Low-affinity Transport System Research Articles

Transport of Sulfonium Compounds: CHARACTERIZATION OF THE S-ADENOSYLMETHIONINE ANDS-METHYLMETHIONINE PERMEASES FROM THE YEASTSACCHAROMYCES CEREVISIAE

We report here the characterization and the molecular analysis of the two high affinity permeases that mediate the transport of S-adenosylmethionine (AdoMet) and S-methylmethionine (SMM) across the plasma membrane of yeast cells. Mutant cells unable to use AdoMet as a sulfur source were first isolated and demonstrated to lack high affinity AdoMet transport capacities. Functional complementation cloning allowed us to identify the corresponding gene (SAM3), which encodes an integral membrane protein comprising 12 putative membrane spanning regions and belonging to the amino acid permease family. Among amino acid permease members, the closest relative of Sam3p is encoded by the YLL061w open reading frame. Disruption of YLL061w was shown to specifically lead to cells unable to use SMM as a sulfur source. Accordingly, transport assays demonstrated that YLL061w disruption mutation impaired the high affinity SMM permease, and YLL061w was therefore renamed MMP1. Further study of sam3Delta and mmp1Delta mutant cells showed that in addition to high affinity permeases, both sulfonium compounds are transported into yeast cells by low affinity transport systems that appear to be carrier-facilitated diffusion.

Inhibition of nitrate uptake by ammonium in barley. Analysis Of component fluxes

NO3- uptake by plant roots is rapidly inhibited by exposure to NH4+. The rapidity of the effect has led to the presumption that the inhibition results from the direct effects of NH4+ at the plasma membrane. The mechanism of this inhibition, however, has been in contention. In the present study we used the radiotracer 13N to determine the relative effects of short-term exposures to NH4+ on the 13NO3- influx, efflux, and partitioning of absorbed 13N in barley (Hordeum vulgare) roots. Plants were grown without NO3- or NO2- (uninduced for NO3- uptake), or with 0.1, 1.0, 10 mM NO3-, or 0. 1 mM NO2- (to generate plant roots induced for NO3- uptake). Exposure to 1 mM NH4+ strongly reduced influx; the effect was most pronounced in plants induced for NO3- uptake when NO3- absorption was measured at low external NO3-. At higher [NO3-] and in uninduced plants the inhibitory effect was much diminished, indicating that NH4+ inhibition of influx was mediated via effects on the inducible high-affinity transport system rather than on the constitutive high-affinity transport system or the low-affinity transport system. Exposure to NH4+ also caused increased NO3- efflux; the largest effect was at low external [NO3-] in uninduced plants. In absolute terms, the reduction of influx made the dominant contribution to the observed reduction of net uptake of NO3-. Differences in response between plants induced with NO3- and those induced with NO2- indicate that NO2- may not be an appropriate analog for NO3- under all conditions.

Glucose transport in a methylotrophic yeast Hansenula polymorpha

Glucose transport was studied in a methylotrophic yeast Hansenula polymorpha. Two kinetically different glucose transport systems were revealed in cells grown under different growth conditions. Glucose-repressed cells exhibited a low-affinity transport system ( K m for glucose 1.75 mM) while glucose-derepressed and ethanol-grown cells had a high-affinity transport system ( K m for glucose 0.05–0.06 mM). The high- and low-affinity transport systems differed in substrate specificity, sensitivity to pH, dinitrophenol and protonophore carbonyl cyanide- m-chlorophenyl-hydrazone. The kinetic rearrangement of the glucose transport system in response to altered growth conditions was dependent on de novo protein synthesis.

Glucose transport in a methylotrophic yeast Hansenula polymorpha.

Glucose transport was studied in a methylotrophic yeast Hansenula polymorpha. Two kinetically different glucose transport systems were revealed in cells grown under different growth conditions. Glucose-repressed cells exhibited a low-affinity transport system (Km for glucose 1.75 mM) while glucose-derepressed and ethanol-grown cells had a high-affinity transport system (Km for glucose 0.05-0.06 mM). The high- and low-affinity transport systems differed in substrate specificity, sensitivity to pH, dinitrophenol and protonophore carbonyl cyanide-m-chlorophenyl-hydrazone. The kinetic rearrangement of the glucose transport system in response to altered growth conditions was dependent on de novo protein synthesis.

ATP-dependent transport of glutathione conjugate of 7beta, 8alpha-dihydroxy-9alpha,10alpha-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene in murine hepatic canalicular plasma membrane vesicles.

Glutathione (GSH) S-transferases (GSTs) have an important role in the detoxification of (+)-anti-7,8-dihydroxy-9,10-oxy-7,8,9, 10-tetrahydrobenzo[a]pyrene [(+)-anti-BPDE], which is the ultimate carcinogen of benzo[a]pyrene. However, the fate and/or biological activity of the GSH conjugate of (+)-anti-BPDE [(-)-anti-BPD-SG] is not known. We now report that (-)-anti-BPD-SG is a competitive inhibitor (Ki 19 microM) of Pi-class isoenzyme mGSTP1-1, which among murine hepatic GSTs is most efficient in the GSH conjugation of (+)-anti-BPDE. Thus the inhibition of mGSTP1-1 activity by (-)-anti-BPD-SG might interfere with the GST-catalysed GSH conjugation of (+)-anti-BPDE unless one or more mechanisms exist for the removal of the conjugate. The results of the present study indicate that (-)-anti-BPD-SG is transported across canalicular liver plasma membrane (cLPM) in an ATP-dependent manner. The ATP-dependent transport of (-)-anti-[3H]BPD-SG followed Michaelis-Menten kinetics (Km 46 microM). The ATP dependence of the (-)-anti-BPD-SG transport was confirmed by measuring the stimulation of ATP hydrolysis (ATPase activity) by the conjugate in the presence of cLPM protein, which also followed Michaelis-Menten kinetics. In contrast, a kinetic analysis of ATP-dependent uptake of the model conjugate S-[3H](2,4-dinitrophenyl)-glutathione ([3H]DNP-SG) revealed the presence of a high-affinity and a low-affinity transport system in mouse cLPM, with apparent Km values of 18 and 500 microM respectively. The ATP-dependent transport of (-)-anti-BPD-SG was inhibited competitively by DNP-SG (Ki 1.65 microM). Likewise, (-)-anti-BPD-SG was found to be a potent competitive inhibitor of the high-affinity component of DNP-SG transport (Ki 6.3 microM). Our results suggest that GST-catalysed conjugation of (+)-anti-BPDE with GSH, coupled with ATP-dependent transport of the resultant conjugate across cLPM, might be the ultimate detoxification pathway for this carcinogen.

Transport mechanisms of a glycoside, p-nitrophenyl- β- d-glucopyranoside, across rat small intestinal brush-border membranes

We examined the mechanism of p-nitrophenyl- β- d-glucopyranoside ( p-NP- β- d-Glc) transport in brush-border membrane vesicles from rat small intestine. The initial uptake rate showed an overshoot phenomenon in the presence of an inwardly directed sodium-ion concentration gradient. The overshoot disappeared when the sodium-ion concentration gradient was replaced with a potassium ion concentration gradient. d-Glucose and p-NP- β- d-Glc analogues inhibited the uptake, whereas uridine, leucine and disaccharide did not. Data on the concentration dependence of p-NP- β- d-Glc uptake indicated that two carrier-mediated systems are involved. The uptake via the high-affinity site required an inwardly directed sodium-ion concentration gradient, while the uptake via the low-affinity site proceeded such a gradient. d-Glucose competitively inhibited the initial uptake of p-NP- β- d-Glc via the high-affinity site with a K i value of 301 μM. The p-NP- β- d-Glc is transported in the small intestine via both the same carrier-mediated transport system that takes up d-glucose and a distinct low-affinity carrier-mediated transport system.

Possible link between a 35 kDa membrane protein and osmolyte transport in Staphylococcus aureus.

Accumulation of proline and glycine betaine occurs via three transport systems in Staphylococcus aureus (Pourkomailian and Booth 1992, 1994). A 35 kDa membrane protein was identified to be missing from a mutant, BP108, which lacks activity of the low affinity osmolyte transport system. The loss in activity of the low affinity transport system may be linked to the loss in expression of the 35 kDa membrane protein as a result of transposon insertion.

Cysteine Uptake for Accumulation of Glutathione by the Cyanobacterium Synechocystis strain PCC 6803.

By incubation with precursor amino acids, L-glutamate, L-cysteine, and glycine, or with L-cysteine, the unicellular cyanobacterium Synechocystis strain PCC 6803 increased intracellular glutathione levels, under illumination with white light. L-Cystine could not serve as the precursor. Two transport systems for L-cysteine were evident in the strain: a high (Ks; L-cysteine concentration giving one-half of the maximum uptake, 21~35 μM) and a low (Ks, 1.0~1.8 mM) affinity transport system. The latter, responsible for glutathione accumulation, was an energy-requiring process. L-Cysteine uptake by the two transport systems were Na(+)-independent, and inhibited by the presence of neutral amino acids and less inhibited by basic or acidic amino acids. These results suggested that the neutral amino acid symport(s) is involved in L-cysteine uptake for the GSH accumulation in this strain.

AtKUP1: A Dual-Affinity K + Transporter from Arabidopsis

Plant roots contain both highand low-affinity transport systems for uptake of K 1 from the soil. In this study, we characterize a K 1 transporter that functions in both highand low-affinity uptake. Using yeast complementation analysis, we isolated a cDNA for a functional K 1 transporter from Arabidopsis (referred to as AtKUP1 for Arabidopsis thaliana K 1 uptake). When expressed in a yeast mutant, AtKUP1 dramatically increased K 1 uptake capacity at both a low and high [K 1 ] range. Kinetic analyses showed that AtKUP1-mediated K 1 uptake displays a “biphasic” pattern similar to that observed in plant roots. The transition from the high-affinity phase ( K m of 44 m M) to the low-affinity phase ( K m of 11 mM) occurred at 100 to 200 m M external K 1 . Both lowand high-affinity K 1 uptake via AtKUP1 were inhibited by 5 mM or higher concentrations of NaCl. In addition, AtKUP1-mediated K 1 uptake was inhibited by K 1 channel blockers, including tetraethylammonium, Cs 1 , and Ba 2 1 . Consistent with a possible function in K 1 uptake from the soil, the AtKUP1 gene is primarily expressed in roots. We conclude that the AtKUP1 gene product may function as a K 1 transporter in Arabidopsis roots over a broad range of [K 1 ] in the soil.

AtKUP1: A Dual-Affinity K+ Transporter from Arabidopsis

Plant roots contain both high- and low-affinity transport systems for uptake of K+ from the soil. In this study, we characterize a K+ transporter that functions in both high- and low-affinity uptake. Using yeast complementation analysis, we isolated a cDNA for a functional K+ transporter from Arabidopsis (referred to as AtKUP1 for Arabidopsis thaliana K+ uptake). When expressed in a yeast mutant, AtKUP1 dramatically increased K+ uptake capacity at both a low and high [K+] range. Kinetic analyses showed that AtKUP1-mediated K+ uptake displays a "biphasic" pattern similar to that observed in plant roots. The transition from the high-affinity phase (K(m) of 44 microM) to the low-affinity phase (K(m) of 11 mM) occurred at 100 to 200 microM external K+. Both low- and high-affinity K+ uptake via AtKUP1 were inhibited by 5 mM or higher concentrations of NaCl. In addition, AtKUP1-mediated K+ uptake was inhibited by K+ channel blockers, including tetraethylammonium, Cs+, and Ba2+. Consistent with a possible function in K+ uptake from the soil, the AtKUP1 gene is primarily expressed in roots. We conclude that the AtKUP1 gene product may function as a K+ transporter in Arabidopsis roots over a broad range of [K+] in the soil.

Escherichia coli is unable to produce pyrroloquinoline quinone (PQQ).

Many bacteria can synthesize the cofactor pyrroloquinoline quinone (PQQ), a cofactor of several dehydrogenases, including glucose dehydrogenase (GCD). Among the enteric bacteria, Klebsiella pneumoniae has been shown to contain the genes required for PQQ biosynthesis. Escherichia coli and Salmonella typhimurium were thought to be unable to synthesize PQQ but it has been reported that strain EF260, a derivative of E. coli FB8, can synthesize PQQ after mutation and can oxidize glucose to gluconate via the GCD/PQQ pathway (F. Biville, E. Turlin & F. Gasser, 1991, J Gen Microbiol 137, 1775-1782). We have re-investigated this claim and conclude that it is most likely erroneous. (i) Strain EF260, isolated originally by Biville and coworkers, was unable to synthesize a holo-enzyme GCD unless PQQ was supplied to the growth medium. No GCD activity could be detected in membrane fractions. (ii) The amount of PQQ detected in the growth medium of EF260 was very low and not very different from that found in a medium with its parent strain or in a medium containing no cells. (iii) EF260 cells were unable to produce gluconate from glucose via the PQQ/GCD pathway. (iv) Introduction of a gcd::Cm deletion in EF260, eliminating GCD, did not affect glucose metabolism. This suggested a pathway for glucose metabolism other than the PQQ/GCD pathway. (v) Glucose uptake and metabolism in EF260 involved a low-affinity transport system of unknown identity, followed most likely by phosphorylation via glucokinase. It is concluded that E. coli cannot synthesize PQQ and that it lacks genes required for PQQ biosynthesis.

Isolation of the putP gene of Corynebacterium glutamicum and characterization of a low-affinity uptake system for compatible solutes

Corynebacterium glutamicum accumulates the compatible solutes proline, glycine betaine, and ectoine under conditions of high osmolality. Uptake of proline is mediated by both a high-affinity and a low-affinity secondary transport system. The low-affinity uptake system also accepts glycine betaine and ectoine as substrates. In the present study, the gene encoding the high-affinity proline uptake system PutP was isolated by heterologous complementation of Escherichia coli mutant strain WG389, which lacks the transport systems BetT, PutP, ProP, and ProU and is unable to synthesize proline and glycine betaine. This gene (putP) encodes a protein of 524 amino acids that shares identity with the proline transport systems PutP of E. coli, Staphylococcus aureus, Salmonella typhimurium, Haemophilus influenzae, and Klebsiella pneumoniae. Functional studies of PutP synthesized in E. coli mutant strain MKH13, which also lacks the transport systems for compatible solutes and is unable to synthesize glycine betaine, revealed that this carrier system is not regulated by the external osmolality on the level of activity. Km values of 7.6 mM for proline and 1.3 mM for sodium as cotransported ion were determined. Deletion of the putP gene allowed the functional characterization of another proline uptake system with low affinity.

Characterization of Methanobacterium thermoautotrophicum Marburg mutants defective in regulation of L-tryptophan biosynthesis.

Three nitrosoguanidine-induced mutants of the archaeon Methanobacterium thermoautotrophicum Marburg resistant to 5-methyltryptophan were isolated and characterized. They were found to take up L-tryptophan, as wild-type cells, via an energy-dependent, low-affinity transport system specific for L-tryptophan, with a Km of 300 microM and a Vmax of 7 nmol/mg (dry weight)/min. Resistance to 5-methyltryptophan was not due to feedback-resistant anthranilate synthase but to constitutive expression of the trp genes, as measured by the specific activities of anthranilate synthase and tryptophan synthase, the enzymes encoded by trpEG and trpB, respectively, of the trpEGCFBAD gene cluster. Estimation of trpE mRNA obtained from mutant cells grown in minimal medium with or without L-tryptophan suggested that constitutive expression resulted from deficient transcriptional regulation. The enhanced expression of the trp genes in the mutants was found to result in intracellular L-tryptophan pools that were two- to fourfold higher than in the wild type. Sequencing of the region upstream of trpE revealed in two mutants point mutations mapping on the 5'-side of the archaeal box A, whereas in the third mutant this region did not differ from that of the wild type. These results suggest that (i) in M. thermoautotrophicum the 5-methyltryptophan-resistant phenotype arises from lesions in components of a regulatory system controlling transcription of the trp genes and (ii) cis-acting sequence elements in front of the trpE promoter may form part of this system.

NO3- and ClO3- fluxes in the chl1-5 mutant of Arabidopsis thaliana. Does the CHL1-5 gene encode a low-affinity NO3- transporter?

The CHL1 gene is considered to encode a low-affinity transport system (LATS) for NO3- in Arabidopsis thaliana (Y.-F. Tsay, J.I. Schroeder, K.A. Feldmann, N.M. Crawford [1993] Cell 72: 705-713). However, the anticipated reduced NO3- uptake by the LATS associated with loss of CHL1 gene activity in chl1-5 deletion mutants was evident only when plants were grown on NH4NO3. When KNO3 was the sole N source, NO3- accumulation and short-term tracer influx (using 13NO3- and 15NO3-) in leaves and roots of wild-type and mutant plants were essentially identical. Nevertheless, root uptake of 36CIO3- by the LATS and CIO3- accumulation in roots and shoots of mutant plants were significantly lower than in wild-type plants when grown on KNO3. One explanation for these results is that a second LATS is able to compensate for the chl1-5 deficiency in KNO3-grown plants. Growth on NH4NO3 may down-regulate the second LATS enough that the anticipated difference in NO3- uptake becomes apparent.

Purification and Characterization of Fet3 Protein, a Yeast Homologue of Ceruloplasmin

The FET3 gene product of Saccharomyces cerevisiae is an essential component of the high affinity iron transport system. Based on FET3 sequence homology to the multicopper oxidase family and iron oxidation studies in spheroplasts (De Silva, D. M., Askwith, C. C., Eide, D., and Kaplan, J. (1995) J. Biol. Chem. 270, 1098-1101), it was hypothesized that the Fet3 protein (Fet3p) was a cell surface ferroxidase. To further characterize the protein, we have isolated Fet3p from yeast membranes and purified the protein to apparent homogeneity. Consistent with its localization at the plasma membrane, Fet3p is a glycosylated protein. SDS-polyacrylamide gel electrophoresis analysis showed that the protein was present in two differentially glycosylated forms of approximately 120 and 100 kDa. Purified Fet3p is a copper-containing protein that is able to catalyze the oxidation of a variety of organic compounds in addition to ferrous iron. Azide and metal chelators strongly inhibited enzyme activity. Iron appeared to be the best substrate for the enzyme, and the apparent Km for ferrous oxidation was 2 microM. Interestingly, Fet3p was able to effectively catalyze the incorporation of iron onto apotransferrin. We conclude that Fet3p is a ferro-O2-oxidoreductase in yeast, homologous to the human plasma protein ceruloplasmin.

Listeria monocytogenes Scott A transports glucose by high-affinity and low-affinity glucose transport systems.

Listeria monocytogenes transported glucose by a high-affinity phosphoenolpyruvate-dependent phosphotransferase system and a low-affinity proton motive force-mediated system. The low-affinity system (Km = 2.9 mM) was inhibited by 2-deoxyglucose and 6-deoxyglucose, whereas the high-affinity system (Km = 0.11 mM) was inhibited by 2-deoxyglucose and mannose but not 6-deoxyglucose. Cells and vesicles artificially energized with valinomycin transported glucose or 2-deoxyglucose at rates greater than those of de-energized cells, indicating that a membrane potential could drive uptake by the low-affinity system.

Ammonium fluxes into plant roots: Energetics, kinetics and regulation

AbstractAmmonium uptake across the plasma membranes of seedling roots of intact rice plants is thermodynamically active at low external concentrations, and consequently, electrogenic uniport is an unlikely mechanism for influx. At higher NH4+ concentrations uptake is passive and electrogenic uniport is a possibility. While passive permeation of NH3 is also possible at high external [NH4+], influx measurements at 10 mM NH4+ demonstrated a pH dependence which was inconsistent with significant NH3 permeation. Kinetic studies using 13NH4+ established that influx at low external [H4+] occurred via high affinity transport systems (HATS) in rice and spruce, while at higher [NH4+], influx was mediated by low affinity transport systems (LATS), that showed linear concentration dependence. Ammonium influx via the HATS was shown to be up‐regulated or down‐regulated in response to changes of N status, whereas influx in the LATS was insensitive to N status. The identity or identities of the regulatory signals responsible for controlling influx are discussed.

Influence of Glycine Betaine on Degradation of Eicosane by Marinobacter hydrocarbonoclasticus at High Salinity

Summary The osmolyte glycine betaine (GB) was accumulated but not metabolized by the marine bacterium Marinobacter hydrocarbonoclasticus possibly via a low-affinity transport system. It exerted a negative infuence on growth, more pronounced in media with the highest NaCl concentrations (≥ 2 M). GB also reduced eicosane metabolisation, possibly as a consequence of a decrease in ectoine synthesis. At low salinity (0.2 M NaCl), however, GB enhanced eicosane degradation. It is hypothesized that this effect was due to the prevention of a decrease in eicosane emulsification, observed in media not supplemented with GB.

MOAT4, a novel multispecific organic-anion transporter for glucuronides and mercapturates in mouse L1210 cells and human erythrocytes.

Glucuronides and mercapturates were examined as possible high-affinity substrates for a low-affinity ATP-dependent transport system for 2,4-dinitrophenyl S-glutathione (DNP-SG) in mouse L1210 cells. Initial inhibitor studies with inside-out vesicles revealed that the low-affinity transport of [3H]DNP-SG (Km 450 microM) exhibits a high sensitivity to N-acetyl 2,4-dinitrophenyl cysteine (NAc-DNP-Cys) (Ki 5.0 microM) and alpha-naphthyl beta-D-glucuronide (naphthyl glucuronide) (Ki 8.5 microM). Direct transport measurements showed the presence of ATP-dependent uptake activities for NAc-DNP-[35S]Cys and naphthyl [14C] glucuronide, and Km values for half-maximal transport were comparable to the Ki values of these compounds for inhibition of [3H]DNP-SG transport. Transport of [3H]DNP-SG, NAc-DNP-[35S]Cys and naphthyl [14C]glucuronide each showed the same sensitivity to various anions and anion conjugates. Inhibition was competitive and was most potent for bilirubin ditaurate, indoprofen, 4-biphenylacetic acid, 4-acridine 4 beta-D-glucuronide, N-acetyl leukotriene E4, 17 beta-oestradiol 3 beta-D-glucuronide and taurolithocholate 3-sulphate. Inside-out vesicles from human erythrocytes contain a comparable ATP-dependent transport system. These results show that NAc-DNP-Cys and naphthyl glucuronide are high-affinity substrates for a single system identified previously as a low-affinity transporter of DNP-SG. Substrate and inhibitor studies identify this system as a novel multispecific organic-anion transport system (MOAT4) that accommodates glucuronides and mercapturates and is distinct from other MOAT transporters. Human erythrocytes contain an additional ATP-dependent system for NAc-DNP-Cys (Km 33 microM) that does not transport monoglucuronides.

Evidence for Substrate Induction of a Nitrate Efflux System in Barley Roots.

Induction of an NO3- efflux system in intact barley (Hordeum vulgare L.) roots was demonstrated. Since the measurement of NO3- efflux is dependent on its accumulation, experiments were devised to facilitate accumulation under noninducing conditions. This was accomplished by incubating seedlings in 10 mM NO3- in the presence of RNA and protein synthesis inhibitors. Under these conditions NO3- uptake is mediated by constitutive high- and low-affinity transport systems. Control roots were incubated with 1.0 mM NO3-. This resulted in the accumulation of similar levels of NO3- in both treated and control roots; however, cytoplasmic NO3- efflux from inhibitor-treated roots was much lower than from control roots. Following a brief lag period, efflux rates increased rapidly in the presence of NO3- for 8 to 12 h. The NO3- efflux system was also induced by ambient NO2-. After induction the efflux system was relatively stable in the presence of RNA and protein synthesis inhibitors as long as NO3- or NO2- was present. These results suggest that NO3- efflux may be an inducible system requiring both RNA and protein synthesis, as does induction of the uptake system. The efflux system, however, has a much slower turnover rate than the uptake system.