Escherichia coli K-12 mutants that allow transport of maltose via the beta-galactoside transport system.

We have isolated mutants of Escherichia coli that have an altered beta-galactoside transport system. This altered transport system is able to transport a sugar, maltose, that the wild-type beta-galactoside transport system is unable to transport. The mutation that alters the specificity of the transport system is in the lacY gene, and we refer to the allele as lacYmal. The lacYmal allele was detected originally in strains in which the lac genes were fused to the malF gene. Thus, as a result of gene fusion and isolation of the lacYmal mutation, a new transport system was evolved with regulatory properties and specificity similar to those of the original maltose transport system. Maltose transport via the lacYmal gene product is independent of all of the normal maltose transport system components. The altered transport system shows a higher affinity than the wild-type transport system for two normal substrates of the beta-galactoside transport system, thiomethyl-beta-D-galactoside and o-nitrophenyl-beta-D-galactoside.     

Approaches used to examine the mechanism and regulation of hexose transport in rat myoblasts

This review discusses some of the approaches and general criteria that we have used to examine the properties of the hexose transport system in undifferentiated L6 rat myoblasts. These approaches include studying the kinetics of hexose transport in whole cells and plasma membrane vesicles, the effects of various inhibitors on hexose transport, the isolation and characterization of hexose transport mutants, and the use of cytochalasin B (CB) to identify the transport component(s). Transport kinetics indicated that two transport systems are present in these cells. 2-Deoxy-D-glucose is transported primarily by the high affinity system, whereas 3-O-methyl-D-glucose is transported by the low affinity system. Furthermore, these two transport systems are inactivated to different extents by CB. CB has a higher binding affinity for the low affinity hexose transport system. The inhibitory effect of various hexose analogues also revealed the presence of two hexose transport systems. The effects of various ionophores and energy uncouplers on hexose transport suggest that the high affinity system is an active transport process, whereas the low affinity system is of the facilitated diffusion type. The high affinity system is also sensitive to sulfhydryl reagents, whereas the low affinity system is not. Further evidence for the presence of two transport systems comes from the characterization of hexose transport mutants. Two of the mutants isolated are shown to be defective in the high affinity transport system, but not in the low affinity transport system. These mutants are also defective in the CB low affinity binding site. Based on our results a tentative working model for hexose transport in L6 rat myoblasts is presented.     

Kinesin‐1/Hsc70‐dependent mechanism of slow axonal transport and its relation to fast axonal transport

Cytoplasmic protein transport in axons (‘slow axonal transport’) is essential for neuronal homeostasis, and involves Kinesin‐1, the same motor for membranous organelle transport (‘fast axonal transport’). However, both molecular mechanisms of slow axonal transport and difference in usage of Kinesin‐1 between slow and fast axonal transport have been elusive. Here, we show that slow axonal transport depends on the interaction between the DnaJ‐like domain of the kinesin light chain in the Kinesin‐1 motor complex and Hsc70, scaffolding between cytoplasmic proteins and Kinesin‐1. The domain is within the tetratricopeptide repeat, which can bind to membranous organelles, and competitive perturbation of the domain in squid giant axons disrupted cytoplasmic protein transport and reinforced membranous organelle transport, indicating that this domain might have a function as a switchover system between slow and fast transport by Hsc70. Transgenic mice overexpressing a dominant‐negative form of the domain showed delayed slow transport, accelerated fast transport and optic axonopathy. These findings provide a basis for the regulatory mechanism of intracellular transport and its intriguing implication in neuronal dysfunction.     

High-Affinity Transport of γ-Aminobutyric Acid, Glycine, Taurine, L-Aspartic Acid, and L-Glutamic Acid in Synaptosomal (P2) Tissue: A Kinetic and Substrate Specificity Analysis

In a cortical P2 fraction, [14C]gamma-aminobutyric acid ([14C]GABA), [14C]glycine, [14C]taurine, and [14C]glutamic and [14C]aspartic acids are transported by four separate high-affinity transport systems with L-glutamic acid and L-aspartic acid transported by a common system. GABA transport in cortical synaptosomal tissue occurs by one high-affinity system, with no second, low-affinity, transport system detectable. Only one high-affinity system is observed for the transport of aspartic/glutamic acids; as with GABA transport, no low-affinity transport is detectable. In the uptake of taurine and glycine (cerebral cortex and pons-medulla-spinal cord) both high- and low-affinity transport processes could be detected. The high-affinity GABA and high-affinity taurine transport classes exhibit some overlap, with the GABA transport system being more specific and having a much higher Vmax value. High-affinity GABA transport exhibits no overlap with either the high-affinity glycine or the high-affinity aspartic/glutamic acid transport class, and in fact they demonstrate somewhat negative correlations in inhibition profiles. The inhibition profiles of high-affinity cortical glycine transport and those of high-affinity cortical taurine and aspartic/glutamic acid transport also show no significant positive relationship. The inhibition profiles of high-affinity glycine transport in the cerebral cortex and in the pons-medulla-spinal cord show a significant positive correlation with each other; however, high-affinity glycine uptake in the pons-medulla-spinal cord is more specific than that in the cerebral cortex. The inhibition profile of high-affinity taurine transport exhibits a nonsignificant negative correlation with that of the aspartic/glutamic acid transport class.     

Negative interactions between amino acid and methylamine/ammonia transport systems of Saccharomyces cerevisiae.

The transport of methylamine (methylammonium ion) and ammonia (ammonium ion) is accomplished in Saccharomyces cerevisiae by means of a specific active transport system. L-Amino acids are noncompetitive inhibitors of methylamine transport. This inhibition is relieved or eliminated in mutant strains that have a reduced ability to transport amino acids. The inhibition of methylamine transport occurs immediately upon the addition of amino acids to the assay system and persists until the external amino acid pool is depleted. The degree of inhibition observed is a direct function of the rate of amino acid transport. Both methylamine and ammonia are capable of inhibiting amino acid transport. The inhibition of amino acid transport is eliminated in mutant strains that cannot transport methylamine and ammonia.     

2-Deoxy-D-glucose resistant yeast with altered sugar transport activity

The transport of glucose and maltose in Saccharomyces cerevisiae was observed to occur by both high and low affinity transport systems. A spontaneously isolated 2-deoxy-D-glucose resistant mutant was observed to transport glucose and maltose only by the high affinity transport systems. Associated with this was an increase in the Vmax values, indicating derepression of the high affinity transport systems. The low affinity transport systems could not be detected. This mutant will be important in examining the repression regulatory and sugar transport mechanisms in yeast.     

Na(+)-dependent, active nucleoside transport in S49 mouse lymphoma cells and loss in AE-1 mutant deficient in facilitated nucleoside transport

S49 murine lymphoma cells were examined for expression of various nucleoside transport systems using a non-metabolized nucleoside, formycin B, as substrate. Nitrobenzylthioinosine (NBTI)-sensitive, facilitated transport was the primary nucleoside transport system of the cells. The cells also expressed very low levels of NBTI-resistant, facilitated nucleoside transport as well as of Na(+)-dependent, concentrative formycin B transport. Concentrative transport was specific for uridine and purine nucleosides, just as the concentrative nucleoside transporters of other mouse and rat cells. A nucleoside transport mutant of S49 cells, AE-1, lacked both the NBTI-sensitive, facilitated and Na(+)-dependent, concentrative formycin B transport activity, but Na(+)-dependent, concentrative transport of alpha-aminoisobutyrate was not affected.     

Transport of the two natural auxins, indole-3-butyric acid and indole-3-acetic acid, in Arabidopsis

Polar transport of the natural auxin indole-3-acetic acid (IAA) is important in a number of plant developmental processes. However, few studies have investigated the polar transport of other endogenous auxins, such as indole-3-butyric acid (IBA), in Arabidopsis. This study details the similarities and differences between IBA and IAA transport in several tissues of Arabidopsis. In the inflorescence axis, no significant IBA movement was detected, whereas IAA is transported in a basipetal direction from the meristem tip. In young seedlings, both IBA and IAA were transported only in a basipetal direction in the hypocotyl. In roots, both auxins moved in two distinct polarities and in specific tissues. The kinetics of IBA and IAA transport appear similar, with transport rates of 8 to 10 mm per hour. In addition, IBA transport, like IAA transport, is saturable at high concentrations of auxin, suggesting that IBA transport is protein mediated. Interestingly, IAA efflux inhibitors and mutations in genes encoding putative IAA transport proteins reduce IAA transport but do not alter IBA movement, suggesting that different auxin transport protein complexes are likely to mediate IBA and IAA transport. Finally, the physiological effects of IBA and IAA on hypocotyl elongation under several light conditions were examined and analyzed in the context of the differences in IBA and IAA transport. Together, these results present a detailed picture of IBA transport and provide the basis for a better understanding of the transport of these two endogenous auxins.     

运输空间容量对断奶仔猪行为和生产性能的影响

为了解运输对断奶仔猪行为与生产性能的影响,针对北京至邯郸的实际仔猪运输,设计两种运输空间容量(0·09m2/头和0·15m2/头),对运输过程断奶仔猪的躺卧、站立行为进行观察,并对运输前后仔猪体重和体表受损程度进行测定,其结果表明:(1)在试验运输条件下,运输温度与仔猪躺卧行为呈明显的负相关(PearsonCorrelationTest,R=-0·324,P<0·01);(2)两种运输空间容量下,仔猪站立行为(Mann-WhitneyUTest,P>0·05)及运输过程中仔猪的活动激烈程度无显著差异(Chi-squareTest,P>0·05),但仔猪的躺卧行为有显著差异(Mann-WhitneyUTest,P<0·05);(3)不同运输时间持续段上,仔猪的站立和躺卧行为所占比例(FriedmanTest,P<0·001)和仔猪的活动激烈程度有显著差异(Chi-squareTest,P<0·01);(4)两种运输空间容量下,仔猪体表受损程度(Mann-WhitneyUTest,P>0·05)和仔猪体重变化无显著差异(tTest,P>0·05)。这些结果提示,将运输空间容量降低到仔猪趴卧时所需的空间容量,对断奶仔猪的躺卧行为有一定的影响,但对仔猪的站立行为、体表受损及体重变化无显著影响。其原因可能是仔猪趴卧的面积并不是仔猪运输空间的最低阈值,这需要进一步研究证实。     

Comparison of diverse transport signals in synthetic peptide-induced nuclear transport

Several investigations have demonstrated the ability of synthetic peptides homologous to the nuclear transport signal of simian virus 40 large T antigen to induce the nuclear transport of nonnuclear carrier proteins. To determine the generality of peptide-induced transport, six peptides with sequences derived from four previously identified nuclear transport signals were synthesized and examined for their ability to induce the transport of mouse immunoglobulin G following microinjection into the cytoplasm of mammalian cells. Peptides containing transport signals from simian virus 40 T antigen, Xenopus nucleoplasmin, and adenovirus E1A proteins were highly efficient at peptide-induced transport, while a peptide homologous to yeast MAT alpha 2 protein was incapable of inducing transport. A short nucleoplasmin peptide that contained only the basic amino acid domain was capable of inducing transport but yielded a much slower rate of transport than a long nucleoplasmin peptide encompassing the previously identified minimal transport signal. The short nucleoplasmin signal exhibited a greater capacity for transport than a peptide homologous to the cytoplasmic mutant T antigen signal when conjugates with a low number of signals coupled per carrier protein were examined. However, the short nucleoplasmin peptide was only marginally more effective than the T antigen mutant peptide when conjugates with a high number of signals coupled per carrier protein were examined.     

Effect of phospholipase A on active transport of amino acids with membrane vesicles of Mycobacterium phlei.

Active transport of proline remained unaffected in phospholipase A-treated electron transport particles from Mycobacterium phlei. However, the steady state level of proline was reduced 50 to 60% in phospholipase A-treated depleted electron transport particles that were devoid of membrane-bound coupling factor-latent ATPase activity. The decrease in the uptake of proline in the phospholipase A-treated depleted electron transport particles was not due to a change in the apparent K-m for proline, but it was related to the amount of phospholipid cleaved from the membranes. Restoration in the level of proline transport in phospholipase A-treated depleted electron transport particles was achieved by reconstituting these vesicles with diphosphatidylglycerol and phosphatidylethanolamine liposomes. Diphosphatidylglycerol was found to be most effective in the restoration of proline uptake. In contrast to the effect of phospholipase A treatment on proline transport, similar treatement of the electron transport particles or depleted electron transport particles failed to inhibit the active transport of either glutamine or glutamic acid. Studies with phospholipase A-treated membrane vesicles confirmed earlier findings that a proton gradient is not required for active transport of amino acids.     

Effect of basic and nonbasic amino acid substitutions on transport induced by simian virus 40 T-antigen synthetic peptide nuclear transport signals.

A previous study demonstrated the ability of a synthetic peptide homologous to the simian virus 40 T-antigen nuclear transport signal to induce the nuclear transport of carrier proteins and the dependence of peptide-induced transport on a positive charge at the lysine corresponding to amino acid 128 of T antigen. In this investigation synthetic peptides were utilized to examine the effect on transport of amino acid substitutions within the T-antigen nuclear transport signal. Nuclear transport was evaluated by immunofluorescence after microinjection of protein-peptide conjugates into the cytoplasm of mammalian cells. Substitution of other basic amino acids at position 128 revealed a hierarchy for nuclear transport. The rate of nuclear transport was most rapid when a lysine was at position 128 followed in descending order by arginine, D-lysine, ornithine, and p-aminophenylalanine. Peptide-induced nuclear transport was dependent upon a positively charged amino acid at positions 128 and 129, since substitutions of neutral asparagines at these positions abolished transport. However, partial transport was observed with the peptide having an asparagine at position 128 when a high number of peptides were conjugated to the carrier protein.     

Use of a genetic variant to study the hexose transport properties of human skin fibroblasts.

Human skin fibroblasts from 'normal' subjects were found to possess at least two hexose transport systems. One system was responsible for the uptake of 2-deoxy-D-glucose (dGlc), D-glucose and D-galactose, whereas the other was responsible primarily for the uptake of 3-O-methyl-D-glucose (MeGlc). The transport of dGlc was the rate-limiting step in the uptake process; over 97% of the internalized dGlc was phosphorylated and the specific activity of hexokinase was several times higher than that for dGlc transport. The dGlc transport system was activated by glucose starvation, and was very sensitive to inhibition by cytochalasin B and energy uncouplers. Fibroblasts isolated from a patient with symptoms of hypoglycaemia were found to differ from their normal counterparts in the dGlc transport system. They exhibited a much higher transport affinity for dGlc, D-glucose and D-galactose, with no change in the respective transport capacity. Transport was not the rate-limiting step in dGlc uptake by these cells. Moreover, the patient's dGlc transport system was no longer sensitive to inhibition by cytochalasin B and energy uncouplers. This suggested that the intrinsic properties of the patient's dGlc transport system were altered. It should be noted that the patient's dGlc transport system could still be activated by glucose starvation. Despite the changes in the dGlc transport system, the MeGlc transport system in the patient's fibroblasts remained unaltered. The observed difference in the properties of the two hexose transport systems in the 'normal' and the patient's fibroblasts strongly suggests that the two transport systems may be coded or regulated by different genes. The present finding provides the first genetic evidence from naturally occurring fibroblasts indicating the presence of two different hexose transport systems.     

Efficiency, selectivity, and robustness of nucleocytoplasmic transport

All materials enter or exit the cell nucleus through nuclear pore complexes (NPCs), efficient transport devices that combine high selectivity and throughput. NPC-associated proteins containing phenylalanine–glycine repeats (FG nups) have large, flexible, unstructured proteinaceous regions, and line the NPC. A central feature of NPC-mediated transport is the binding of cargo-carrying soluble transport factors to the unstructured regions of FG nups. Here, we model the dynamics of nucleocytoplasmic transport as diffusion in an effective potential resulting from the interaction of the transport factors with the flexible FG nups, using a minimal number of assumptions consistent with the most well-established structural and functional properties of NPC transport. We discuss how specific binding of transport factors to the FG nups facilitates transport, and how this binding and competition between transport factors and other macromolecules for binding sites and space inside the NPC accounts for the high selectivity of transport. We also account for why transport is relatively insensitive to changes in the number and distribution of FG nups in the NPC, providing an explanation for recent experiments where up to half the total mass of the FG nups has been deleted without abolishing transport. Our results suggest strategies for the creation of artificial nanomolecular sorting devices.     

Adenosine transport in liver before and after organ preservation.

The effect of 24-h cold storage of liver on nucleoside transport was investigated. Nucleoside transport was estimated under conditions when both known types of nucleoside transport, facilitated diffusion and Na+/nucleoside cotransport, were active and when one of these transport mechanisms was inhibited. The rate of adenosine transport was not decreased after long-term cold storage of the liver. Inhibition of one of the transport systems decreased the rate of adenosine uptake before and after preservation of the liver to about the same extent. The adenosine transport rate was maintained during long-term (100 min) liver perfusion ex vivo. Slight activation of energy-dependent transport in the beginning of reperfusion and the slower recovery of this transport after the second transition from Na+-free to Na+-containing perfusion are not regarded as physiologically important because they were observed after changing the ionic content of the extracellular medium. We conclude that the nucleoside transport systems in liver are quite well preserved after 24-h cold storage of the organ.     

Ethoxyzolamide Differentially Inhibits CO2 Uptake and Na+-Independent and Na+-Dependent HCO3- Uptake in the Cyanobacterium Synechococcus sp. UTEX 625

The effects of ethoxyzolamide (EZ), a carbonic anhydrase inhibitor, on the active CO2 and Na+-independent and Na+-dependent HCO3- transport systems of the unicellular cyanobacterium Synechococcus sp. UTEX 625 were examined. Measurements of transport and accumulation using radiochemical, fluorometric, and mass spectrometric assays indicated that active CO2 transport and active Na+-independent HCO3- transport were inhibited by EZ. However, Na+-independent HCO3- transport was about 1 order of magnitude more sensitive to EZ inhibition than was CO2 transport (50% inhibition = 12 [mu]M versus 80 [mu]M). The data suggest that both the active CO2 (G.D. Price, M.R. Badger [1989] Plant Physiol 89: 37-43) and the Na+ -independent HCO3 - transport systems possessed carbonic anhydrase-like activity as part of their mechanism of action. In contrast, Na+-dependent HCO3- transport was only partially (50% inhibition = 230 [mu]M) and noncompetitively inhibited by EZ. The collective evidence suggested that EZ inhibition of Na+ -dependent HCO3- transport was an indirect consequence of the action of EZ on the CO2 transport system, rather than a direct effect on HCO3- transport. A model is presented in which the core of the inorganic carbon translocating system is formed by Na+-dependent HCO3- transport and the CO2 transport system. It is argued that the Na+-independent HCO3 - utilizing system was not directly involved in translocation, but converted HCO3- to CO2 for use in CO2 transport.     

Hexose transport in L6 rat myoblasts. II. The effects of sulfhydryl reagents

The importance of sulfhydryl groups for hexose transport in undifferentiated L6 rat myoblasts was investigated. N-ethylmaleimide (NEM) and p-chloromer-curibenzenesulfonic acid (pCMBS) inhibited 2-deoxy-D-glucose (2-DOG) transport in a time and concentration-dependent manner. The inhibition produced by both reagents was virtually complete within 5 min, although neither reagent inhibited transport more than 70–80% regardless of the concentrations or incubation times used. Furthermore, the inhibition of 2-DOG transport by pCMBS or NEM could not be prevented by simultaneous preincubation of cells with 20 mM D-glucose or 20 mM 2-DOG. This suggests that sulfhydryl groups required for transport are separate from the hexose binding and transport site. By comparing the effects of the membrane impermeant pCMBS to those of the membrane permeant NEM, cell surface sulfhydryl groups were shown to be essential for hexose binding and transport. In contrast to the inhibition of 2-DOG transport, pCMBS and NEM had much less of an effect on 3-O-methyl-D-glucose (3-OMG) transport. For example, 1 mM NEM inhibited 2-DOG transport by 66%, whereas 3-OMG transport was inhibited by only 7%. This supports the suggestion that these hexose analogues may be transported by different carriers. Kinetic analysis of transport shows that treatment of cells with 1 mM NEM or 1 pCMBS results in inactivation of the high affinity 2-DOG transport system, whereas the low affinity transport system is unaffected. 3-OMG is preferentially transported by the low affinity system.     

Arginine transport in mitochondria of Neurospora crassa.

Transport of arginine into mitochondria of Neurospora crassa has been studied. Arginine transport was found to be saturable (Km = 6.5 mM) and to have a pH optimum of pH 7.5. Mitochondrial arginine transport appeared to be facilitated transport rather than active transport because: (i) the arginine concentration within the mitochondrial matrix after transport was similar to that of the reaction medium, and (ii) uncouplers and substrates of oxidative phosphorylation did not affect the transport rate. The basic amino acids ornithine, lysine, and D-arginine inhibited arginine transport. The arginine transport system could be irreversibly blocked by treating mitochondria with the reactive arginine derivative, N-nitrobenzyloxycarbonyl-arginyl diazomethane.     

Relation between red cell anion exchange and water transport

A new distilbene compound, 4',4'-dichloromercuric-2,2,2',2'-bistilbene tetrasulfonic acid (DCMBT), has been synthesized for use in studies of anion and water transport in the human red cell. DCMBT combines features of both the specific stilbene anion transport inhibitor, DIDS, and the mercurial water transport inhibitor, pCMBS. This new compound inhibits anion transport almost completely with a Ki of 15 microM. DCMBT also inhibits water transport by about 15-20% with a Ki of about 8 microM. Treatment of red cells with DIDS inhibits the effect of DCMBT on water transport, suggesting that anion transport and water transport are mediated by the same protein.     

Aboral changes in D-glucose transport by human intestinal brush-border membrane vesicles.

D-Glucose transport was investigated in isolated brush-border membrane vesicles from human small intestine. Characteristics of D-glucose transport from the jejunum were compared with that in the mid and terminal ileum. Jejunal and mid-ileal D-glucose transport was Na+-dependent and electrogenic. The transient overshoot of jejunal D-glucose transport was significantly greater than corresponding values in mid-ileum. The terminal ileum did not exhibit Na+-dependent D-glucose transport, but did exhibit Na+-dependent taurocholate transport. Na+-glucose co-transport activity as measured by tracer-exchange experiments was greatest in the jejunum, and diminished aborally. We conclude that D-glucose transport in man is Na+-dependent and electrogenic in the proximal intestine and directly related to the activity of D-glucose-Na+ transporters present in the brush-border membranes. D-Glucose transport in the terminal ileum resembles colonic transport of D-glucose.