Ferrous iron transport mutants in Escherichia coli K12

A ferrous iron transport system in Escherichia coli is described. Mutants in this transport system were isolated using the antibiotic streptonigrin. The gene locus feo (for ferrous iron transport) was mapped near pncA at 38.5 min on the genetic map of E. coli K12. The transport of ferrous iron was regulated by fur as the siderophore transport systems.     

Cell-free Transport to Distinct Golgi Cisternae Is Compartment Specific and ARF Independent

The small GTPase ADP-ribosylation factor (ARF) is absolutely required for coatomer vesicle formation on Golgi membranes but not for anterograde transport to the medial-Golgi in a mammalian in vitro transport system. This might indicate that the in vivo mechanism of intra-Golgi transport is not faithfully reproduced in vitro, or that intra-Golgi transport occurs by a nonvesicular mechanism. As one approach to distinguishing between these possibilities, we have characterized two additional cell-free systems that reconstitute transport to the trans-Golgi (trans assay) and trans-Golgi network (TGN assay). Like in vitro transport to the medial-Golgi (medial assay), transport to the trans-Golgi and TGN requires cytosol, ATP, and N-ethylmaleimide–sensitive fusion protein (NSF). However, each assay has its own distinct characteristics of transport. The kinetics of transport to late compartments are slower, and less cytosol is needed for guanosine-5′-O-(3-thiotriphosphate) (GTPγS) to inhibit transport, suggesting that each assay reconstitutes a distinct transport event. Depletion of ARF from cytosol abolishes vesicle formation and inhibition by GTPγS, but transport in all assays is otherwise unaffected. Purified recombinant myristoylated ARF1 restores inhibition by GTPγS, indicating that the GTP-sensitive component in all assays is ARF. We also show that asymmetry in donor and acceptor membrane properties in the medial assay is a unique feature of this assay that is unrelated to the production of vesicles. These findings demonstrate that characteristics specific to transport between different Golgi compartments are reconstituted in the cell-free system and that vesicle formation is not required for in vitro transport at any level of the stack.     

l-Aspartate Transport into Pea Chloroplasts : Kinetic and Inhibitor Evidence for Multiple Transport Systems

The kinetics of l-aspartate transport into pea chloroplasts was studied in the presence and absence of transport inhibitors to determine whether multiple aspartate carriers exist. Transport was measured by the silicone oil centrifugation technique. Reciprocal plots of concentration-dependent transport rates were biphasic, indicating the presence of two transport components, distinguishable on the basis of their affinity for aspartate. These transport components, called high affinity and low affinity transport could also be distinguished on the basis of their apparent substrate saturability and their sensitivity to media pH. The apparent K_m for high affinity transport was 30 micromolar. The K_m for low affinity transport was not determined. To test whether these transport components could also be distinguished on the basis of inhibitor sensitivity and to assess the value of inhibitors for distinguishing multiple aspartate translocators, a survey of several classes of potential inhibitors was conducted. High affinity aspartate transport was inhibited by p-chloromercuribenzenesulfonate and mersalyl, both sulfhydryl-reactive reagents; diethyl pyrocarbonate, a histidine-reactive reagent; and nigericin and carbonyl cyanide m-chlorophenylhydrazone, both ionophores. Low affinity aspartate transport was not inhibited by p-chloromercuribenzenesulfonate or nigericin, but preliminary results suggest it was sensitive to diethyl pyrocarbonate. Because the high and low affinity transport components could be distinguished not only by their sensitivity to media pH and substrate saturability, but also by their sensitivity to various inhibitors, we concluded that they may represent different transport systems or carriers.     

The Vesicle Protein SAM-4 Regulates the Processivity of Synaptic Vesicle Transport

Axonal transport of synaptic vesicles (SVs) is a KIF1A/UNC-104 mediated process critical for synapse development and maintenance yet little is known of how SV transport is regulated. Using C. elegans as an in vivo model, we identified SAM-4 as a novel conserved vesicular component regulating SV transport. Processivity, but not velocity, of SV transport was reduced in sam-4 mutants. sam-4 displayed strong genetic interactions with mutations in the cargo binding but not the motor domain of unc-104. Gain-of-function mutations in the unc-104 motor domain, identified in this study, suppress the sam-4 defects by increasing processivity of the SV transport. Genetic analyses suggest that SAM-4, SYD-2/liprin-α and the KIF1A/UNC-104 motor function in the same pathway to regulate SV transport. Our data support a model in which the SV protein SAM-4 regulates the processivity of SV transport.     

Succinate transport in Bacillus subtilis. Dependence on inorganic anions

Cations were generally ineffective in stimulating succinate transport in a succinate dehydrogenase mutant of Bacillus subtilis unless accompanied by polyvalent anions; phosphate and sulfate being particularly active. The K_m values for the phosphate or sulfate requirement were approx. 3 mM.Biphasic kinetics were characteristic of both the succinate (K_m values 0.1 and 1 mM), and inorganic phosphate (K_m values 0.1 and 3 mM) transport system(s). The phosphate transport system(s) was repressed by high inorganic phosphate and a coordinate increase in the transport of phosphate, arsenate, and phosphate-stimulated succinate transport accompanied growth in low phosphate media.A class of arsenate resistant mutants were simultaneously defective in the transport of arsenate, phosphate and succinate when cells were repressed for phosphate transport, however, the transport of these ions was regained in these mutants when grown in low phosphate media. Organic phosphate esters did not stimulate succinate transport in arsenate resistant mutants but were effective after growth in low phosphate media. Growth under phosphate limitation permitted the simultaneous regain of both phosphate and sulfate dependent succinate transport activities whereas sulfate limitation alone was ineffective.Succinate was not transported by an anion exchange diffusion mechanism since phosphate efflux was low or absent during succinate transport.The transport of C₄-dicarboxylates in B. subtilis is strongly stimulated by intracellular polyvalent anions. The absence of an anion permeability mechanism precludes succinate transport but partial escape from this restriction is mediated by the derepression of a phosphate transport system.     

Dual Mechanisms of Metabolite Acquisition by the Obligate Intracytosolic Pathogen Rickettsia prowazekii Reveal Novel Aspects of Triose Phosphate Transport

Rickettsia prowazekii is an obligate intracytosolic pathogen and the causative agent of epidemic typhus fever in humans. As an evolutionary model of intracellular pathogenesis, rickettsiae are notorious for their use of transport systems that parasitize eukaryotic host cell biochemical pathways. Rickettsial transport systems for substrates found only in eukaryotic cell cytoplasm are uncommon among free-living microorganisms and often possess distinctive mechanisms. We previously reported that R. prowazekii acquires triose phosphates for phospholipid biosynthesis via the coordinated activities of a novel dihydroxyacetone phosphate transport system and an sn-glycerol-3-phosphate dehydrogenase (K. M. Frohlich et al., J. Bacteriol. 192:4281–4288, 2010). In the present study, we have determined that R. prowazekii utilizes a second, independent triose phosphate acquisition pathway whereby sn-glycerol-3-phosphate is directly transported and incorporated into phospholipids. Herein we describe the sn-glycerol-3-phosphate and dihydroxyacetone phosphate transport systems in isolated R. prowazekii with respect to kinetics, energy coupling, transport mechanisms, and substrate specificity. These data suggest the existence of multiple rickettsial triose phosphate transport systems. Furthermore, the R. prowazekii dihydroxyacetone phosphate transport systems displayed unexpected mechanistic properties compared to well-characterized triose phosphate transport systems from plant plastids. Questions regarding possible roles for dual-substrate acquisition pathways as metabolic virulence factors in the context of a pathogen undergoing reductive evolution are discussed.     

Transport of C4-dicarboxylic acids in salmonella typhimurium.

C₄-Dicarboxylic acids are transported into Salmonella typhimurium by stereospecific systems of both high and low affinity. Succinate and l-malate are accumulated in a tricarboxylic acid cycle mutant as was d(+)-malate in induced wild-type cells. Accumulated dicarboxylates are exchangeable with exogenous dicarboxylates. The trichloroacetic acid cycle dicarboxylates are the best inducers of their own transport. Specific mutants devoid of dicarboxylate transport activity (dct) were isolated and differed from tricarboxylate transport mutants (tct) with respect to growth and transport. A mutant devoid of α-ketoglutarate dehydrogenase was unable to transport dicarboxylic acids but citrate transport remained unaffected. Tricarboxylic acid cycle mutants were markedly dependent on an exogenous energy source for the transport of succinate, proline, or leucine. Dicarboxylate transport was largely inhibited by various metabolic inhibitors but could only be inhibited by N,N'-dicyclohexylcarbodiimide anaerobically. ATPase mutants were unimpaired in their ability to transport succinate or proline aerobically.     

In Vivo Colocalisation of oskar mRNA and Trans-Acting Proteins Revealed by Quantitative Imaging of the Drosophila Oocyte

Efficient mRNA transport in eukaryotes requires highly orchestrated relationships between nuclear and cytoplasmic proteins. For oskar mRNA, the Drosophila posterior determinant, these spatio-temporal requirements remain opaque during its multi-step transport process. By in vivo covisualization of oskar mRNA with Staufen, its putative trafficking protein, we find oskar mRNA to be present in particles distinct from Staufen for part of its transport. oskar mRNA stably associated with Staufen near the posterior pole. We observe oskar mRNA to oligomerize as hundreds of copies forming large particles which are necessary for its long range transport and localization. We show the formation of these particles occurs in the nurse cell nucleus in an Hrp48-dependent manner. We present a more refined model of oskar mRNA transport in the Drosophila oocyte.     

O-GlcNAc modification of nuclear pore complexes accelerates bidirectional transport

Macromolecular transport across the nuclear envelope depends on facilitated diffusion through nuclear pore complexes (NPCs). The interior of NPCs contains a permeability barrier made of phenylalanine-glycine (FG) repeat domains that selectively facilitates the permeation of cargoes bound to nuclear transport receptors (NTRs). FG-repeat domains in NPCs are a major site of O-linked N-acetylglucosamine (O-GlcNAc) modification, but the functional role of this modification in nucleocytoplasmic transport is unclear. We developed high-throughput assays based on optogenetic probes to quantify the kinetics of nuclear import and export in living human cells. We found that increasing O-GlcNAc modification of the NPC accelerated NTR-facilitated transport of proteins in both directions, and decreasing modification slowed transport. Superresolution imaging revealed strong enrichment of O-GlcNAc at the FG-repeat barrier. O-GlcNAc modification also accelerated passive permeation of a small, inert protein through NPCs. We conclude that O-GlcNAc modification accelerates nucleocytoplasmic transport by enhancing the nonspecific permeability of the FG-repeat barrier, perhaps by steric inhibition of interactions between FG repeats.     

Citrate transport in Salmonella typhimurium.

Citrate was rapidly metabolized in wild-type cells of Salmonella typhimurium but actively accumulated in both aconitase mutants and fluorocitrate-poisoned cells. In aconitase mutants citrate was transported by a single high affinity system (K_m 23 μm, V_max 27.2 nmol min^?1 mg^?1), characterized by a single pH optimum of 7.0 and a Q₁₀ of 3.0, and was stimulated by Na⁺. cis-Aconitate, tricarballylate, trans-aconitate, and dl-fluorocitrate were weak competitive inhibitors of citrate transport whereas various other tricarboxylic acid cycle intermediates and carboxylates were ineffective. Spontaneous citrate transport mutants were unable to oxidize citrate, cis-aconitate, or tricarballylate. Such mutants were specific for citrate and transported dicarboxylates normally whereas dicarboxylate transport mutants transported and oxidized citrate normally. In whole cells of an aconitase mutant citrate transport was strongly dependent on an energy source. d(?)-Lactate dehydrogenase mutants were singularly defective in energization by d(?)-lactate. Membrane vesicles of wild-type cells were capable of energized transport by d(?)-lactate or ascorbate-phenyl-methyl sulfonate. Citrate transport in whole cells was primarily energized aerobically, and ATPase deficient mutants were still able to transport citrate in whole cells.     

Transport of succinate by Pseudomonas putida

Induced succinate uptake and transport (defined as transport of a compound followed by its metabolism and transport in the absence of subsequent metabolism) by Pseudomonas putida are active processes resulting in intracellular succinate concentrations 10-fold that of the initial extracellular concentration. Uptake was studied with the wild-type strain P. putida P2 and transport with a mutant deficient in succinate dehydrogenase activity. Addition of succinate, fumarate, or malate to the growth medium induces both processes above a basal level. Induction is dependent on protein synthesis and subject to catabolite repression. When extracts of induced and noninduced wild-type cells were assayed for succinate dehydrogenase, fumarase, and malate dehydrogenase only malate dehydrogenase increased in specific activity. Transport is inhibited by iodoacetamide, KCN, NaN₃, and 2,4-dinitrophenol and shows pH and temperature optima of 6.2 and 30 °C. Kinetic parameters are: basal uptake (cells grown on glutamate) K_m 11.6 μm, v 0.32 nmoles per min per mg dry cell mass; induced uptake (cells grown on succinate plus NH₄Cl) K_m 12.5 μm, v 5.78 nmoles per min per mg dry cell mass; induced transport (cells grown on nutrient broth plus succinate) K_m 10 μm, V 0.98 nmoles per min per mg dry cell mass. It was not possible to determine the kinetic parameters of basal transport. Malate and fumarate were the only compounds exhibiting competitive inhibition of uptake and transport suggesting common transport system for all three compounds. The K_i values for competitive inhibition and the K_m for succinate indicate the order of affinity for both uptake and transport are succinate > malate > fumarate. Data from kinetic parameters of uptake and transport and studies on succinate metabolism provide evidence consistent with concurrent increases in transport and metabolism to account for induced succinate uptake by P. putida.     

Fasting and refeeding modulate neutral amino acid transport activity in the basolateral membrane of the rat exocrine pancreatic epithelium: fasting-induced insulin insensitivity

The effects of fasting and refeeding on amino acid transport in the perfused rat exocrine pancreas were investigated using a rapid dual tracer dilution technique. Unidirectional amino acid influx (15 s) was quantified (relative to the extracellular tracer d-mannitol) over a wide range of perfusate concentrations in pancreata isolated frm fed and 24 h, 48 h, and 72 h fasted and 72 h fasted and refed (24 h) animals. In fed animals transport of phenylalamine (1–24 mM) and l-serine (1–50 mM) was saturable and weighted non-linear regression analyses of the overall transport indicated an apparent K_t=10±3mM and V_max=7.0±1.0 μmol/min per g (n = 7) for phenylalanine and K_t=16±3 mM and V_max=20.6±2.1 μmol/min per g (n = 5) for serine. Fasting animals for 24 h or 48 h did not change the kinetics of either phenylalanine or serine transport. After a 72 h fast the rate of phenylalanine transport (V_max=15.9±2.9 μmol/min per g, (n = 5) was enhanced whereas the transport affinity (K_t=11±3 mM) remained unaltered. l-Serine transport was essentially unaltered. When 72 h fasted animals were refed for 24 h the V_max for the phenylalanine transport was reduced to values observed in fed animals. In parallel experiments refeeding had no significant effect on serine transport. Perfusion of pancreata isolated from 72 h fasted animals with bovine insulin (1 mU/ml or 1 μU/ml) did not stimulate either phenylalanine or serine transport. The fasting-induced stimulation of transport may provide a mechanism by which the extracellular supply of essential amino acids as phenylalanine is increased to meet the demands of continued proteolytic and lipolytic enzyme synthesis.     

An improved method for the production of Neurospora crassa spheroplasts and a new method for measuring transport in fragile cells

An improved method for the production of Neurospora spheroplasts and a procedure for measuring transport by these fragile structures are described. Using these techniques, the activity of the glucose active transport system in spheroplasts has been measured. The results indicate that removal of the Neurospora cell wall does not affect the activity of this transport system. In light of this finding, it is unlikely that soluble periplasmic components play a role in the glucose active transport system of Neurospora.     

Starch Synthetase, Phosphorylase, ADPglucose Pyrophosphorylase, and UDPglucose Pyrophosphorylase in Developing Maize Kernels

Three types of whole plant experiments are presented to substantiate the concept that an important function of ethylene in abscission is to reduce the transport of auxin from the leaf to the abscission zone. (a) The inhibitory effect of ethylene on auxin transport, like ethylene-stimulated abscission, persists only as long as the gas is continuously present. Cotton (Gossypium hirsutum L. cv. Stoneville 213) and bean (Phaseolus vulgaris L. cv. Resistant Black Valentine) plants placed in 14 μl/l of ethylene for 24 or 48 hours showed an increase in leaf abscission and a reduced capacity to transport auxin; but when returned to air, auxin transport gradually increased and abscission ceased. (b) Ethylene-induced abscission and auxin transport inhibition show similar sensitivities to temperature. A 24-hour exposure of cotton plants to 14 μl/l of ethylene at 8 C resulted in no abscission and no significant inhibition of auxin transport. Increasing the temperature during ethylene treatment resulted in a progressively greater reduction in auxin transport with abscission occurring at [unk]27 C where auxin transport was inhibited over 70%. (c) Auxin pretreatment reduced both ethylene-induced abscission and auxin transport inhibition. No abscission occurred, and auxin transport was inhibited only 18% in cotton plants which were pretreated with 250 mg/l of naphthalene acetic acid and then placed in 14 μl/l of ethylene for 24 hours. In contrast, over 30% abscission occurred, and auxin transport was inhibited 58% in the corresponding control plants.     

Sulfate Transport in Cultured Tobacco Cells : EFFECTS OF CALCIUM AND SULFATE CONCENTRATION

Sulfate transport by tobacco cells (var. Xanthi) cultured in liquid medium was investigated. Monophasic uptake was observed over a sulfate concentration range from 0.01 to 10 millimolar, and the K_m was 20 micromolar. A time-dependent stimulation of transport was observed when cells were incubated in medium containing 0.5 millimolar Ca²⁺. Calcium stimulation was dependent on the culture cycle and was maximal during the early exponential phase. It was not observed in sulfur-deficient cells with high transport rates and was relatively small in sulfate-loaded cells with low transport rates. A kinetic analysis showed that Ca²⁺ increased the maximum rate of transport without affecting the K_m.     

Proper Fatty Acid Composition Rather than an Ionizable Lipid Amine Is Required for Full Transport Function of Lactose Permease from Escherichia coli

Energy-dependent uphill transport but not energy-independent downhill transport by lactose permease (LacY) is impaired when expressed in Escherichia coli cells or reconstituted in liposomes lacking phosphatidylethanolamine (PE) and containing only anionic phospholipids. The absence of PE results in inversion of the N-terminal half and misfolding of periplasmic domain P7, which are required for uphill transport of substrates. Replacement of PE in vitro by lipids with no net charge (phosphatidylcholine (PC), monoglucosyl diacylglycerol (GlcDAG), or diglucosyl diacylglycerol (GlcGlcDAG)) supported wild type transmembrane topology of the N-terminal half of LacY. The restoration of uphill transport in vitro was dependent on LacY native topology and proper folding of P7. Support of uphill transport by net neutral lipids in vitro (PE > PC ≫ GlcDAG ≠ GlcGlcDAG provided that PE or PC contained one saturated fatty acid) paralleled the results observed previously in vivo (PE = PC > GlcDAG ≠ GlcGlcDAG). Therefore, a free amino group is not required for uphill transport as previously concluded based on the lack of in vitro uphill transport when fully unsaturated PC replaced E. coli-derived PE. A close correlation was observed in vivo and in vitro between the ability of LacY to carry out uphill transport, the native conformation of P7, and the lipid headgroup and fatty acid composition. Therefore, the headgroup and the fatty acid composition of lipids are important for defining LacY topological organization and catalytically important structural features, further illustrating the direct role of lipids, independent of other cellular factors, in defining membrane protein structure/function.     

Phosphate transport in membrane vesicles from Escherichia coli

Escherichia coli strain AN710 possesses only the PIT system for phosphate transport. Membrane vesicles from this strain, which contain phosphate internally, perform exchange and active transport of phosphate. The energy for active transport is supplied by the respiratory chain with ascorbate-phenazine methosulphate as electron donor. To a lesser extent also the oxidation of d-lactate energizes phosphate transport; the oxidation of succinate is only marginally effective. Phosphate transport is driven by the proton-motive force and in particular by the pH gradient across the membrane. This view is supported by the observation that phosphate transport is stimulated by valinomycin, inhibited by nigericin and abolished by the uncoupler carbonyl cyanide m-chlorophenylhydrazone. Neither inhibitor affects phosphate exchange. The phosphate analogue arsenate inhibits both the exchange reaction and active transport. Both processes are stimulated by K⁺ and Mg²⁺, the highest activities being observed with both ions present.Membrane vesicles have also been isolated from Escherichia coli K10, a strain which possesses only a functional PST phosphate transport system. These vesicles perform neither exchange nor active transport of phosphate, although active transport of amino acids is observed in the presence of ascorbate-phenazine methosulphate or d-lactate.     

Gas transport during high-frequency ventilation: the significance of direct wash-out

Gas transport during high-frequency oscillation was investigated in vitro using CO₂ elimination from the lung surrogate as a measure of gas transport efficiency. The length of the connecting tube between the piston pump and the three-port connector did not affect gas transport efficiency if the oscillatory volume (V_DEL) was constant; inserting an additional tube between the three-port connector and the endotracheal tube decreased gas transport efficiency dramatically. In contradistinction, increasing V_DEL caused a steep rise in gas transport efficiency as soon as V_DEL surpassed the volume of the tubes connecting the lung surrogate with its surroundings. As gas transport effiency was found to be very sensitive to the net oscillatory volume, i.e. V_DEL minus the volume of the tubes connecting the lung and the surroundings, direct wash-out was considered to be an effective gas transport mechanism during high frequency oscillation. Two preliminary experiments on dogs allowed us to substantiate this hypothesis in vivo.     

Respiration in energy-transducing membranes of the thermophilic cyanobacterium Mastigocladus laminosus: I. Relation of the respiratory and photosynthetic electron transport

The thermophilic cyanobacterium Mastigocladus laminosus was grown at different CO₂ concentrations and temperatures. Respiratory and photosynthetic electron transport in isolated membranes were measured and their activities were compared. Cells grown at low CO₂ concentration showed respiratory electron transport, whereas Photosystem-II-dependent transport was optimal in cells grown at high CO₂ concentrations. The respiratory electron transport from NADH and succinate were KCN-sensitive, whereas NADPH-dependent O₂ uptake was not. It could be shown that NADH and succinate donate electrons in the photosynthetic electron pathway via Photosystem I. In cytochrome-c-553-depleted membranes added cytochrome c-553 could stimulate photosynthetic and respiratory electron transport. A common electron transport pathway between the quinone and cytochrome c is postulated.     

Electron transport driven phosphorylation catalyzed by proteoliposomes containing hydrogenase,fumarate reductase and ATP synthase

Liposomes containing the hydrogenase complex and the fumarate reductase complex isolated from Wolinella (formerly Vibrio) succinogenes, together with vitamin K₁ catalyzed the electron transport from H₂ to fumarate. With the fumarate reductase complex present in excess the activity of electron transport was close to that of the hydrogenase complex. Liposomes containing an ATP synthase in addition to the electron transport components catalyzed the phosphorylation of ADP driven by the electron transport.