Maintenance of Asymmetric Cellular Localization of an Auxin Transport Protein through Interaction with the Actin Cytoskeleton

In shoots, polar auxin transport is basipetal (that is, from the shoot apex toward the base) and is driven by the basal localization of the auxin efflux carrier complex. The focus of this article is to summarize the experiments that have examined how the asymmetric distribution of this protein complex is controlled and the significance of this polar distribution. Experimental evidence suggests that asymmetries in the auxin efflux carrier may be established through localized secretion of Golgi vesicles, whereas an attachment of a subunit of the efflux carrier to the actin cytoskeleton may maintain this localization. In addition, the idea that this localization of the efflux carrier may control both the polarity of auxin movement and more globally regulate developmental polarity is explored. Finally, evidence indicating that the gravity vector controls auxin transport polarity is summarized and possible mechanisms for the environmentally induced changes in auxin transport polarity are discussed.     

Imaging Carrier Dynamics and Transport in Hybrid Perovskites with Transient Absorption Microscopy

In this review, the recent progress in using transient absorption microscopy to image charge transport and dynamics in semiconducting hybrid organic–inorganic perovskites is discussed. The basic principles, instrumentation, and resolution of transient absorption microscopy are outlined. With temporal resolution as high as 10 fs, sub‐diffraction‐limit spatial resolution, and excited‐state structural resolution, these experiments have provided crucial details on charge transport mechanisms that have been previously obscured in conventional ultrafast spectroscopy measurements. Morphology‐dependent mapping unveils spatial heterogeneity in carrier recombination and cooling dynamics. By spatially separating the pump and probe beams, carrier transport across grain boundaries has been directly visualized. Further, femtosecond temporal resolution allows for the examination of nonequilibrium transport directly, revealing extraordinarily long‐range hot carrier migration. The application of transient absorption microscopy is not limited to hybrid perovskites but can also be useful for other polycrystalline materials in which morphology plays an important role in carrier transport.     

Transport and accumulation of flavonoids in grapevine (Vitis vinifera L.)

Flavonoids are a group of secondary metabolites widely distributed in plants that represent a huge portion of the soluble phenolics present in grapevine (Vitis vinifera L.). These compounds play different physiological roles and are often involved in protection against biotic and abiotic stress. Even if the flavonoid biosynthetic pathways have been largely characterized, the mechanisms of their transport and accumulation in cell wall and vacuole are still not completely understood. This review analyses the known mechanisms of flavonoid uptake and accumulation in grapevine, with reference to the transport models and membrane carrier proteins described in other plant species. The effect of different environmental factors on flavonoid biosynthesis and transporters is also discussed.Key words: ABC proteins, active transport, bilitranslocase, biotic and abiotic stress, flavonoid, secondary metabolites     

Transport of glutamine into isolated pea chloroplasts

Abstract. Uptake of [¹⁴C] glutamine into isolated pea chloroplasts has been examined by using a centrifugal filtration technique. Competition experiments showed that glutamine uptake is mediated by a dicarboxylate carrier with K_m 1.10 mM and V _max. 118 nmol of glutamine min⁻¹ per mg of chlorophyll. Isolated pea chloroplasts accumulated glutamine in the sucrose-impermeable space to concentrations higher than that present in the external solution when the latter was below 0.5 mM. It is suggested that glutamine accumulation is driven by exchange (utilizing the dicarboxylate carrier) with the endogenous pool of dicarboxylates in the chloroplasts. Increasing pH stimulated glutamine uptake but inhibited that of glutamate and 2-oxoglu-tarate. The hypothesis is advanced that when molecules of different charge are exchanged across the chloroplast envelope via the dicarboxylate carrier, electroneutrality is maintained by transport of protons, and that this explains the observed effects of increasing pH. The low rates of glutamine transport coupled with the strong competition of other dicarboxylates for the carrier suggest that export in vivo from the chloroplast of nitrogen in the form of glutamine is not of major importance.     

Transport and action of ascorbate at the plant plasma membrane

The plasmalemma is both a bridge and a barrier between the cytoplasm and the outside world. It is a dynamic interface that perceives and transmits information concerning changes in the environment to the nucleus to modify gene expression. In plants, ascorbate is an essential part of this dialogue. The concentration and ratio of reduced to oxidized ascorbate in the apoplast, for example, possibly modulates cell division and growth. The leaf apoplast contains millimolar amounts of ascorbate that protect the plasmalemma against oxidative damage. The apoplastic ascorbate-dehydroascorbate redox couple is linked to the cytoplasmic ascorbate-dehydroascorbate redox couple by specific transporters for either or both metabolites. Although evidence about the mechanisms driving ascorbate or dehydroascorbate transport remains inconclusive, these carrier proteins potentially regulate the level and redox status of ascorbate in the apoplast. The redox coupling between compartments facilitated by these transport systems allows coordinated control of key physiological responses to environmental cues.     

Transport catalysis

Carrier linked solute transport through biomembranes is analysed with the viewpoint of catalysis. Different from enzymes, in carriers the unchanged substrate induces optimum fit in the transition state. The enhanced intrinsic binding energy pays for the energy required of the global conformation changes, thus decreasing the activation energy barrier. This “induced transition fit” (ITF) explains several phenomena of carrier transport, e.g., high or low affinity substrate requirements for unidirectional versus exchange, external energy requirement for “low affinity” transport, the existence of side specific inhibitors to ground states of the carrier, the requirement of external energy in active transport to supplement catalytic energy in addition to generate electrochemical gradients.     

Transport catalysis

Carrier linked solute transport through biomembranes is analysed with the viewpoint of catalysis. Different from enzymes, in carriers the unchanged substrate induces optimum fit in the transition state. The enhanced intrinsic binding energy pays for the energy required of the global conformation changes, thus decreasing the activation energy barrier. This "induced transition fit" (ITF) explains several phenomena of carrier transport, e.g., high or low affinity substrate requirements for unidirectional versus exchange, external energy requirement for "low affinity" transport, the existence of side specific inhibitors to ground states of the carrier, the requirement of external energy in active transport to supplement catalytic energy in addition to generate electrochemical gradients.     

Transport of L-glucose by Rhodotorula glutinis

The kinetics of L-glucose transport by Rhodotorula glutinis were studied over a 720-fold range of sugar concentrations. Analysis of the saturation isotherm revealed the presence of a one-carrier system for L-glucose in the plasma membrane of Rhodotorula glutinis. This carrier exhibited a km of 3.7 +/- 0.3 mM. D-Ribose was found to be a competitive inhibitor with a Ki of 19 +/- 1 mM. The results suggest that L-glucose is transported by the high-Km, D-ribose carrier. L-Glucose was transported against a concentration gradient and the transport was inhibited by the proton conductor 2,4-dinitrophenol.     

Specificity of the Glucose Transport System in Leishmania tropica Promastigotes*

SYNOPSIS. The glucose transport system in Leishmania tropica promastigotes was characterized by the use of labeled 2-deoxy-D-glucose (2-DOG), a nonmetabolizable glucose analog. The uptake system has a Q₁₀ of 2 and a heat of activation of 10.2 kcal/mole. The glucose transport system is subject to competitive inhibition by 2-DOG, glucosamine, N-acetyl glucosamine, mannose, galactose, and fructose which suggests that substitutions in the hexose chain at carbons 2 and 4 do not affect carrier specificity. In contrast, changes at carbon 1 (α-methyl-D-glucoside, 1,5-anhydroglucitol) and carbon 3 (3–0-methyl glucose) lead to loss of carrier affinity since these sugars do not compete for the glucose carrier. Sugars that compete with the glucose carrier have one common feature—they all exist in the pyranose form in solution. The carrier for D-glucose does not interact with L-glucose or any of the pentose sugars tested. Uptake of 2-DOG is inhibited by glycerol. This inhibition, however, is noncompetitive; it is evident, therefore, that glucose and glycerol do not compete for the same carrier. Glycerol does not repress the glucose carrier since cells grown in presence of glycerol transport the sugar normally.     

Transport viewed as a catalytic process

Transport catalysis is analysed in terms of the "induced transition fit" (ITF) concept. The essentials of ITF are briefly elucidated, emphasizing the difference of substrate-protein interactions between enzymes and carriers exemplified by the paradigm ADP/ATP carrier (AAC). Two of the numerous applications of the ITF are discussed in more detail: unidirectional passive and active transport and the relation of substrate site type inhibitors to the carrier conformations. According to ITF in most cases of unidirectional transport intrinsic binding energies may be insufficient for transport catalysis and requires additional energy from ATP or electrochemical gradients. The impacts of external energy on the carrier cycle are examined for ABC transporters (mdr) and for cation-substrate co-transporters (LacY). The relations of inhibitors to the binding site of the carrier are discussed, given the paradigm examples of side specific inhibitors of the AAC. Results with the AAC suggest the induction of an abortive ground state by inhibitors, representing extreme side specific conformation of the binding center.     

A Unified Kinetic Hypothesis of Carrier-Mediated Transport: Its Applications

A model analysis of the process of carrier mediated membrane transport is presented, wherein the carrier is present in two forms of differing affinity for substrate. The two forms of carrier undergo interconversion by asymmetric metabolic reactions on each side of the membrane. From this model system expressions are derived for the steady-state distribution ratio for substrate, for the unidirectional fluxes of substrate and hence for the initial velocity of uptake of substrate, and for the effect of preloading cells upon the initial velocity of uptake of labeled substrate. These expressions are applied to published data for glycine transport in Ehrlich ascites tumor cells to obtain numerical values for the parameters of a concentrative membrane carrier system. Concentrative uptake is shown to be consequent to the differing affinities of the two forms of carrier. When the affinities of the two forms are equal, equilibrative uptake occurs. The model analysis is applied to the phenomena of metabolic and competitive inhibition.     

Limits on the Tightness of Coupling in Active Transport

Control of the coupled reaction sequence in active transport depends on systematic changes in the properties of the carrier protein as the reaction proceeds. These changes would have to be brought about by specific interactions with the substrate, the binding forces being used to stabilize either (i) a carrier state with altered properties or (ii) the transition state in a carrier transformation. In the first case the tightness of coupling (the ratio of the coupled rate to slippage) will at first rise with the increment in binding energy in the altered state but will approach an upper limit when overly strong binding forces retard substrate dissociation in a subsequent step in the coupled reaction sequence. Primary and secondary active transport are subject to this limitation because the coupling mechanism necessarily involves intermediates in which the substrate is strongly bound. Exchange-only transport is not necessarily subject to the same limitation because the mechanism can involve only a substrate-catalyzed change in carrier state. The available data, although scant, agree with these conclusions. Received: 3 June 1998/Revised: 22 September 1998     

Transport of aspartic acid, arginine, and tyrosine by the opportunistic protist Pneumocystis carinii

In order to improve culture media and to discover potential drug targets, uptake of an acidic, a basic, and an aromatic amino acid were investigated. Current culture systems, axenic or co-cultivation with mammalian cells, do not provide either the quantity or quality of cells needed for biochemical studies of this organism. Insight into nutrient acquisition can be expected to lead to improved culture media and improved culture growth. Aspartic acid uptake was directly related to substrate concentration, Q(10) was 1.10 at pH 7.4. Hence the organism acquired this acidic amino acid by simple diffusion. Uptake of the basic amino acid arginine and the aromatic amino acid tyrosine exhibited saturation kinetics consistent with carrier-mediated mechanisms. Kinetic parameters indicated two carriers (K(m)=22.8+/-2.5 microM and K(m)=3.6+/-0.3 mM) for arginine and a single carrier for tyrosine (K(m)=284+/-23 microM). The effects of other L-amino acids showed that the tyrosine carrier was distinct from the arginine carriers. Tyrosine and arginine transport were independent of sodium and potassium ions, and did not appear to require energy from ATP or a proton motive force. Thus facilitated diffusion was identified as the mechanism of uptake. After 30 min of incubation, these amino acids were incorporated into total lipids and the sedimentable material following lipid extraction; more than 90% was in the cellular soluble fraction.     

Hitching a Ride: Mechanics of Transport Initiation through Linker-Mediated Hitchhiking

In contrast to the canonical picture of transport by direct attachment to motor proteins, recent evidence shows that a number of intracellular “cargos” navigate the cytoplasm by hitchhiking on motor-driven “carrier” organelles. We describe a quantitative model of intracellular cargo transport via hitchhiking, examining the efficiency of hitchhiking initiation as a function of geometric and mechanical parameters. We focus specifically on the parameter regime relevant to the hitchhiking motion of peroxisome organelles in fungal hyphae. Our work predicts the dependence of transport initiation rates on the distribution of cytoskeletal tracks and carrier organelles, as well as the number, length, and flexibility of the linker proteins that mediate contact between the carrier and the hitchhiking cargo. Furthermore, we demonstrate that attaching organelles to microtubules can result in a substantial enhancement of the hitchhiking initiation rate in tubular geometries such as those found in fungal hyphae. This enhancement is expected to increase the overall transport rate of hitchhiking organelles and lead to greater efficiency in organelle dispersion. Our results leverage a quantitative physical model to highlight the importance of organelle encounter dynamics in noncanonical intracellular transport.     

Transport of D-beta-hydroxybutyrate across rat liver mitochondrial membranes

1. D-beta-hydroxybutyrate, a major ketone body, is produced or converted in mitochondria from various animal tissues. 2. It is an easy permeate anion of the inner mitochondrial membrane. However, its translocation is not a passive diffusion process since it is inhibited by pyruvate transport inhibitors like alpha-cyanocinnamate and derivatives. 3. This carrier mediated process is associated with proton movements. Besides, dicarboxylate anions strongly inhibit the penetration into mitochondria. 4. This is in agreement with the existence of a second transport process related to the dicarboxylate carrier.     

Transport of proteins into mitochondria. Posttranslational transfer of ADP/ATP carrier into mitochondria in vitro

The mitochondrial ADP/ATP carrier is an integral transmembrane protein of the inner membrane. It is synthesized on cytoplasmic ribosomes. Kinetic data suggested that this protein is transferred into mitochondria in a posttranslational manner. The following results provide further evidence for such a mechanism and provide information on its details. 1. In homologous and heterologous translation systems th newly synthesized ADP/ATP carrier protein is present in the postribosomal supernatant. 2. Analysis by density gradient centrifugation and gel filtration shows, that the ADP/ATP carrier molecules in the postribosomal fraction are present as soluble complexes with apparent molecular weights of about 120 000 and 500 000 or larger. The carrier binds detergents such as Triton X-100 and deoxycholate forming mixed micelles with molecular weights of about 200 000-400 000. 3. Incubation of a postribosomal supernatant of a reticulocyte lysate containing newly synthesized ADP/ATP carrier with mitochondria isolated from Neurospora spheroplasts results in efficient transfer of the carrier into mitochondria. About 20-30% of the transferred carrier are resistant to proteinase in whole mitochondria. The authentic mature protein is also largley resistant to proteinase in whole mitochondria and sensitive after lysis of mitochondria with detergent. Integrity of mitochondria is a preprequisite for translocation into proteinase resistant position. 4. The transfer in vitro into a proteinase-resistant form is inhibited by the uncoupler carbonyl-cyanide m-chlorophenylhydrazone but not the proteinase-sensitive binding. These observations suggest that the posttranslational transfer of ADP/ATP carrier occurs via the cytosolic space through a soluble oligomeric precursor form. This precursor is taken up by intact mitochondria into an integral position in the membrane. These findings are considered to be of general importance for the intracellular transfer of insoluble membrane proteins. They support the view that such proteins can exist in a water-soluble form as precursors and upon integration into the membrane undergo a conformational change. Uptake into the membrane may involve the cleavage of an additional sequence in some proteins, but this appears not to be a prerequisite as demonstrated by the ADP/ATP carrier protein.     

Grain Boundary Scattering of Charge Transport in n‐Type (Hf,Zr)CoSb Half‐Heusler Thermoelectric Materials

High thermoelectric figure of merit zT of ≈1.0 has been reported in both n‐ and p‐type (Hf,Zr)CoSb‐based half‐Heusler compounds, and further improvement of thermoelectric performance relies on the insightful understanding of electron and phonon transport mechanisms. In this work, the thermoelectric transport features are analyzed for (Hf_0.3Zr_0.7)_1?xNb_xCoSb (x = 0.02–0.3) with a wide range of carrier concentration. It is found that, although both temperature and energy dependencies of charge transport resemble ionized impurity scattering, the grain boundary scattering is the dominant scattering mechanism near room temperature. With increasing carrier concentration and grain size, the influence of the grain boundary scattering on electron transport weakens. The dominant scattering mechanism changes from grain boundary scattering to acoustic phonon scattering as temperature rises. The lattice thermal conductivity decreases with increasing Nb doping content due to the increased strain field fluctuations. These results provide an in‐depth understanding of the transport mechanisms and guidance for further optimizing thermoelectric properties of half‐Heusler alloys and other thermoelectric systems.     

Cellular Mechanisms of Organic Anion Transport in Crustacean Renal Tissue

Decapod Crustacea appear to utilize two overall strategies forexcretion of anionic wasteproducts and xenobiotics. One strategyutilizes a potent renal secretory system; the other utilizesboth modest renal secretion and strong gut secretion (possiblyaided by midgut gland conjugation). In crabs, the level of participationof the renal system in overall excretion is primarily determinedbythe location of an anion pump and a carrier protein. The bladder of Cancer borealis, which strongly secretes themodel organic anion, p-aminohippuric acid (PAH), has a serosalorganic anion pump and a luminal facilitated carrier. The bladderof Cancer trroratus, which reabsorbs PAH from bladder urine,has a luminal organic anion pump and a serosal facilitated carrier.     

Transport by reconstituted lactose carrier from parental and mutant strains ofEscherichia coli

Summary The lactose transport carrier from parental (X71/F'W3747) and mutant cells (54/F'5441) was reconstituted into proteoliposomes. Transport by the counterflow assay showed slightly greater activity in proteoliposomes prepared from extracts of the mutant membranes compared with that for the parental cell. The mutant carrier showed a threefold lowerK _m but similarV _max compared to the parent. On the other hand proteoliposomes from the mutant showed a defect in protonmotive force-driven accumulation, compared with the parent. With a pH gradient (inside alkaline) plus a membrane potential (inside negative) the parental proteoliposomes accumulated lactose 25-fold over the medium concentration while the mutant proteoliposomes accumulated sixfold. In a series of experiments proteoliposomes were exposed to proteolytic enzymes. Chrymotrypsin treatment resulted in 30% inhibition of counterflow activity for the reconstituted carrier from both parent and mutant. Papain produced 84% inhibition of transport by the reconstituted parental carrier but only 41% of that of the mutant. Trypsin and carboxypeptidase Y treatment had no effect on counterflow activity of either parent or mutant. Exposure of purified lactose carrier in proteoliposomes to carboxypeptidase Y resulted in the release of alanine and valine, the two C-terminal amino acids predicted from the DNA sequence.