Membrane-related processes and overall energy metabolism inTrypanosoma brucei and other kinetoplastid species

An electrochemical proton gradient exists across the plasma membrane and the mitochondrial membrane of the bloodstream form ofTrypanosoma brucei. The membrane potential across the plasma membrane and the regulation of the internal pH depend on the temperature.Leishmania donovani regulates its internal pH and maintains a constant electrochemical proton gradient across its plasma membrane under all conditions examined. The mitochondrion of theT. brucei bloodstream form is energized, even though the reactions taking place in it do not result in net ATP synthesis and the Kreb's cycle and the respiratory chain are absent. Glucose is transported across the plasma membrane ofT. brucei by a facilitated diffusion carrier, that can transport a wider range of substrates than its mammalian counterparts. Pyruvate exits the cell via a facilitated diffusion transporter as well. Conflicting evidence exists for the mechanism of glucose transport inL. donovani; biochemical evidence suggests proton/glucose symport, while facilitated diffusion is indicated by physiological data.     

Plasma-membrane rosettes in root hairs of Equisetum hyemale

Particle arrangement in the plasma membrane during cell wall formation was investigated by means of the double-replica technique in root hairs of Equisetum hyemale. Particle density in the protoplasmic fracture face of the plasma membrane was higher than in the extraplasmic fracture face. Apart from randomly distributed particles, particle rosettes were visible in the PF face of the plasma membrane. The rosettes consisted of six particles arranged in a circle and had an outer diameter of approx. 26 nm. No gradient in the number of rosettes was found, which agrees with micrifibril deposition taking place over the whole hair. The particle rosettes were found individually, which might indicate that they spin out thin microfibrils as found in higher-plant cell walls. Indeed microfibril width in these walls, measured in shadowed preparations, is 8.5±1.5 nm. It is suggested that the rosettes are involved in microfibril synthesis. Non-turgid cells lacked microfibril imprints in the plasma membrane and no particle rosettes were present on their PF face. Fixation with glutaraldehyde caused, probably as a result of plasmolysis, the microfibril imprints to disappear together with the particle rosettes. The PF face of the plasma membrane of non-turgid hairs sometimes showed domains in which the intramembrane particles were aggregated in a hexagonal pattern. Microfibril orientation during deposition will be discussed.Abbreviations EF extraplasmic fracture face - PF protoplasmic fracture face     

Characterization of amino-acid transport in Ricinus communis roots using isolated membrane vesicles

The mechanism and specificity of amino-acid transport at the plasma membrane of Ricinus communis L. roots was investigated using membrane vesicles isolated by phase partitioning. The transport of glutamine, isoleucine, glutamic acid and aspartic acid was driven by both a pH gradient and a membrane potential (internally alkaline and negative), created artificially across the plasma membrane. This is consistent with transport via a proton symport. In contrast, the transport of the basic amino acids, lysine and arginine, was driven by a negative internal membrane potential but not by a pH gradient, suggesting that these amino acids may be taken up via a voltage-driven uniport. The energized uptake of all of the amino acids tested showed a saturable phase, consistent with carrier-mediated transport. In addition, the membrane-potential-driven transport of all the amino acids was greater at pH 5.5 than at pH 7.5, which suggests that there could be a direct pH effect on the carrier. Several amino-acid carriers could be resolved, based on competition studies: a carrier with a high affinity for a range of neutral amino acids (apart from asparagine) but with a low affinity for basic and acidic amino acids; a carrier which has a high affinity for a range of neutral amino acids except isoleucine and valine, but with a low affinity for basic and acidic amino acids; and a carrier which has a higher affinity for basic and some neutral amino acids but has a lower affinity for acidic amino acids. The existence of a separate carrier for acidic amino acids is discussed.Abbreviations PM plasma membrane - TPP⁺ tetraphenylphosphonium ion - pH pH gradient - membrane potential This work was supported by the Agricultural and Food Research Council and The Royal Society. We would like to thank Mrs. Sue Nelson for help with some of the membrane preparations.     

Comparison of calcium release from sarcoplasmic reticulum of slow and fast twitch muscles

Summary It is shown that equations developed to analyze the contributions of secondary active transport processes to symmetrical cells (Gordon, L. G. M., Macknight, A. D. C., 1991,J. Membrane Biol. 120: 139–152) can be used, with minor modifications, to analyze the steady-state membrane potential in epithelia under the unique situation of short circuiting. Only under such conditions is there a single intracellular potential relative to both the mucosal and serosal media. The equations are investigated in relation to a model tight epithelium—the toad urinary bladder. It is shown that the properties of the membrane transport pathways are such that the intracellular potential under short-circuit conditions must be more negative than often reported. Given measurements of membrane potential and of voltage-divider ratio, it is possible to use the equations to estimate the absolute values of the membrane permeabilities and conductances under shortcircuit conditions.     

MSL1 is a mechanosensitive ion channel that dissipates mitochondrial membrane potential and maintains redox homeostasis in mitochondria during abiotic stress

Mitochondria must maintain tight control over the electrochemical gradient across their inner membrane to allow ATP synthesis while maintaining a redox‐balanced electron transport chain and avoiding excessive reactive oxygen species production. However, there is a scarcity of knowledge about the ion transporters in the inner mitochondrial membrane that contribute to control of membrane potential. We show that loss of MSL1, a member of a family of mechanosensitive ion channels related to the bacterial channel MscS, leads to increased membrane potential of Arabidopsis mitochondria under specific bioenergetic states. We demonstrate that MSL1 localises to the inner mitochondrial membrane. When expressed in Escherichia coli, MSL1 forms a stretch‐activated ion channel with a slight preference for anions and provides protection against hypo‐osmotic shock. In contrast, loss of MSL1 in Arabidopsis did not prevent swelling of isolated mitochondria in hypo‐osmotic conditions. Instead, our data suggest that ion transport by MSL1 leads to dissipation of mitochondrial membrane potential when it becomes too high. The importance of MSL1 function was demonstrated by the observation of a higher oxidation state of the mitochondrial glutathione pool in msl1‐1 mutants under moderate heat‐ and heavy‐metal‐stress. Furthermore, we show that MSL1 function is not directly implicated in mitochondrial membrane potential pulsing, but is complementary and appears to be important under similar conditions.     

Proton-coupled organic cation transport in renal brush-border membrane vesicles

We had previously proposed that organic cations are transported across the brush-border membrane in the canine kidney by a H⁺ exchange (or antiport) system (Holohan, P.D. and Ross, C.R. (1981) J. Pharmacol. Exp. Ther. 216, 294–298). In the present report, we demonstrate that in brush-border membrane vesicles the transport of organic cations is chemically coupled to the countertransport of protons, by showing that the uphill or concentrative transport of a prototypic organic cation, N¹-methylnicotinamide (NMN), is chemically coupled to the flow of protons down their chemical gradient. In a reciprocal manner, the concentrative transport of protons is coupled to the counterflow of organic cations down their concentration gradient. The transport of organic cations is monitored by measuring [³H]NMN while the transport of protons is monitored by measuring changes in acridine orange absorbance. The functional significance of the coupling is that a proton gradient lowers the K_m and increases the V_max for NMN transport.     

Nitrogen transfer in the interface between the symbionts in pea root nodules

Transport mechanisms for transfer of nitrogen from the bacteroid side across the symbiosome membrane of pea (Pisum sativum L.) root nodules were identified by the use of energised bacteroid side-out symbiosome membrane vesicles. Such membrane vesicles were used to study a mechanism with high capacity for transport of ammonium and another mechanism capable of transporting aspartate. Both transport mechanisms are voltage driven and the rate of transport relates positively to the magnitude of the imposed membrane potentials. Competition for transport between ammonium and aspartate was not observed. The ammonium transporter has been identified as a voltage-driven channel whereas the symbiosome membrane aspartate transporter appears to be a H⁺/aspartate symport. The results suggest that nitrogen transfer between the symbionts in pea root nodules involves transfer of amino acids as well as ammonium. In the symbiosome subfraction, which represents the interface between the symbionts, specific aspartate aminotransferase activity was more than four times as high as in the bacteroid cytosol. This finding supports a hypothesis that transamination cycles operating between the symbionts may constitute a component of the transfer of nitrogen between the symbionts.     

Developmental features of calcium oxalate crystal sand deposition inBeta vulgaris L. Leaves

Summary Sugar beet (Beta vulgaris L.) leaf has a layer of cells extended laterally between the palisade parenchyma and spongy mesophyll that develop numerous small crystals (crystal sand) within their vacuoles. Solubility studies and histochemical staining indicate the crystals are calcium oxalate. The crystals are deposited within the vacuoles early during leaf development, and at maturity the cells are roughly spherical in shape and 2 to 3 times larger than other mesophyll cells. Crystal deposition is preceeded by formation of membrane vesicles within the vacuole. The membranes are synthesizedde novo in the vacuole and have a typical trilaminate structure as viewed with the TEM. The membranes are formed within paracrystalline aggregates of tubular particles (6–8nm outer diameter) as membrane sheets, but are later organized into chambers or vesicles. Calcium oxalate is then precipitated within the membrane chambers. The tubular particles involved in membrane synthesis are usually present in the vacuoles of mature crystal cells, but in very small amounts.     

Involvement of ascorbic acid and ab-type cytochrome in plant plasma membrane redox reactions

Summary Higher plant plasma membranes contain ab-type cytochrome that is rapidly reduced by ascorbic acid. The affinity towards ascorbate is 0.37 mM and is very similar to that of the chromaffin granule cytochromeb ₅₆₁. High levels of cytochromeb reduction are reached when ascorbic acid is added either on the cytoplasmic or cell wall side of purified plasma membrane vesicles. This result points to a transmembrane organisation of the heme protein or alternatively indicates the presence of an effective ascorbate transport system. Plasma membrane vesicles loaded by ascorbic acid are capable of reducing extravesicular ferricyanide. Addition of ascorbate oxidase or washing of the vesicles does not eliminate this reaction, indicating the involvement of the intravesicular electron donor. Absorbance changes of the cytochromeb -band suggest the electron transfer is mediated by this redox component. Electron transport to ferricyanide also results in the generation of a membrane potential gradient as was demonstrated by using the charge-sensitive optical probe oxonol VI. Addition of ascorbate oxidase and ascorbate to the vesicles loaded with ascorbate results in the oxidation and subsequent re-reduction of the cytochromeb. It is therefore suggested that ascorbate free radical (AFR) could potentially act as an electron acceptor to the cytochrome-mediated electron transport reaction. A working model on the action of the cytochrome as an electron carrier between cytoplasmic and apoplastic ascorbate is discussed.Abbreviations AFR ascorbate free radical - AO ascorbate oxidase - DTT dithiothreitol - FCCP carbonylcyanidep-trifluorome-thoxyphenylhydrazon - Hepes N-(2-hydroxyethyl)-piperazine-N-(2-ethanesulfonic acid) - Oxonol VI bis(3-propyl-5-oxoisoxazol-4-yl) penthamethine oxonol - PMSF phenylmethylsulfluoride     

Leucine Transport Is Affected by Bacillus thuringiensis Cry1 Toxins in Brush Border Membrane Vesicles from Ostrinia nubilalis Hb (Lepidoptera: Pyralidae) and Sesamia nonagrioides Lefebvre (Lepidoptera: Noctuidae) Midgut

The pore-forming activity of Cry1Ab, Cry1Fa and Cry1Ca toxins and their interaction with leucine transport mediated by the K⁺/leucine cotransporter were studied in brush border membrane vesicles (BBMVs) isolated from the midgut of Ostrinia nubilalis and Sesamia nonagrioides. In both species, as in other Lepidoptera, leucine uptake by BBMVs can take place in the absence of cations, but it can also be driven by a K⁺ gradient. Experiments with the voltage-sensitive fluorescent dye 3,3′-diethylthiacarbocyanine iodide proved that Cry1Ab, a Bacillus thuringiensis toxin active in vivo, enhanced the membrane permeability to potassium in O. nubilalis BBMVs. This result is in agreement with similar effects observed in S. nonagrioides BBMV incubated with various Cry1 toxins active in vivo. The effect of the above toxins was tested on the initial rate of 0.1 mM leucine influx. Instead of an increase in leucine influx, a reduction was observed with the Cry1 toxins active in vivo. Cry1Ab and Cry1Fa, but not the inactive toxin Cry1Da, inhibited in a dose-dependent manner leucine uptake both in the absence and in the presence of a K⁺ gradient, a clear indication that their effect is independent of the channel formed by the toxins and that this effect is exerted directly on the amino acid transport system.     

Protamine alters structure and conductance ofNecturus gallbladder tight junctions without major electrical effects on the apical cell membrane

Summary Protamine is a naturally occurring basic protein (pI; 9.7 to 12.0). We have recently reported that protamine dissolved in the mucosal bath (2 to 20 m), induces about a twofold increase in transepithelial resistance inNecturus gallbladder within 10 min. Conductance decreased concomitantly with cation selectivity.In this leaky epithelium, where >90% of an applied current passes between cells, an increment in resistance of this magnitude suggests a paracellular actiona priori. To confirm this, ionic conductance across the apical cell membrane was studied with microelectrodes. Protamine increased transepithelial resistance without changing apical cell membrane voltage or fractional membrane resistance. Variation in extracellular K concentration (6 to 50mm) caused changes in apical membrane voltage not different from control.To determine if protamine-induced resistance changes were associated with structural alteration of tight junctions, gallbladders were fixedin situ at peak response and analyzed by freeze-fracture electron microscopy. According to a morphometrical analysis, the tight junctional intramembranous domain expands vertically due to incorporation of new strands (fibrils) into the main compact fibrillar meshwork.Since morphologic changes are complete within 10 min, strands are probably recycled into and out of the tight junctional membrane domain possibly by the cytoskeleton either from cytoplasmic vesicles or from intramembranous precursors. Regulation of tight junctional permeability by protamine and other perturbations may constitute a common mechanism by which leaky epithelia regulate transport, and protamine, in concentrations employed in this study, seems reasonably specific for the tight junction.     

Endocytic transit inDictyostelium discoideum

Summary The cells ofDictyostelium discoideum are soil amoebae with a simple endocytic pathway: Particles or fluid are taken up at the plasma membrane in a process dependent on the actin cytoskeleton. After rapid acidification and subsequent neutralisation of the food vacuoles during which breakdown of the contents occurs, indigestible remnants are exocytosed. This tight coupling between endocytosis and exocytosis is thought to maintain membrane homeostasis. In spite of the apparent overall difference between the endocytic pathways of mammalian cells andD. discoideum, conserved proteins are involved in individual steps of endocytic transport, possibly indicating that in mammalian cells it is only the routing of marker that has evolved from a simple transit to a complex, branched pathway.     

Periplaststruktur und Organisation der Plasmamembran vonRhodomonas spec. (Cryptophyceae)

Summary The periplast of the CryptophyceaeRhodomonas has a hexagonal substructure. This substructure is caused by periplast plates. In freeze fracture replicas of the plasma membrane, there are corresponding hexagonal areas with numerous particles. These areas are separated by regions with less particles. Aggregates of particles, partly rosette-like, indicate insertion sites of ejectisomes.

     

Effect of growth factors and antitumor drugs on normal and Vall2Ha-ras transformed C3H 10T1/2 cell transplasma membrane electron transport and growth

Summary Bothras and the transplasma membrane electron transport system have been implicated in the control of cell growth. Since the loss of growth regulation is at the heart of the cancerous phenotype, we have investigated the effect of factors like antitumor drugs, which inhibit cell growth, and growth factors which either cause or prepare the cells for growth. We have found that growth factors increase the plasma membrane electron transport of the normal cells but not that of the cells which have been transfected with oncogenicras (which have a 2–5-fold faster basal rate). In the presence of adriamycin the activity of the electron transport system is and cell numbers are decreased more in normal cells than in the cells which have been transfected with the oncogenic version of the Harveyras gene. Cisplatin inhibits cell growth but does not inhibit ferricyanide reduction. Ferricyanide alone or in conjunction with EGF or TPA does not stimulate cell proliferation with either cell line in the absence of fetal calf serum, so simple activation of plasma membrane electron transport is not sufficient to activate a complete growth response.Abbreviations EGF epidermal growth factor - TPA phorbol myristate acetate - PDGF platelet derived growth factor - MTT 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromides - SDS sodium dodecyl sulfate     

Cytochemistry and freeze-fracture of membranes isolated from the electrocyte of Electrophorus electricus (L.)

Summary Membranes were isolated from the main electric organ of Electrophorus electricus and studied by means of cytochemistry and freezefracture. The membrane fractions consisted of vesicles inside-in as determined by localization of anionic sites using colloidal iron and cationized ferritin particles. The anionic sites were not homogeneously distributed on the surface of the vesicle. Freeze-fracture showed the presence of intramembranous particles associated with either protoplasmic (P) or extracellular (E) faces of the membrane. Regions of the membrane without particles were observed. The results are discussed in relation to the existence of association between intramembranous particles and membrane receptors.For all correspondence     

Unusual features of intercellular junctions in the larvacean Oikopleura dioica

Summary In the pelagic larvacean Oikopleura dioica, the epithelium lining the alimentary tract consists of ciliated and unciliated cell types. The ciliated cells also exhibit an apical border of long microvilli. Between the microvilli, the cellular membrane often projects deeply down into the cytoplasm; the membranes of these invaginations and those of apicolateral interdigitations may be associated with one another by tight junctions. Some of these junctions may be autocellular. The tight junctions are seen by freeze-fracture to be very simple in construction, composed of a single row of intramembranous particles, which may be fused into a P-face ridge. There is a dense cytoplasmic fuzz associated with these tight junctions which may extend into adjoining zonula adhaerens-like regions. The invaginations of the apical membranes are, in addition, associated by gap junctions which may also be autocellular. More conventional homocellular and heterocellular tight and gap junctions occur along the lateral borders of ciliated cells and between ciliated and unciliated cells. These gap junctions possess a reduced intercellular cleft and typical P-face connexons arranged in macular plaques, with complementary E-face pits. Both cell types exhibit extensive stacks of basal and lateral interdigitations. The tight junctions found here are unusual in that they are associated with a dense cytoplasmic fuzz which is normally more characteristic of zonulae adhaerentes.     

Oxytalan fibres in the epidermal basement membrane of Lineus atrocaeruleus (Nemertia)

The basement membrane of the epidermis in Lineus atrocaeruleus (Schmarda 1859) (Nemertia: Heteronemertea) is formed by a tight network of oxytalan fibres which send projections into the epidermis and branch towards the proximal dermis, in close association with the subepidermal musculature. The histophysiology of this relationship is discussed.     

Glucose transport into the extremely halophilic archaebacteria

Penetration of glucose into cells of several extremely halophilic archaebacteria of the Halobacterium and Haloferax genera (Halobacterium saccharovorum and Halobacterium salinarium, Haloferax volcanii and Haloferax mediterranei) has been studied. Some characteristics of transport systems of carbohydrate-utilizing halobacteria Halobacterium saccharovorum, Haloferax mediterranei and Haloferax volcanii (pH and temperature optima, stereospecificity, kinetic parameters) have been determined. Inability of H. salinarium cells for active glucose transport has been shown. The dependence of glucose transport on the Na⁺ ions gradient (on the whole cells and membrane vesicles) has been demonstrated. Cells or membrane vesicles of carbohydrate-utilizing halobacteria grown in media containing this sugar indicated the activation of glucose transport, whereas cells grown in media without sugars did not. This fact has allowed us to conclude that corresponding transport systems are inducible.     

Transport of iron across the outer membrane

Summary The TonB protein is involved in energy-coupled receptor-dependent transport processes across the outer membrane. The TonB protein is anchored in the cytoplasmic membrane but exposed to the periplasmic space. To fulfill its function, it has to couple the energy-providing metabolism in the cytoplasmic membrane with regulation of outer membrane receptor activity. Ferrichrome and albomycin transport, uptake of colicin M, and infection by the phages T1 and80 occur via the same receptor, the FhuA protein in the outer membrane. Therefore, this receptor is particularly suitable for the study of energy-coupled TonB-dependent transport across the outer membrane. Ferrichrome, albomycin and colicin M bind to the FhuA receptor but are not released into the periplasmic space of unenergized cells, ortonB mutants. In vivo interaction between FhuA and TonB is suggested by the restoration of activity of inactive FhuA proteins, bearing amino acid replacements in the TonB box, by TonB derivatives with single amino acid substitutions. Point mutations in thefhuA gene are suppressed by point mutations in thetonB gene. In addition, naturally occurring degradation of the TonB protein and its derivatives is preferentially prevented in vivo by FhuA and FhuA derivatives where functional interaction takes place. It is proposed that in the energized state, TonB induces a conformation in FhuA which leads to the release of the FhuA-bound compounds into the periplasmic space. Activation of FhuA by TonB depends on the ExbBD proteins in the cytoplasmic membrane. They can be partially replaced by the TolQR proteins which show strong sequence similarity to the ExbBD proteins. A physical interaction of these proteins with the TonB protein is suggested by TonB stabilization through ExbB and TolQR. We propose a permanent or reversible complex in the cytoplasmic membrane composed of the TonB protein and the ExbBD/TolQR proteins through which TonB is energized.