Spheroplast-derived membrane vesicles from Rhodobacter capsulatus cells catalyzing nucleotide transport

Rodobacter capsulatus cells, which were cultured anaerobically in high light intensity, had fewer foldings in the cytoplasmic membrane than those which were grown in lower light intensities. Spheroplast-derived membrane fractions obtained from cells cultured under high light intensity contained a high yield of large right-side-out membrane vesicles. The right-side-out vesicles catalyzed reversible light-induced proton efflux as did intact cells. Nucleotide transport activity was also catalyzed by these membrane vesicles. This activity was indirectly monitored by measurement of photophosphorylation or hydrolysis of externally added diphospho- and triphosphonucleosides. These enzymatic activities occur inside the cytoplasmic membrane of spheroplasts and membrane vesicles and therefore require the transport of the externally added reagents. The indirect measurements of transport were complemented by the demonstration of direct uptake of radiolabeled nucleotides into the membrane vesicles. These data support the suggestion that a nucleotide transporter located in the cytoplasmic membrane of R. capsulatus bacteria mediates these activities.     

Nucleotide exchange in membrane vesicles from the photosynthetic bacterium Rhodopseudomonas capsulata

Membrane vesicles (heavy chromatophores) prepared from the photosynthetic bacteria Rhodopseudomonas capsulata catalyze photophosphorylation of exogenous ADP and also take up [³H]ADP from the external medium. The rate of uptake depends on the concentration of external ADP reaching half-maximal velocity at 2.7 mm. The rate increases also with the increase in the concentration of internal ADP. Vesicles, preloaded with [³H]ADP release the radioactive nucleotide when ADP is included in the external medium. Regular chromatophores, which are inside-out membrane vesicles also take up [³H]ADP from the external medium when preloaded with ADP. These results are interpreted to indicate the existence of nucleotide transport across the cytoplasmic membrane of these bacteria which is catalyzed by an ADP exchange carrier.     

Effects of advanced glycation end-products on the proliferation and differentiation of osteoblast-like cells

Dextran M20 was added to isolated rat liver mitochondria to mimic cytosolic macromolecules. Under these conditions, the morphological changes in the mitochondrial periphery that occur upon isolation of the organelle are restored, i.e. the volume of the intermembrane space decreases and the contact site frequency increases. The ADP routing from mitochondrial kinases at various locations was investigated by using the activities of oxidative phosphorylation and externally added pyruvate kinase as sensors for ADP transport into the matrix and extramitochondrial compartment, respectively. The studies reveal that a significant fraction of the ADP generated by either adenylate kinase in the intermembrane space or by outer membrane bound hexokinase isozyme I, is not accessible to extramitochondrial pyruvate kinase. Quantitative information on the ADP compartmentation in rat liver mitochondria was obtained by comparing the ADP supply from mitochondrial kinases to oxidative phosphorylation with that of non-bound, extramitochondrially located kinases. This approach allowed us to estimate the ADP diffusion gradients which were present across the outer membrane and between the compartment formed by bound hexokinase and the extramitochondrial compartment. In the presence of 10% dextran M20 these ADP gradients amounted to approximately 12 µM. The possible role of mitochondrial kinases in ADP transport into mitochondria in vivo is discussed. (Mol Cell Biochem 174: 43–51, 1997)     

Control of energy transformation of mitochondria. Analysis by a quantitative model

A mathematical model of control of energy transformation in mitochondria is presented. The considered processes are: the proton translocation by the respiratory chain, the production of ATP by ATPase, the translocation of adenine nucleotides and of phosphate by their translocators, and a passive backflow of protons through the mitochondrial membrane. The mathematical equations expressing the steady-state kinetics of these processes and the relations between them were derived on the basis of current experimental data. The model predicts fairly well the values of the proton electrochemical gradient, of the ATP/ADP ratios within and outside mitochondria and of the distribution of phosphate between both compartments in different metabolic states of mitochondria. From the general agreement of model computations with experimental data, it is suggested that the electron flux through the respiratory chain is immediately controlled by the energy back-pressure of the proton electrochemical gradient, that the ATPase reaction is near equilibrium in phosphorylating mitochondria but that the adenine nucleotide exchange across the mitochondrial membrane requires some loss of energy. The latter is caused by an inhibition of the translocator by ATP from the outer side or by ADP from the inner side depending on the actual ATP/ADP in both compartments. It explains that no fixed relation exists between the rate of respiration and the phosphorylation state of extramitochondrial adenine nucleotides. The relation is modified by the concentration of phosphate and by intramitochondrial energy utilization.     

Tightly bound nucleotides of the energy-transducing ATPase of chloroplasts and their role in photophosphorylation.

1. Like other energy-transducing membranes, chloroplast membranes bear a coupling ATPase with especially tight binding sites for adenine nucleotides. Membranes washed several times still contain 2.5 nmol ATP and 1.3 nmol ADP bound per mg chlorophyll, which is equivalent to 1.9 ATP and 1.0 ADP per coupling ATPase. 2. In de-energized membranes, these nucleotides exchange to only a limited extent with added nucleotides. In membranes illuminated in the presence of pyocyanine, however, complete exchange of the bound nucleotides occurs rapidly, irrespective of whether ATP or ADP is present in the medium. 3. Pi can exchange into these nucleotided at both the beta and gamma positions when the membranes are energized in the presence of Mg-2+. Equilibrium with the beta and gamma groups of th ebound nucleotides is, however, not complete. 4. The inhibitors and uncouplers Dio-9, S13 and EDTA have different effects on the exchange of nucleotides, the exchange of inorganic phosphate and photophosphorylation. 5. The bound ATP level on the membrane is stable to a wide variety of conditions. The ADP level, however, drops to near zero under conditions of maximal activation of the emmbrane ATPase.     

TonB-dependent receptors—structural perspectives

Plants, bacteria, fungi, and yeast utilize organic iron chelators (siderophores) to establish commensal and pathogenic relationships with hosts and to survive as free-living organisms. In Gram-negative bacteria, transport of siderophores into the periplasm is mediated by TonB-dependent receptors. A complex of three membrane-spanning proteins TonB, ExbB and ExbD couples the chemiosmotic potential of the cytoplasmic membrane with siderophore uptake across the outer membrane. The crystallographic structures of two TonB-dependent receptors (FhuA and FepA) have recently been determined. These outer membrane transporters show a novel fold consisting of two domains. A 22-stranded antiparallel β-barrel traverses the outer membrane and adjacent β-strands are connected by extracellular loops and periplasmic turns. Located inside the β-barrel is the plug domain, composed primarily of a mixed four-stranded β-sheet and a series of interspersed α-helices. Siderophore binding induces distinct local and allosteric transitions that establish the structural basis of signal transduction across the outer membrane and suggest a transport mechanism.     

Exchange of the actin-bound nucleotide in intact arterial smooth muscle

The actin-bound ADP was separated from cytoplasmic nucleotides by treatment of intact arterial smooth muscle with 50% ethanol. In (32)P-labeled smooth muscle the actin-bound ADP and phosphate readily exchanged with the cytoplasmic [gamma,beta-(32)P]ATP; the specific radioactivity of actin-bound ADP was equal to that of the beta-phosphate of cytoplasmic ATP and the specific radioactivity of actin-bound phosphate was equal to that of the gamma-phosphate of cytoplasmic ATP. In contrast, the exchange of the actin-bound ADP in skeletal muscle was very slow. The presence of cytoplasmic ATP was required for the exchange of the actin-bound ADP and phosphate; if ATP synthesis was inhibited the exchange was also inhibited. The extent of exchange was reduced in muscles contracted by histamine or K(+), as compared with resting muscles. The exchange was also shown in other mammalian smooth muscles, uterus, urinary bladder, and stomach. The data indicate a dynamic state of actin in smooth muscle. The data also suggest that polymerization-depolymerization of actin is part of the contraction-relaxation cycle of smooth muscle.     

Tightly bound nucleotides of the energy-transducing ATPase, and their role in oxidative phosphorylation. I. The Paracoccus denitrificans system.

1. The coupling ATPase of Paracoccus denitrificans can be removed from the membrane by washing coupled membrane fragments at low salt concentrations. 2. This ATPase resembles coupling ATPases of mitochondria, chloroplasts and other bacteria. It is a negatively charged protein of molecular weight about 300,000. An inhibitor protein in bound tightly to the ATPase in vivo, and can be destroyed by trypsin treatment. 3. ATP and ADP are found tightly bound to the coupling ATPase of P. denitrificans, both in its membrane-bound and isolated state. The ATP/ADP ratio on the enzyme is greater than one. 4. Under de-energised condtions, the bound nucleotides are not available to the suspending medium. When the membrane is energised however, the bound nucleotides can exchange with added nucleotides and incorporate 32Pi. 32Ppi is incorporated into the beta and gamma positions of the bound nucleotides, but beta-labelling probably does not occur on the coupling ATPase. 5. Uncouplers inhibit the exchange of the free nucleotides or 32Pi into the bound nucleotides, while venturicidin (an energy transfer inhibitor) and aurovertin stimulate the exchange. 6. The response of the bound nucleotides to energisation is consistent with their being involved directly in the mechanism of oxidative phosphorylation.     

Function of the mitochondrial outer membrane as a diffusion barrier in health and diseases

The mitochondrial outer membrane separates the intermembrane space from the cytosol. The whole exchange of metabolites, cations and information between mitochondria and the cell occurs through the outer membrane. Experimental evidence is reviewed supporting the hypothesis of dynamic ADP compartmentation within the intermembrane space. The outer membrane creates a diffusion barrier for small molecules (adenine nucleotides, creatine phosphate, creatine etc.) causing rate-dependent concentration gradients as a prerequisite for the action of ADP shuttles via creatine kinases or adenylate kinases. If the outer membrane becomes leaky, cytochrome c and apoptosis-inducing factor can be released, leading to apoptosis, and as a bioenergetic consequence the cytosolic phosphorylation potential decreases. Leaky outer membranes can be detected in saponin-skinned fibres with spectrophotometric and oxygraphic methods. This is of special interest in respect to acute impairment of mitochondria during ischaemia/reperfusion.     

Bcl-xL promotes the open configuration of the voltage-dependent anion channel and metabolite passage through the outer mitochondrial membrane

The diffusion of metabolites across the outer mitochondrial membrane is essential for coupled cellular respiration. The outer membrane of mitochondria isolated from growth factor-deprived cells is impaired in its ability to exchange metabolic anions. When added to mitochondria, recombinant Bcl-x(L) restores metabolite exchange across the outer membrane without inducing the loss of cytochrome c from the intermembrane space. Restoration of outer membrane permeability to anionic metabolites does not occur directly through Bcl-x(L) ion channels. Instead, recombinant Bcl-x(L) maintains the outer mitochondrial membrane channel, VDAC, in an open configuration. Consistent with these findings, when ADP-induced oxidative phosphorylation is limited by exogenous beta-NADH, recombinant Bcl-x(L) can sustain outer mitochondrial membrane permeability to ADP. beta-NADH limits respiration by promoting the closed configuration of VDAC. Together these results demonstrate that following an apoptotic signal, Bcl-x(L) can maintain metabolite exchange across the outer mitochondrial membrane by inhibiting VDAC closure.     

High phosphate requirement for oxidative phosphorylation and low affinity for phosphate transport in newborn rat liver mitochondria

Rat liver mitochondria are not fully functional at birth. The relationship between this deficiency and the affinity for phosphate, in oxidative phosphorylation or in phosphate transport, have been studied.The phosphate concentration necessary to observe maximal rate of succinate oxidation in the presence of ADP was higher for newborn than for adult rat liver mitochondria. After preincubation of newborn rat liver mitochondria with ATP, the rate of succinate oxidation in the presence of ADP increased with phosphate concentration similarly for newborn and adult rat liver mitochondria. The maximal rate of phosphate-acetate exchange, which is an indirect measure of the rate of phosphate transport across the mitochondrial membrane, was not significantly different for adult and newborn rat liver mitochondria. On the contrary the apparent affinity for phosphate was about ten-fold lower for newborn than for adult mitochondria.     

The influence of the cytosolic oncotic pressure on the permeability of the mitochondrial outer membrane for ADP: implications for the kinetic properties of mitochondrial creatine kinase and for ADP channelling into the intermembrane space

Summary Cytosolic proteins as components of the physiological mitochondrial environment were substituted by dextrans added to media normally used for incubation of isolated mitochondria. Under these conditions the volume of the intermembrane space decreases and the contact sites between the both mitochondrial membranes increase drastically. These morphological changes are accompanied by a reduced permeability of the mitochondrial outer compartment for adenine nucleotides as it was shown by extensive kinetic studies of mitochondrial enzymes (oxidative phosphorylation, mi-creatine kinase, mi-adenylate kinase). The decreased permeability of the mitochondrial outer membrane causes increased rate dependent concentration gradients in the micromolar range for adenine nucleotides between the intermembrane space and the extramitochondrial space. Although all metabolites crossing the outer membrane exhibit the same concentration gradients, considerable compartmentations are detectable for ADP only due to its low extramitochondrial concentration. The consequences of ADP-compartmentation in the mitochondrial intermembrane space for ADP-channelling into the mitochondria are discussed.     

High phosphate requirement for oxidative phosphorylation and low affinity for phosphate transport in newborn rat liver mitochondria

Rat liver mitochondria are not fully functional at birth. The relationship between this deficiency and the affinity for phosphate, in oxidative phosphorylation or in phosphate transport, have been studied. The phosphate concentration necessary to observe maximal rate of succinate oxidation in the presence of ADP was higher for newborn than for adult rat liver mitochondria. After preincubation of newborn rat liver mitochondria with ATP, the rate of succinate oxidation in the presence of ADP increased with phosphate concentration similarly for newborn and adult rat liver mitochondria. The maximal rate of phosphate-acetate exchange, which is an indirect measure of the rate of phosphate transport across the mitochondrial membrane, was not significantly different for adult and newborn rat liver mitochondria. On the contrary the apparent affinity for phosphate was about ten-fold lower for newborn than for adult mitochondria.     

New substrates for TonB-dependent transport: do we only see the 'tip of the iceberg'?

TonB-dependent transport is a mechanism for active uptake across the outer membrane of Gram-negative bacteria. The system promotes transport of rare nutrients and was thought to be restricted to iron complexes and vitamin B12. Recent experimental evidence of TonB-energized transport of nickel and different carbohydrates, in addition to bioinformatic-based predictions, challenges this notion and reveals that the number and variety of TonB-dependent substrates is underestimated. It is becoming clear that the chemical nature of the substrates, the energetic requirements for transport and the subsequent translocation across the cytoplasmic membrane can differ from those of the well-studied systems for iron complexes and vitamin B12. These findings question the understanding of TonB-dependent uptake and provide insights into the adaptation of bacteria to their environments.     

Adenine nucleotide translocase-dependent anion transport in pea chloroplasts

Pea chloroplasts were found to take up actively ATP and ADP and exchange the external nucleotides for internal ones. Using carrier-free [14C]ATP, the rate of nucleotide transport in chloroplasts prepared from 12-14-day-old plants was calculated to be 330 mumol ATP/g chlorophyll/min, and the transport was not affected by light or temperature between 4 and 22 degrees C. Adenine nucleotide uptake was inhibited only slightly by carboxyatractylate, whereas bongkrekic acid was nearly as effective an inhibitor of the translocator in pea chloroplasts as it was in mammalian mitochondria. There was no counter-transport of adenine nucleotides with substrates carried on the phosphate translocator including inorganic phosphate, 3-phosphoglycerate and dihydroxyacetone phosphate. However, internal or external phosphoenolpyruvate, normally considered to be transported on the phosphate carrier in chloroplasts, was able to exchange readily with adenine nucleotides. Furthermore, inorganic pyrophosphate which is not transported by the phosphate carrier initiated efflux of phosphoenolpyruvate as well as ATP from the chloroplast. These findings illustrate some interesting similarities as well as differences between the various plant phosphate and nucleotide transport systems which may relate to their role in photosynthesis.     

TonB-dependent receptors-structural perspectives

Plants, bacteria, fungi, and yeast utilize organic iron chelators (siderophores) to establish commensal and pathogenic relationships with hosts and to survive as free-living organisms. In Gram-negative bacteria, transport of siderophores into the periplasm is mediated by TonB-dependent receptors. A complex of three membrane-spanning proteins TonB, ExbB and ExbD couples the chemiosmotic potential of the cytoplasmic membrane with siderophore uptake across the outer membrane. The crystallographic structures of two TonB-dependent receptors (FhuA and FepA) have recently been determined. These outer membrane transporters show a novel fold consisting of two domains. A 22-stranded antiparallel beta-barrel traverses the outer membrane and adjacent beta-strands are connected by extracellular loops and periplasmic turns. Located inside the beta-barrel is the plug domain, composed primarily of a mixed four-stranded beta-sheet and a series of interspersed alpha-helices. Siderophore binding induces distinct local and allosteric transitions that establish the structural basis of signal transduction across the outer membrane and suggest a transport mechanism.     

Lipid trafficking to the outer membrane of Gram-negative bacteria

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.     

Recognition and transport of ferric enterobactin in Escherichia coli.

The specificity of the outer membrane protein receptor for ferric enterobactin transport in Escherichia coli and the mechanism of enterobactin-mediated transport of ferric ions across the outer membrane have been studied. Transport kinetic and inhibition studies with ferric enterobactin and synthetic structural analogs have mapped the parts of the molecule important for receptor binding. The ferric complex of the synthetic structural analog of enterobactin, 1,3,5-N,N',N'-tris-(2,3-dihydroxybenzoyl)triaminomethylbenzene (MECAM), was transported with the same maximum velocity as was ferric enterobactin. A double-label transport assay with [59Fe, 3H]MECAM showed that the ligand and the metal are transported across the outer membrane at an identical rate. Under the growth conditions used, large fractions of the transported complexes were available for exchange across the outer membrane when a large excess of extracellular complex was added to the cell suspension; at least 60% of the internalized [59Fe]enterobactin exchanged with extracellular [55Fe]enterobactin. Internalized [59Fe, 3H]MECAM was released from the cell as the intact complex when either unlabeled Fe-MECAM or Fe-enterobactin was added extracellularly. The results suggest a mechanism of active transport of unmodified coordination complex across the outer membrane with possible accumulation in the periplasm.     

Adenine nucleotide translocase-dependent anion transport in pea chloroplasts

Pea chloroplasts were found to take up actively ATP and ADP and exchange the external nucleotides for internal ones. Using carrier-free [¹⁴C]ATP, the rate of nucleotide transport in chloroplasts prepared from 12–14-day-old plants was calculated to be 330 μmol ATP/g chlorophyll/min, and the transport was not affected by light or temperature between 4 and 22°C. Adenine nucleotide uptake was inhibited only slightly by carboxyatractylate, whereas bongkrekic acid was nearly as effective an inhibitor of the translocator in pea chloroplasts as it was in mammalian mitochondria. There was no counter-transport of adenine nucleotides with substrates carried on the phosphate translocator including inorganic phosphate, 3-phosphoglycerate and dihydroxyacetone phosphate. However, internal or external phosphoenolpyruvate, normally considered to be transported on the phosphate carrier in chloroplasts, was able to exchange readily with adenine nucleotides. Furthermore, inorganic pyrophosphate which is not transported by the phosphate carrier initiated efflux of phosphoenolpyruvate as well as ATP from the chloroplast. These findings illustrate some interesting similarities as well as differences between the various plant phosphate and nucleotide transport systems which may relate to their role in photosynthesis.