively, P < 0.05). The transepithelial transport of potassium was similar for both groups. The relative SGLT1 mRNA levels of the anemic rats in the intermediate (1.796 +/- 0.659 AU) and distal (1.901 +/- 0.766 AU) segments were significantly higher than the values for the control rats (intermediate 1.262 +/- 0.450 AU, distal 1.244 +/- 0.407 AU). No significant difference was observed for the relative SLGT1 mRNA levels in the proximal segment or for the GLUT2 mRNA levels in all segments. Conclusion: Iron deficiency decreases the absorption of glucose, sodium, and water and increases SGLT1 mRNA in the intermediate and distal segments of the small intestine of rats. (C) 2011 Elsevier Inc. All rights reserved.