Methionine transport in Pseudomonas aeruginosa.

A high-affinity (Km = 2.7 x 10(-7) M) energy-requiring methionine-transport system has been characterized in RM 46 and RM 48, two different PAO methionine auxotrophs of Pseudomonas aeruginosa. After 8 s of transport 40--60% of the methionine label in the alcohol extract appears in S-adenosyl-L-methionine (SAM) with the remaining activity in free methionine. Methionine transport required a high degree of structural specificity for transport. Stimulation of transport occurred by addition of glucose or organic acids. The ability of a given substrate to stimulate transport was related to the type of carbon source used for growth. Transport was sensitive to sulfhydryl reagents and required oxidative phosphorylation, as indicated by the inhibitory effects of anaerobiosis, cyanide, and arsenate. The degree of inhibition by arsenate correlated with the level of ATP in the cell. Rapid transport in a SAM-deficient mutant (TM 1) and inhibition by arsenate of transport in this mutant suggested that SAM formation was not directly linked to transport and that ATP supplied energy for transport. Inhibition by arsenate was more severe in glucose- compared to citrate-stimulated cells. This result was also observed with proline transport indicating that this was not a peculiarity of the methionine-transport system. These data emphasize the close link between glucose metabolism, ATP levels, and transport. This ATP level is not so critical for transport in cells metabolizing citrate.     

Characterization of the D-allose-mediated regulation of sugar transport in Chinese hamster fibroblasts

Exposure to D-allose has been demonstrated to lead to decreased 2-deoxy-D-glucose (2-DG) and 3-0-methyl-D-glucose transport in the V79 Chinese hamster lung fibroblast cell line. The effect of D-allose 1) was maximal after 4 hours exposure to the cells; 2) was optimal between 2.77 and 5.55 mM D-allose; and 3) led to a decreased Vmax for 2-DG transport with no change in the transport Km value. The decrease in 2-DG transport induced by D-allose was reversible and the reversal was differentially affected by cycloheximide, being blocked by a low concentration of cycloheximide (0.05 micrograms/ml) but not a high concentration of the inhibitor (5 micrograms/ml). D-allose did not competitively inhibit the transport of 2-DG while D-glucose under similar conditions yielded a Kl for 2-DG transport inhibition of 1.7 mM. Additionally, D-allose did not affect the phosphorylation of 2-DG by hexokinase in cell-free cytosol. The data indicate that D-allose has significant lowering effects on sugar transport activity. Additionally, while the sugar itself may be the active component in sugar transport regulation, the effect is not blocked by inhibition of protein synthesis but the synthesis of a regulatory protein(s) may be involved in the return of sugar transport following D-allose removal.     

Inhibition by forskolin of insulin-stimulated glucose transport in L6 muscle cells.

The cardioactive diterpene forskolin is a known activator of adenylate cyclase, but recently a specific interaction of this compound with the glucose transporter has been identified that results in the inhibition of glucose transport in several human and rat cell types. We have compared the sensitivity of basal and insulin-stimulated hexose transport to inhibition by forskolin in skeletal muscle cells of the L6 line. Forskolin completely inhibited both basal and insulin-stimulated hexose transport when present during the transport assay. The inhibition of basal transport was completely reversible upon removal of the diterpene. In contrast, insulin-stimulated hexose transport did not recover, and basal transport levels were attained instead. This effect of inhibiting (or reversing) the insulin-stimulated fraction of transport is a novel effect of the diterpene. Forskolin treatment also inhibited the stimulated fraction of transport when the stimulus was by 4 beta-phorbol 12,13-dibutyrate, reversing back to basal levels. Half-maximal inhibition of the above-basal insulin-stimulated transport was achieved with 35-50 microM-forskolin, and maximal inhibition with 100 microM. Forskolin did not inhibit 125I-insulin binding under conditions where it caused significant inhibition of insulin-stimulated hexose transport. Forskolin significantly elevated the cyclic AMP levels in the cells; however its inhibitory effect on the above basal, insulin-stimulated fraction of hexose transport was not mediated by cyclic AMP since: (i) 8-bromo cyclic AMP and cholera toxin did not mimic this effect of the diterpene, (ii) significant decreases in cyclic AMP levels caused by 2',3'-dideoxyadenosine in the presence of forskolin did not prevent inhibition of insulin-stimulated hexose transport, (iii) isobutylmethylxanthine did not potentiate forskolin effects on glucose transport but did potentiate the elevation in cyclic AMP, and (iv) 1,9-dideoxyforskolin, which does not activate adenylate cyclase, inhibited hexose transport analogously to forskolin. We conclude that forskolin can selectively inhibit the insulin- and phorbol ester-stimulated fraction of hexose transport under conditions where basal transport is unimpaired. The results are compatible with the suggestions that glucose transporters operating in the stimulated state (insulin or phorbol ester-stimulated) differ in their sensitivity to forskolin from transporters operating in the basal state, or, alternatively, that a forskolin-sensitive signal maintains the stimulated transport rate.     

Antigen-specific stimulation of amino acid transport in bovine lymphocytes

Treatment of bovine lymphocytes isolated from animals which were either infected with Mycobacterium bovis or sensitized to a purified protein derivative (PPD-B) from this organism induced an increase in the transport of α-aminoisobutyric acid (AIB) and α-methylaminoisobutyric acid (MeAIB). PPD-B did not stimulate these transport activities in lymphocytes from nonsensitized animals. The transport stimulation was first measurable after about 7 hours of treatment, reached about a two-fold enhancement after 20 hours, and continued to increase to 30- to 40-fold after 6 days. The stimulation of AIB transport was inhibited by both ouabain and cycloheximide. Experiments to determine transport system specificities in nonstimulated lymphocytes showed that MeAIB transport was primarily by the Na⁺-dependent, A-system, and leucine transport was mostly by Na⁺-independent system(s). In contrast, AIB transport was about 25% by the A-system, 25% by at least one Na⁺-dependent, non-A-system, and 50% by one or more Na⁺-independent system(s). Analysis of the three components of AIB transport after treatment with PPD-B showed that: (1) transport by both the A-system and the Na⁺-independent system(s) was stimulated; (2) A-system transport was stimulated to a larger extent than Na⁺-independent transport; and (3) Na⁺-dependent, non-A-system transport was not stimulated significantly.     

Sequences in the nonconsensus nucleotide-binding domain of ABCG5/ABCG8 required for sterol transport

ATP-binding cassette transporters ABCG5 (G5) and ABCG8 (G8) form a heterodimer that transports cholesterol and plant sterols from hepatocytes into bile. Mutations that inactivate G5 or G8 cause hypercholesterolemia and premature atherosclerosis. We showed previously that the two nucleotide-binding domains (NBDs) in the heterodimer are not functionally equivalent; sterol transport is abolished by mutations in the consensus residues of NBD2 but not of NBD1. Here, we examined the structural requirements of NBD1 for sterol transport. Substitutions of the D-loop aspartate and Q-loop glutamine in either NBD did not impair sterol transport. The H-loop histidine of NBD2 (but not NBD1) was required for sterol transport. Exchange of the signature motifs between the NBDs did not interfere with sterol transport, whereas swapping the Walker A, Walker B, and signature motifs together resulted in failure to transport sterols. Selected substitutions within NBD1 altered substrate specificity: transport of plant sterols by the heterodimer was preserved, whereas transport of cholesterol was abolished. In summary, these data indicate that NBD1, although not required for ATP hydrolysis, is essential for normal function of G5G8 in sterol transport. Both the position and structural integrity of NBD2 are essential for sterol transport activity.     

FAPP2 is involved in the transport of apical cargo in polarized MDCK cells

Phosphatidylinositol-4-phosphate (PI(4)P) is the main phosphoinositide in the Golgi complex and has been reported to play a pleiotropic role in transport of cargo from the trans-Golgi network to the plasma membrane (PM) in polarized Madin-Darby canine kidney (MDCK) cells. Overexpression of the chimeric fluorescent protein encoding the pleckstrin homology domain, which is specific for PI(4)P, inhibited both apical and basolateral transport pathways. The transport of apical cargo from the Golgi was shown to be specifically decreased by adenovirus-mediated RNA interference directed against PI(4)P adaptor protein (FAPP) 2. FAPP1 depletion had no effect on transport. On the other hand, FAPP2 was not involved in the Golgi-to-PM transport of cargo that was targeted to the basolateral membrane domain. Thus, we conclude that FAPP2 plays a specific role in apical transport in MDCK cells.     

Serine and glycine transport in fetal ovine hepatocytes

The role of hepatic serine and glycine transport in the regulation of the biosynthesis of serine by the fetal liver has not been studied. The goal of this study was to characterize serine and glycine transport and utilization at physiologic concentrations in primary cultures of fetal ovine hepatocytes. Primary culture of hepatocytes from mid gestation ( approximately 90 days) and term ( approximately 135 days) fetal sheep were studied after overnight serum free culture. At both gestational ages, the initial rate for sodium dependent 300 microM serine transport (1697+/-131 pmoles/min/mg protein at mid, 1765+/-544 at term) was fourfold greater than sodium dependent 300 microM glycine transport (309+/-54 at mid, 579+/-252 at term). At physiologic concentrations (300 microM), 69+/-7% of serine and 49+/-8% of glycine transport was sodium dependent. At term, sodium dependent serine transport has a V(max) of 1751+/-348 pmoles/min/mg protein and a K(m) of 159+/-111 microM. Sodium independent serine transport has a V(max) of 904+/-185 and a K(m) of 416+/-188 microM. Sodium dependent glycine transport has a V(max) of 410+/-69 and a K(m) of 2290+/-895 microM while sodium independent glycine transport exhibits non-saturable kinetics. Glycine (300 microM) sodium dependent transport was not inhibited by methyl-AIB while sodium dependent 300 microM serine transport was inhibited (31%). n-Ethylmaleimide inhibited sodium dependent serine and glycine transport by 36+/-9% and 37+/-2% respectively in term hepatocytes. Cysteine inhibited sodium dependent serine transport by 37%. Sodium independent glycine transport at 300 microM was higher in low glucose (1.1 mM) medium (881+/-76 pmoles/min/mg protein) compared to high glucose (5.5 mM) medium (510+/-60 P=0.004). There were no significant differences in serine or glycine incorporation into RNA, DNA, glycogen or lipid and protein. The predominance of serine transport over glycine at physiologic concentrations suggests that inward cellular amino acid transport of serine and glycine is not likely to be a regulatory mechanism that would favor serine biosynthesis in fetal ovine hepatocytes.     

Transport of a fluorescent cAMP analog in teleost proximal tubules

Previous studies have shown that killifish (Fundulus heteroclitus) renal proximal tubules express a luminal membrane transporter that is functionally and immunologically analogous to the mammalian multidrug resistance-associated protein isoform 2 (Mrp2, ABCC2). Here we used confocal microscopy to investigate in killifish tubules the transport of a fluorescent cAMP analog (fluo-cAMP), a putative substrate for Mrp2 and Mrp4 (ABCC4). Steady-state luminal accumulation of fluo-cAMP was concentrative, specific, and metabolism-dependent, but not reduced by high K+ medium or ouabain. Transport was not affected by p-aminohippurate (organic anion transporter inhibitor) or p-glycoprotein inhibitor (PSC833), but cell-to-lumen transport was reduced in a concentration-dependent manner by Mrp inhibitor MK571, leukotriene C4 (LTC4), azidothymidine (AZT), cAMP, and adefovir; the latter two compounds are Mrp4 substrates. Although MK571 and LTC4 reduced transport of the Mrp2 substrate fluorescein-methotrexate (FL-MTX), neither cAMP, adefovir, nor AZT affected FL-MTX transport. Fluo-cAMP transport was not reduced when tubules were exposed to endothelin-1, Na nitroprusside (an nitric oxide generator) or phorbol ester (PKC activator), all of which signal substantial reductions in cell-to-lumen FL-MTX transport. Fluo-cAMP transport was reduced by forskolin, and this reduction was blocked by the PKA inhibitor H-89. Finally, in membrane vesicles from Spodoptera frugiperda (Sf9) cells containing human MRP4, ATP-dependent and specific uptake of fluo-cAMP could be demonstrated. Thus, based on inhibitor specificity and regulatory signaling, cell-to-lumen transport of fluo-cAMP in killifish renal tubules is mediated by a transporter distinct from Mrp2, presumably a teleost form of Mrp4.     

The effect of catecholamines and adenosine deaminase on the glucose transport system in rat adipocytes

2-Deoxyglucose uptake (3 min) and 3-O-methylglucose transport (2 s) was measured in rat adipocytes preincubated with 5 microM epinephrine plus adenosine deaminase as described by Green (Green, A. (1983) FEBS Lett. 152, 261-264). 2-Deoxyglucose uptake was about 95% depressed in insulin-treated, but not in 'basal', cells preincubated with epinephrine plus adenosine deaminase for 60 min in broad agreement with Green's report. However, this depression was caused by a decrease in sugar phosphorylation rather than transport. In similarly incubated cells, transport of 3-O-methylglucose, a sugar analogue not phosphorylated in the adipocytes, was not affected by catecholamine plus adenosine deaminase. However, a decrease in transport of about 60% was observed both in the absence and the presence of insulin when the albumin concentration was high enough and the cell concentration low enough to prevent accumulation of free fatty acids in the medium. In addition, the insulin sensitivity with regard to hexose transport was markedly reduced. Transport was approximately doubled in cells incubated with 5 microM epinephrine in the absence of adenosine deaminase. Thus, epinephrine at a high concentration stimulates hexose transport in the absence of adenosine deaminase (presence of adenosine) whereas it inhibits both basal and insulin-stimulated transport in the presence of adenosine deaminase (absence of adenosine).     

Regulation of sugar transport in cultured diploid human skin fibroblasts

The regulation of hexose transport under glucose-starvation conditions was studied in cultured human skin fibroblasts. Glucose starvation enhanced the transport of 2-DG and 3-O-methyl-D-glucose (3-OMG) but not of L-glucose. Glucose-starvation enhanced transport was inhibited by cytochalasin B (10 μM). The starvation-induced change in 2-DG transport was due to an increase in the V_max of both the high and low affinity transport sites (2.8- and 2.4-fold, respectively) with no effect on their K_ms. The presence of 5.55 mM galactose, fructose, or L-glucose in the medium resulted in transport increases similar to those seen in glucose-starved cells, while the presence of 5.55 mM glucose, mannose, or 3-OMG repressed 2-DG transport. Glucose-starvation enhancement of 2-DG transport was blocked by cycloheximide (20 μg/ml) but not by actinomycin D (0.03 μg/ml) or α-amanitin (3.5 μM). Readdition of glucose (5.55 mM) for six hours to glucose-starved cells led to a rapid decrease in hexose transport that could be blocked by cycloheximide but not actinomycin D. Although readdition of 3-OMG to glucose-starved cells had little effect on reversing the transport increases, glucose plus 3-OMG were more effective than glucose alone. Serum containing cultures (10% v/v) of glucose-fed or glucose-starved cells exhibited rapid decreases in 2-DG transport when exposed to glucose-containing serum-free medium. These decreases were prevented by employing glucose-free, serum-free medium. The data indicate that hexose transport regulation in cultured human fibrob asts involves protein synthesis of hexose carriers balanced by interactions of glucose with a regulatory protein(s) and glucose metabolism as they affect the regulation and/or turnover of the carrier molecules.     

Sodium-dependent transport of L-leucine in membrane vesicles prepared from Pseudomonas aeruginosa.

Membrane vesicles were prepared by osmotic lysis of spheroplasts of Pseudomonas aeruginosa strain P14, and the active transport of amino acids was studied. D-Glucose, gluconate, and L-malate supported active transport of various L-amino acids. The respiration-dependent leucine transport was markedly stimulated by Na+. Moreover, without any respiratory substrate, leucine was also transported transiently by the addition of Na+ alone. This transient uptake of leucine was not inhibited either by carbonyl cyanide p-trifluoromethyoxyphenylhydrazone or by valinomycin, but was completely abolished by gramicidin D. Increase in the concentration of Na+ of the medium resulted in a decrease of the Km for L-leucine transport, whereas the Vmax was not significnatly affected. Active transport of leucine was inhibited competitively by isoleucine or by valine, whose transport was also stimulated by Na+. On the other hand, Na+ was not required for the uptake of other L-amino acids tested, but rather was inhibitory for some of them. These results show (i) that a common transport system for branched-chain amino acids exists in membrane vesicles, (ii) that the system requires Na+ for its activity, and (iii) that an Na+ gradient can drive the system.     

Evidence for modulation by ATP of amino acid transport in human placenta

The effect of ATP on placental amino acid transport was studied by measuring the uptake of alpha-(methylamino)-isobutyrate in brush border microvillous plasma membrane vesicles prepared from human full-term placental syncytiotrophoblasts which were incubated with or without ATP. The presence of a Na+ gradient from the outside to the inside of the vesicles prepared after incubation with ATP resulted in a higher initial rate and an increased transport of alpha-(methylamino)-isobutyrate, while Na+ gradient-independent alpha-(methylamino)-isobutyrate uptake was not different in either type of membrane vesicle. The increase in transport activity was not inhibited by cycloheximide. Kinetic analysis showed that ATP enhanced transport activity by increasing the maximal velocity (Vmax) of transport, without significant changes in the affinity (Km) of the carrier for the substrate, suggesting an increase in carrier number in placental syncytiotrophoblasts incubated with ATP.     

Energy coupling of facilitated transport of inorganic ions in Rhodopseudomonas sphaeroides.

Within the scope of a study on the effects of changes in medium composition on the proton motive force in Rhodopseudomonas sphaeroides, the energy coupling of sodium, phosphate, and potassium (rubidium) transport was investigated. Sodium was transported via an electroneutral exchange system against protons. The system functioned optimally at pH 8 and was inactive below pH 7. The driving force for the phosphate transport varied with the external pH. At pH 8, Pi transport was dependent exclusively on delta psi (transmembrane electrical potential), whereas at pH 6 only the delta pH (transmembrane pH gradient) component of the proton motive force was a driving force. Potassium (rubidium) transport was facilitated by a transport system which catalyzed the electrogenic transfer of potassium (rubidium) ions. However, in several aspects the properties of this transport system were different from those of a simple electrogenic potassium ionophore such as valinomycin: (i) accumulated potassium leaked very slowly out of cells in the dark; and (ii) the transport system displayed a threshold in the delta psi, below which potassium (rubidium) transport did not occur.     

Phospho-dependent association of neurofilament proteins with kinesin in situ

Recent studies demonstrate co-localization of kinesin with neurofilament (NF) subunits in culture and suggest that kinesin participates in NF subunit distribution. We sought to determine whether kinesin was also associated with NF subunits in situ. Axonal transport of NF subunits in mouse optic nerve was perturbed by the microtubule (MT)-depolymerizing drug vinblastine, indicating that NF transport was dependent upon MT dynamics. Kinesin co-precipitated during immunoprecipitation of NF subunits from optic nerve. The association of NFs and kinesin was regulated by NF phosphorylation, since (1) NF subunits bearing developmentally delayed phospho-epitopes did not co-purify in a microtubule motor preparation from CNS while less phosphorylated forms did; (2) subunits bearing these phospho-epitopes were selectively not co-precipitated with kinesin; and (3) phosphorylation under cell-free conditions diminished the association of NF subunits with kinesin. The nature and extent of this association was further examined by intravitreal injection of (35)S-methionine and monitoring NF subunit transport along optic axons. As previously described by several laboratories, the wave of NF subunits underwent a progressive broadening during continued transport. The front, but not the trail, of this broadening wave of NF subunits was co-precipitated with kinesin, indicating that (1) the fastest-moving NFs were associated with kinesin, and (2) that dissociation from kinesin may foster trailing of NF subunits during continued transport. These data suggest that kinesin participates in NF axonal transport either by directly translocating NFs and/or by linking NFs to transporting MTs. Both Triton-soluble as well as cytoskeleton-associated NF subunits were co-precipitated with kinesin; these data are considered in terms of the form(s) in which NF subunits undergo axonal transport.     

Effect of sphingoid bases on basal and insulin-stimulated 2-deoxyglucose transport in skeletal muscle.

Incubation of rat soleus muscles with 50 microM sphingosine or 50 microM sphinganine augmented basal 2-deoxy-D-glucose (2DG) transport 32%, but reduced the response to 0.1 and 1.0 mU insulin/ml by 17 and 27%, respectively. When the muscles were incubated with 50 microM phytosphingosine, a 63-93% increase in basal 2DG transport was observed. However, this treatment had no effect on insulin-stimulated 2DG transport. The phytosphingosine-induced increase in basal 2-DG transport was inhibited 93 and 98% with 35 and 70 microM cytochalasin B, respectively, suggesting that it is mediated by glucose transporters. Cellular accumulation of L-glucose, which is not mediated by glucose transporters, was not affected by phytosphingosine. It is concluded that (a) both sphingosine and sphinganine increase basal 2DG transport in muscle but diminish insulin-stimulated transport, and (b) phytosphingosine stimulates basal 2DG transport in muscle by a mechanism involving glucose transporters.     

Segmental differences in short-chain fatty acid transport in rabbit colon: Effect of pH and Na

Short-chain fatty acids (SCFAs) are the predominant luminal anion in the mammalian colon. Although they are rapidly absorbed in vivo, little is known about the mechanisms of transepithelial transport in vitro. Previous studies have suggested that SCFA transport may be linked to Na absorption or an anion exchange mechanism. We compared the transport of propionate under short-circuit conditions in rabbit proximal and distal colon to determine whether there were segmental differences, how SCFAs may be linked to either Na absorption or anion transport, and whether SCFAs, as weak electrolytes, may be affected by transepithelial pH gradients. In distal colon, propionate transport was not significantly altered by stimulation of electrogenic Na absorption, epinephrine or Cl removal. However, a modest transepithelial pH gradient (luminal 6.8/serosal 7.4) stimulated propionate absorption. In proximal colon, propionate transport was significantly altered by manuevers that either stimulated (lowered [Na] in the bathing media) or inhibited (theophylline) apical Na−H exchange. Neither Cl removal, nor the anion exchange inhibitor DIDS, nor a transepithelial bicarbonate gradient, altered propionate transport. A transepithelial pH gradient inhibited propionate secretion, but not in a manner entirely consistent with the effect of pH on the distribution of a weak electrolyte. These results suggest that there is significant segmental heterogeneity in colonic SCFA transport; that transepithelial propionate fluxes are altered by changes in pH or electroneutral Na absorption (Na−H exchange), but not by chloride removal, bicarbonate gradients or electrogenic Na absorption. Regulation of SCFA transport may be an important factor in the physiology of colonic fluid balance.     

Stimulation of glucose transport by guanine nucleotides in permeabilized rat adipocytes.

Effects of guanine nucleotides on glucose transport were studied in permeabilized rat epididymal fat cells. GTP gamma S and Gpp(NH)p, but not App(NH)p, stimulated 3-O-methylglucose transport. Effect of GTP gamma S was dose-dependent, being detectable at 0.1 mM, and 1.0 mM GTP gamma S stimulated glucose transport to the same extent as insulin. GTP gamma S (0.3 mM) enhanced insulin-stimulated glucose transport while 1 mM GTP gamma S did not affect insulin-mediated transport. GDP beta S had no effect on glucose transport by itself but rather enhanced insulin action. NaF, which is known to activate trimeric G proteins, increased glucose transport to the same extent as insulin. Likewise, mastoparan augmented glucose transport. These results indicate that a certain type of trimeric G protein(s) is involved in the regulation of glucose transport.     

Characterization of L-threonine and L-glutamine transport in murine P388 leukemia cells in vitro. Presence of an N-like amino acid transport system

The transport of L-threonine and L-glutamine into murine P388 leukemia cells has been characterized. Threonine appears to be a specific substrate for a Na+-dependent amino acid transport system similar to system ASC of the HTC hepatoma cell. Threonine transport is uninhibited by 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid and alpha-(methylamino)isobutyric acid, shows a pattern of transport similar to that seen in HTC hepatoma cells over the pH range of 5.5-7.5, and is inhibited by L-serine and L-cysteine. Approximately two-thirds of glutamine transport into P388 cells also appears to enter P388 cells via this ASC-analogous system. However, based upon (a) inhibition studies with threonine (where the K1 of threonine inhibition of glutamine transport was 7-fold the Km of threonine transport), (b) inhibition analysis of glutamine transport with various amino acids and amino acid analogues, and (c) different patterns of transport between threonine and glutamine over the pH range of 5.5-7.5, approximately one-third of glutamine transport can be attributed to a second Na+-dependent amino acid transport system. This system appears to be similar to the system N of rat hepatocytes. Glutamine and threonine do not appear to enter P388 cells via systems A or L to any significant degree. P388 cells do not appear to exhibit 'adaptive regulation' of amino acid transport. Differences in 'adaptive regulation' could therefore not be utilized for comparing threonine and glutamine transport.     

Development of Salt-Resistant Active Transport in a Moderately Halophilic Bacterium

The moderately halophilic bacterium Vibrio costicola accumulates α-aminoisobutyric acid (AIB) by active transport. Substantial amounts of Na⁺ ions are needed for this transport. This is not due to an ionic requirement for respiration; cells respire as well as KCl as in NaCl but do not transport AIB in KCl. In cells grown in the presence of 1.0 or 2.0 M NaCl, AIB transport took place in higher NaCl concentrations than in cells grown in the presence of 0.5 M NaCl. The latter cells developed salt-resistant transport when they were exposed to 1.0 M NaCl in the presence of chloramphenicol and other antibiotics that inhibit protein synthesis. Two levels of salt-resistant transport were observed. One level (resistance to 3.0 M NaCl) developed in 1.0 M NaCl without the addition of nutrients, did not seem to require an increase in internal solute concentration, and was not lost when cells grown in 1.0 M NaCl were suspended in 0.5 M NaCl. The second level (resistance to 4.0 M NaCl) developed in 1.0 M NaCl only when nutrients were added, may have required an increased internal solute concentration, and was lost when 1.0 M NaCl-grown cells were suspended in 0.5 M NaCl or KCl. Among the substances that stimulated the development of salt-resistant AIB transport, betaine was especially active. Furthermore, direct addition of betaine permitted cells to transport AIB at higher NaCl concentrations. High salt concentrations inhibited endogenous respiration to a lesser extent than AIB transport, especially in 0.5 M NaCl-grown cells. Thus, these concentrations of salt did not inhibit AIB transport by inhibiting respiration. However, oxidation of glucose and oxidation of succinate were at least as sensitive to high salt concentrations as AIB transport, suggesting that a salt-sensitive transport step(s) is involved in the oxidation of these substrates.     

Amino acid transport in a polyaromatic amino acid auxotroph of Saccharomyces cerevisiae

The initiation of growth of a polyaromatic auxotrophic mutant of Saccharomyces cerevisiae was inhibited by several amino acids, whereas growth of the parent prototroph was unaffected. A comparative investigation of amino acid transport in the two strains employing (14)C-labeled amino acids revealed that the transport of amino acids in S. cerevisiae was mediated by a general transport system responsible for the uptake of all neutral as well as basic amino acids. Both auxotrophic and prototrophic strains exhibited stereospecificity for l-amino acids and a K(m) ranging from 1.5 x 10(-5) to 5.0 x 10(-5) M. Optimal transport activity occurred at pH 5.7. Cycloheximide had no effect on amino acid uptake, indicating that protein synthesis was not a direct requirement for amino acid transport. Regulation of amino acid transport was subject to the concentration of amino acids in the free amino acid pool. Amino acid inhibition of the uptake of the aromatic amino acids by the aromatic auxotroph did not correlate directly with the effect of amino acids on the initiation of growth of the auxotroph but provides a partial explanation of this effect.