Methionine sulfoxide is transported by high-affinity methionine and glutamine transport systems in Salmonella typhimurium.

Three lines of evidence indicated that methionine sulfoxide is transported by the high-affinity methionine and glutamine transport systems in Salmonella typhimurium. First, methionine-requiring strains (metE) which have mutations affecting both of these transport systems (metP glnP) were unable to use methionine sulfoxide as a source of methionine. These strains could still grow on L-methionine because they possessed a low-affinity system (or systems) which transported L-methionine but not the sulfoxide. A methionine auxotroph with a defect only in the metP system, which was dependent upon the glnP+ system for the transport of methionine sulfoxide, was inhibited by L-glutamine because glutamine inhibited the transport of the sulfoxide by the glnP+ system. Second, a metE metP glnP strain could be transduced at either the metP or glnP genes to restore its ability to grow on methionine sulfoxide. Third, the transport of [14C]methionine sulfoxide was inhibited by methionine and by glutamine in the metP+ glnP+ strain. No transport was detected in the metP glnP double-mutant strain.     

Glycine betaine transport by Staphylococcus aureus: evidence for two transport systems and for their possible roles in osmoregulation.

The transport of glycine betaine by Staphylococcus aureus was investigated. Two transport systems were found that could be differentiated on the basis of their affinity for glycine betaine and their activation by osmotic pressure. The high-affinity system was relatively independent of osmotic pressure and exhibited a Km of approximately 3 microM. This system was not inhibited by proline, for which a separate high-affinity transport system has been recently discovered. The low-affinity system was activated approximately 35-fold by an increase in osmotic pressure and exhibited a Km of approximately 130 microM for glycine betaine. This system is partially inhibited by excess proline and may be identical to the low-affinity system recently described for proline. Both glycine betaine transport systems are Na(+)-dependent.     

Methionine and glutamine transport systems in D-methionine utilising revertants of Salmonella typhimurium

Summary In Salmonella typhimurium, methionine auxotrophs such as metB can use D-methionine as a methionine source. MetP mutations prevent this growth since D-methionine can enter only via the metP high-affinity methionine transport system. D-methionine utilising revertants (Dmu⁺) were selected from metB23 metP760 (HU76) following nitrosoguanidine mutagenesis. The properties of two such revertants, HU206 and HU415, indicated that reversion was not due to backmutation of the metP760 mutation. Genetic analysis indicated that each strain possessed two mutations, designated dmu and gln, in addition to the original metB23 and metP760 mutations.The dmu mutation restores ability to grow on D-methionine, partly restores D- and L-methionine transport activity, and makes the cells particularly sensitive to inhibition by L-glutamine while growing on D but not L-methionine. The growth inhibition by L-glutamine was shown to be caused by competition by L-glutamine for D-methionine transport by the high-affinity methionine system. The gln mutation greatly reduces activity of the high-affinity glutamine transport system. The Dmu⁺ strains are also partly defective in the glutamine low-affinity transport system, possibly because the partially-restored methionine high-affinity system, or a component of this system, functions in the transport of glutamine by its low-affinity system.     

Glutamine and glutamate transport by Anabaena variabilis

Anabaena variabilis, a dinitrogen-fixing cyanobacterium, has high- and low-affinity systems for the transport of glutamine and glutamate. The high-affinity systems have Km values of 13.8 and 100 microM and maximal rates of 13.2 and 14.4 nmol X min-1 X mg of chlorophyll a-1 for glutamine and glutamate, respectively. The low-affinity systems have Km values of 1.1 and 1.4 mM and maximal rates of 125 and 100 nmol X min-1 X mg of chlorophyll a-1 for glutamine and glutamate, respectively. Glutamine was unable to support growth of A. variabilis in the absence of any other nitrogen source, and glutamate alone at 500 microM was inhibitory to its growth. The analog L-methionine-DL-sulfoximine (MSX) was transported by a high-affinity system with a Km of 34 microM. Competition experiments and the transport characteristics of a specific class of MSX-resistant mutants imply that glutamine, glutamate, and MSX share a common component for transport. A second class of MSX-resistant mutants had a glutamine synthetase activity with altered affinity constants for glutamine and glutamate relative to the wild-type enzyme.     

Kinetic evaluation, using 13N, reveals two assimilatory nitrate transport systems in Klebsiella pneumoniae.

A kinetic evaluation of initial rates of nitrate transport at concentrations between 1 microM and 1 mM indicated the presence of two transport processes. Analysis of the contribution of each process to the total activity permitted the determination of kinetic constants (Km) of 4.9 microM and 4.2 mM for the high-and low-affinity systems, respectively. The ratio of the maximal velocity of the high-affinity system to that of an apparent low-affinity system was about 0.3. Both systems were inhibited by the presence of NH4+ in the transport assay. Growth in the presence of equimolar NO3- and NH4+ repressed the synthesis of both systems when compared with growth in NO3- alone.     

Transport of basic amino acids by the dinitrogen-fixing cyanobacterium Anabaena PCC 7120

Two transport systems for L-arginine were evident in Anabaena sp. strain PCC 7120: a high-affinity one (Km, 1.7 microM) that accumulated arginine within the cells through an energy-requiring process and another one that exhibited low affinity for L-arginine (Km, 0.75 mM) and was unable to accumulate the substrate. Both systems were inhibited by L-canavanine, L-lysine, and L-ornithine. Two systems were also evident for L-lysine uptake (Km, 1.9 and 110 microM, respectively). After selection for resistance to canavanine or hydroxylysine, independent mutants were isolated which were impaired in the high-affinity uptake of arginine and lysine. A common permease appears, therefore, to be involved in the high-affinity transport of these basic amino acids. Both the high- and the low-affinity systems can contribute to the growth of Anabaena sp. on L-arginine. However, arginine did not effectively repress either nitrogenase or nitrate reductase.     

Low-affinity gamma-aminobutyric acid transport in rat brain

The low-affinity (Km = 100-200 microM) gamma-aminobutyric acid (GABA) transporter from membrane vesicles from rat brain has been characterized and found to be in many aspects similar to the well-known sodium- and chloride-coupled high-affinity gamma-aminobutyric acid transporter (Km = 2-4 microM). Influx by this system is sodium and chloride dependent and stimulated by an interior negative membrane potential. Steady-state levels obtained by both systems are lowered by the sodium channel openers veratridine and aconitine. However, while the channel blocker tetrodotoxin fully reverses this inhibition with the high-affinity system, this is not the case for its low-affinity counterpart. Furthermore, the toxin from the scorpion Androctonus australis Hector inhibited high-affinity transport only. Efflux of gamma-aminobutyric acid taken up by the high-affinity system displayed a Km of about 100 microM. Exchange catalyzed by the low-affinity system was observed in the absence of external sodium and chloride. Furthermore, both activities copurified in the fractionation procedure developed to purify the high-affinity transporter. All these observations are consistent with the idea that both activities are manifestations of only one gamma-aminobutyric acid transporter. The high-affinity binding site represents the extracellular and the low-affinity site the cytosolic aspect of the transporter. In addition, it was found that right-side-out synaptosomes also contain a low-affinity GABA transporter. This apparently represents a different transport protein.     

Repression and inhibition of transport systems for branched-chain amino acids in Salmonella typhimurium.

Kinetics of the transport systems common for entry of L-isoleucine, L-leucine, and L-valine in Salmonella typhimurium LT2 have been analyzed as a function of substrateconcentration in the range of 0.5 to 45 muM. The systems of transport mutants, KA203 (ilvT3) and KA204 (ilvT4), are composed of two components; apparent Km values for uptake of isoleucine, leucine, and valine by the low Km component are 2 muM, 2 to 3 muM, and 1 muM, respectively, and by the high Km component 30 muM, 20 to 40 muM, and 0.1 mM, respectively. The transport system(s) of the wild type has not been separated into components but rather displays single Km values of 9 muM for isoleucine, 10 muM for leucine, and 30 muM for valine. The transport activity of the wild type was repressed by L-leucine, alpha ketoisocaproate, glycyl-L-isoleucine, glycyl-L-leucine, and glycyl-L-methionine. That for the transport mutants was repressed by L-alanine, L-isoleucine, L-methionine, L-valine, alpha-ketoisovalerate, alpha-keto-beta-methylvalerate, glycyl-L-alanine, glycyl-L-threonine, and glycyl-L-valine, in addition to the compounds described above. Repression of the mutant transport systems resulted in disappearance of the low Km component for valine uptake, together with a decrease in Vmax of the high Km component; the kinetic analysis with isoleucine and leucine as substrates was not possible because of poor uptake. The maximum reduction of the transport activity for isoleucine was obtained after growing cells for two to three generations in a medium supplemented with repressor, and for the depression, protein synthesis was essential after removal of the repressor. The transport activity for labeled isoleucine in the transport mutant and wild-type strains was inhibited by unlabeled L-alanine, L-cysteine, L-isoleucine, L-leucine, L-methionine, L-threonine, and L-valine. D-Amino acids neither repressed nor inhibited the transport activity of cells for entry of isoleucine.     

Third system for neutral amino acid transport in a marine pseudomonad.

Uptake of leucine by the marine pseudomonad B-16 is an energy-dependent, concentrative process. Respiratory inhibitors, uncouplers, and sulfhydryl reagents block transport. The uptake of leucine is Na+ dependent, although the relationship between the rate of leucine uptake and Na+ concentration depends, to some extent, on the ionic strength of the suspending assay medium and the manner in which cells are washed prior to assay. Leucine transport can be separated into at least two systems: a low-affinity system with an apparent Km of 1.3 X 10(-5) M, and a high-affinity system with an apparent Km of 1.9 X 10(-7) M. The high-affinity system shows a specificity unusual for bacterial systems in that both aromatic and aliphatic amino acids inhibit leucine transport, provided that they have hydrophobic side chains of a length greater than that of two carbon atoms. The system exhibits strict stereospecificity for the L form. Phenylalanine inhibition was investigated in more detail. The Ki for inhibition of leucine transport by phenylalanine is about 1.4 X 10(-7) M. Phenylalanine itself is transported by an energy-dependent process whose specificity is the same as the high-affinity leucine transport system, as is expected if both amino acids share the same transport system. Studies with protoplasts indicate that a periplasmic binding protein is not an essential part of this transport system. Fein and MacLeod (J. Bacteriol. 124:1177-1190, 1975) reported two neutral amino acid transport systems in strain B-16: the DAG system, serving glycine, D-alanine, D-serine, and alpha-aminoisobutyric acid; and the LIV system, serving L-leucine, L-isoleucine, L-valine, and L-alanine. The high-affinity system reported here is a third neutral amino acid transport system in this marine pseudomonad. We propose the name "LIV-II" system.     

Inorganic cation transport and the effects on C4 dicarboxylate transport in Bacillus subtilis

The complex interrelationships between the transport of inorganic cations and C4 dicarboxylate were examined using mutants defective in potassium transport and retention, divalent cation transport, or phosphate transport. The potassium transport system, studied using 86Rb+ as a K+ analogue, kinetically appeared as a single system (Km 200 microM for Rb+, Ki 50 microM for K+), the activity of which was only slightly reduced in K+ retention mutants. Divalent cation transport, studied using 54Mn2+, 60Co2+, and 45Ca2+, was more complex being represented by at least two systems, one with a high affinity for Mn2+ (Km 2.5 microM) and a more general one of low affinity (Km 1.3-10 mM) for Mg2+, Mn2+, Ca/2+, and Co2+. Divalent cation transport was repressed by Mg2+, derepressed in K+ retention mutants, and defective in Co2+-resistant mutants. Phosphate was required for both divalent cation and succinate transport, and phosphate transport mutants (arsenate resistant) were found to be defective in both divalent cation and succinate transport. Divalent cations, especially Mg2+ and Co2+, decreased Km for succinate transport approximately 20-fold over that achieved with K+; neither cation was required stoichiometrically for succinate transport. The loss of divalent cation transport in cobalt-resistant mutants has been correlated with the loss of a 55,000 molecular weight membrane protein. Similarly, the loss of phosphate transport in arsenate-resistant mutants has been correlated with the loss of a 35,000 molecular weight membrane component.     

Low- and high-Km transport of dinitrophenyl glutathione in inside out vesicles from human erythrocytes.

Kinetic studies on the low- and high-Km transport systems for S-2,4-dinitrophenyl glutathione (DNP-SG) present in erythrocyte membranes were performed using inside-out plasma membrane vesicles. The high-affinity system showed a Km of 3.9 microM a Vmax of 6.3 nmol/mg protein per h, and the low-affinity system a Km of 1.6 mM and a Vmax of 131 nmol/mg protein per h. Both uptake components were inhibited by fluoride, vanadate, p-chloromercuribenzoate (pCMB) and bis(4-nitrophenyl)dithio-3,3'-dicarboxylate (DTNB). The low-Km uptake process was less sensitive to the inhibitory action of DTNB as compared to the high-Km process. N-Ethylmaleimide (1 mM) inhibited the high-Km process only. The high-affinity uptake of DNP-SG was competitively inhibited by GSSG (Ki = 88 microM). Vice versa, DNP-SG inhibited competitively the low-Km component of GSSG uptake (Ki = 3.3 microM). The high-Km DNP-SG uptake system was not inhibited by GSSG. The existence of a common high-affinity transporter for DNP-SG and GSSG in erythrocytes is suggested.     

Activation of a new proline transport system in Salmonella typhimurium.

Proline uptake can be mediated by three different transport systems in wild-type Salmonella typhimurium: a high-affinity proline transport system encoded by the putP gene and two glycine-betaine transport systems with a low affinity for proline encoded by the proP and proU genes. However, only the PutP permease transports proline well enough t allow growth on proline as a sole carbon or nitrogen source. By selecting for mutations that allow a putP mutant to grow on proline as a sole nitrogen source, we isolated mutants (designated proZ) that appeared to activate a cryptic proline transport system. These mutants enhanced the transport of proline and proline analogs but did not require the function of any of the known proline transport genes. The mutations mapped between 75 and 77.5 min on the S. typhimurium linkage map. Proline transport by the proZ mutants was competitively inhibited by isoleucine and leucine, which suggests that the ProZ phenotype may be due to unusual mutations that alter the substrate specificity of the branched-chain amino acid transport system encoded by the liv genes.     

Phosphate transport in Pseudomonas aeruginosa. Involvement of a periplasmic phosphate-binding protein

A binding protein for inorganic phosphate was purified to apparent homogeneity from the shock fluids of phosphate-limited Pseudomonas aeruginosa. The purified protein bound one molecule of phosphate per molecule of binding protein with an average Kd of 0.34 microM. Arsenate, pyrophosphate and polyphosphates up to 15 units long could inhibit the binding of phosphate to the binding protein, although organic phosphates, such as glucose 6-phosphate, glycerol 3-phosphate and adenosine 5'-monophosphate could not. Mutants lacking the phosphate-binding protein were isolated and shown to be deficient in phosphate transport compared with wild-type cells. Two kinetically distinct systems for phosphate uptake could be observed in wild-type cells, with apparent Km values of 0.46 +/- 0.10 microM (high affinity) and 12.0 +/- 1.6 microM (low affinity). In contrast, only a single low-affinity transport system was observable in mutants lacking the binding protein (Km apparent = 19.3 +/- 1.4 microM Pi), suggesting the involvement of the binding protein in the inducible high-affinity phosphate-uptake system of P. aeruginosa.     

Genetic and biochemical studies of transport systems for branched-chain amino acids in Escherichia coli.

Mutants of Escherichia coli K-12 requiring high concentrations of branched-chain amino acids for growth were isolated. One of the mutants was shown to be defective in transport activity for branched-chain amino acids. The locus of the mutation (hrbA) was mapped at 8.9 min on the E. coli genetic map by conjugational and transductional crosses. The gene order of this region is proC-hrbA-tsx. The hrbA system was responsible for the uptake activity of cytoplasmic membrane vesicles. It was not repressed by leucine. The substrate specificities and kinetics of the uptake activities were studied using cytoplasmic membrane vesicles and intact cells of the mutants grown in the presence or absence of leucine. Results showed that there are three transport systems for branched-chain amino acids, LIV-1, -2, and -3. The LIV-2 and -3 transport systems are low-affinity systems, the activities of which are detectable in cytoplasmic membrane vesicles. The systems are inhibited by norleucine but not by threonine. The LIV-2 system is also repressed by leucine. The LIV-1 transport system is a high-affinity system that is sensitive to osmotic shock. When the leucine-isoleucine-valine-threonine-binding protein is derepressed, the high-affinity system can be inhibited by threonine.     

Genetic and biochemical studies of transport systems for branched-chain amino acids in Escherichia coli K-12: isolation and properties of mutants defective in leucine-repressible transport activities.

The characteristics of a mutant (hrbA) of Escherichia coli K-12 that is defective in a leucine-nonrepressible transport system, the LIV-3 system, for branched-chain amino acids were described previously (I. Yamato et al., J. Bacteriol 138:24-32, 1979). New mutants requiring a high concentration of isoleucine for growth were isolated from strain B763 (hrbA ileA) after mutagenesis with ethyl methane sulfonate. These mutants had a defect of the leucine-repressible transport activities for branched-chain amino acids of the parental strain. One of these mutants, strain B7634, had defects of two independent genetic loci (hrbBC and hrbD). The genes hrbBC were mapped at min 76 near malT, and the gene hrbD mapped at min 77 near xyl on the E. coli genetic map. The substrate specificity, kinetic properties, and source of coupling energy of the transport system coded for by each of these genes were studied using cytoplasmic membrane vesicles and intact cells. The results identified three transport systems with characteristic features other than the LIV-3 system. The hrbB and hrbC systems are responsible for the uptake activites of the LIV-2 system, with a high Km value, and the LIV-1 system, with a low Km value, respectively. Both activities are repressed by leucine and inhibited by threonine and the b(--) isomer of 2-aminobicycloheptyl-2-carboxylic acid. They both utilize adenosine 5'-triphosphate as coupling energy and are not detected in cytoplasmic membrane vesicles. The hrbD system is responsible for the LIV-4 system, with a high Km value. Its activity is repressed by leucine and partially inhibited by threonine. It is detected in cytoplasmic membrane vesicles with a proton motive force as the driving energy.     

Mutations Affecting the Different Transport Systems for Isoleucine, Leucine, and Valine in Escherichia coli K-12

Uptake of isoleucine, leucine, and valine in Escherichia coli K-12 is due to several transport processes for which kinetic evidence has been reported elsewhere. A very-high-affinity transport process, a high-affinity transport process, and three different low-affinity transport processes were described. In this paper the existence of these transport processes is confirmed by the isolation and preliminary characterization of mutants altered in one or more of them. The very-high-affinity transport process is missing either in strains carrying the brnR6(am) mutation or in strains carrying the brn-8 mutation. This appears to be a pleiotropic effect since other transport systems are also missing. Mutant analysis shows that more than one transport system with high affinity is present. One of them, high-affinity 1, which needs the activity of a protein produced by the brnQ gene, transports isoleucine, leucine, and valine and is unaffected by threonine. The other, high-affinity 2, which needs the activity of a protein produced by the brnS gene, transports isoleucine, leucine, and valine; this uptake is inhibited by threonine which probably is a substrate. Another protein, produced by the brnR gene, is required for uptake through both high-affinity 1 and high-affinity 2 transport systems. The two systems therefore appear to work in parallel, brnR being a branching point. The brnQ gene is located close to phoA at 9.5 min on the chromosome of E. coli, the brnR gene is located close to lac at 9.0 min, and the brnS gene is close to pdxA at 1 min. A mutant lacking the low-affinity transport system for isoleucine was isolated from a strain in which the high-affinity system was missing because of a brnR mutation. This strain also required isoleucine for growth because of an ilvA mutation. The mutant lacking the low-affinity transport system was unable to grow on isoleucine but could grow on glycylisoleucine. This mutant had lost the low-affinity transport for isoleucine, whereas those for leucine and valine were unaffected. A pleiotropic consequence of this mutation (brn-8) was a complete absence of the very-high-affinity transport system due either to the alteration of a common gene product or to any kind of secondary interference which inhibits it. Mutants altered in isoleucine-leucine-valine transport were isolated by taking advantage of the inhibition that valine exerts on the K-12 strain of E. coli. Mutants resistant both to valine inhibition (Val(r)) and to glycylvaline inhibition are regulatory mutants. Val(r) mutants that are sensitive to glycylvaline inhibition are transport mutants. When the very-high-affinity transport process is repressed (for example by methionine) the frequency of transport mutants among Val(r) mutants is higher, and it is even higher if the high-affinity transport process is partially inhibited by leucine.     

Sugar transport in Coprinus cinereus.

Two transport systems for glucose were detected: a high affinity system with a Km of 27 muM, and a low affinity system with a Km of 3.3 mM. The high affinity system transported glucose, 2-deoxy-D-glucose (Km = 26 muM), 3-O-methylglucose (Km = 19 muM), D-glucosamine (Km = 652 muM), D-fructose (Km = 2.3 mM) and L-sorbose (Km = 2.2 mM). All sugars were accumulated against concentration gradients. The high affinity system was strongly or completely inhibited by N-ethylmaleimide, quercetin, 2,4-dinitrophenol and sodium azide. The system had a distinct pH optimum (7.4) and optimum temperature (45 degrees C). The low affinity system transported glucose, 2-deoxy-D-glucose (Km = 7.5 mM), and 3-O-methylglucose (Km = 1.5 mM). Accumulation again occurred against a concentration gradient. The low affinity system was inhibited by N-ethylmaleimide, quercetin and 2,4-dinitrophenol, but not by sodium azide. The rate of uptake by the low affinity system was constant over a wide temperature range (30--50 degrees C) and was not much affected by pH; but as the pH of the medium was altered from 4.5 to 8.9 a co-ordinated increase in affinity for 2-deoxy-D-glucose (from 52.1 mM to 0.3 mM) and decrease in maximum velocity (by a factor of five) occurred. Both uptake systems were present insporelings germinated in media containing sodium acetate as sole carbon source. Only the low affinity system could initially be demonstrated in glucose-grown tissue, although the high affinity system was restored by starvation inglucose-free medium. The half-ti me for restoration of high affinity activity was 3.5 min and the process was unaffected by cycloheximide. Addition of glucose to an acetate-grown culture inactivated the high affinity system with a half-life of 5--7.5 s. Addition of cycloheximide to an acetate-grown culture caused decay of the high affinity system with a half-life of 80 min. Regulation is thus thought to depend on modulation of protein activity rather than synthesis, and the kinetics of glucose, 2-deoxy-D-glucose and 3-O-methylglucose uptake would be consistent with there being a single carrier showing negative co-operativity. Analysis of transport defective mutants revealed defects in both transport systems although the mutants used were alleles of a single gene. It is concluded that this gene (the ftr cistron) is the structural gene for an allosteric molecule which serves both transport systems.     

Proline transport in Staphylococcus aureus: a high-affinity system and a low-affinity system involved in osmoregulation.

L-Proline enhanced the growth of Staphylococcus aureus in high-osmotic-strength medium, i.e., it acted as an osmoprotectant. Study of the kinetics of L-[14C]proline uptake by S. aureus NCTC 8325 revealed high-affinity (Km = 1.7 microM; maximum rate of transport [Vmax] = 1.1 nmol/min/mg [dry weight]) and low-affinity (Km = 132 microM; Vmax = 22 nmol/min/mg [dry weight]) transport systems. Both systems were present in a proline prototrophic variant grown in the absence of proline, although the Vmax of the high-affinity system was three to five times higher than that of the high-affinity system in strain 8325. Both systems were dependent on Na+ for activity, and the high-affinity system was stimulated by lower concentrations of Na+ more than the low-affinity system. The proline transport activity of the low-affinity system was stimulated by increased osmotic strength. The high-affinity system was highly specific for L-proline, whereas the low-affinity system showed a broader substrate specificity. Glycine betaine did not compete with proline for uptake through either system. Inhibitor studies confirmed that proline uptake occurred via Na(+)-dependent systems and suggested the involvement of the proton motive force in creating an Na+ gradient. Hyperosmotic stress (upshock) of growing cultures led to a rapid and large uptake of L-[14C]proline that was not dependent on new protein synthesis. It is suggested that the low-affinity system is involved in adjusting to increased environmental osmolarity and that the high-affinity system may be involved in scavenging low concentrations of proline.     

Basic and neutral amino acid transport in Aspergillus nidulans.

Arginine and methionine transport by Aspergillus nidulans mycelium was investigated. A single uptake system is responsible for the transport of arginine, lysine and ornithine. Transport is energy-dependent and specific for these basic amino acids. The Km value for arginine is 1 X 10(-5) M, and Vmax is 2-8 nmol/mg dry wt/min; Km for lysine is 8 X 10(-6) M; Kt for lysine as inhibitor of arginine uptake is 12 muM, and Ki for ornithine is mM. On minimal medium, methionine is transported with a Km of 0-I mM and Vmax about I nmol/mg dry wt/min; transport is inhibited by azide. Neutral amnio acids such as serine, phenylalanine and leucine are probably transported by the same system, as indicated by their inhibition of methionine uptake and the existence of a mutant specifically impaired in their transport. The recessive mutant nap3, unable to transport neutral amino acids, was isolated as resistant to selenomethionine and p-fluorophenylanine. This mutant has unchanged transport of methionine by general and specific sulphur-regulated permeases.