Melibiose permease of Escherichia coli: mutation of aspartic acid 55 in putative helix II abolishes activation of sugar binding by Na+ ions

An aspartic residue (Asp55) located in the putative transmembrane alpha-helix II of the melibiose(mel) permease of Escherichia coli was replaced by Cys using oligonucleotide-directed, site-specific mutagenesis. Although D55C permease is expressed at 0.7 times the level of wild type permease, the mutated mel permease loses the ability to catalyse Na+ or H+ coupled melibiose transport against a concentration gradient. (3H) p-nitrophenyl-alpha-D-galactoside (NPG) binding studies demonstrated that D55C permease binds the sugar co-substrate but Na+ (or Li+) ions do no longer enhance the affinity of D55C permease for the co-transported sugar. In addition sugar binding on D55C permease but not on wild type permease is inactivated by sulfhydryl reagents and the inhibition protected by an excess of melibiose. These observations suggest 1) that the negatively-charged Asp55 residue, expected to be within the membrane embedded domain near the NH2 extremity of mel permease, is in or near the Na(+)-binding site and 2) that the cation and sugar binding sites may be overlapping.     

Na+-dependent methyl beta-thiogalactoside transport in Salmonella typhimurium.

We have studied the role of sodium ions in methyl beta-thiogalactoside (TMG) transport via the melibiose permease (TMG II) in Salmonella typhimurium. TMG uptake via TMG II in anaerobic, straved and metabolically poisoned cells is dependent on an inward-directed Na+ gradient. Cells which have been partially depleted of endogenous substrates show H+ extrusion upon sodium-stimulated TMG influx. Measurements of the electrochemical H+ gradient in cells, starved in different ways for endogenous substrates, suggest that this proton extrusion is probably not linked to the actual translocation mechanism but is the result of metabolism induced by TMG plug Na+ uptake.     

Kinetics of the intestinal brush border proline (Imino) carrier

The kinetics of L-proline transport across intestinal brush borders via the Imino carrier were studied using membrane vesicles. The Imino carrier is defined as the agent responsible for L-alanine insensitive. Na+-dependent uptake of L-proline. Initial rate measurements were made under voltage clamped conditions (pD = 0) to investigate L-proline transport as a function of cis and trans Na+ and proline concentrations. Under zero-trans conditions, increasing cis Na+ activated proline uptake with a Hill coefficient of 1.7 and decreased the apparent Kt with no change in Jimax. The Jimax was approximately 60 pmol mg-1 s-1 and the apparent Kt ranged from 0.25 mM at cis Na = 100 to 1.0 mM at cis Na+ = 30 mM. Trans Na inhibited proline uptake via a reduction in Jimax. Trans proline had no significant effect in the absence of trans Na+, but it relieved the trans Na+ inhibition. Under equilibrium exchange conditions, the Jimax was twice that observed under zero-trans conditions. These kinetics of L-proline transport suggest a model in which uptake occurs by a rapid equilibrium iso-ordered ter ter system. Two Na+ ions bind first to the carrier on the cis face of the membrane to increase the affinity of the carrier for proline. The fully loaded complex then isomerizes to release the substrates to the trans side. The partially loaded Na+-only forms are unable to translocate across the membrane. A rate-limiting step appears to be the isomerization of unloaded carrier from the trans to the cis side of the membrane.     

Facilitated diffusion properties of melibiose permease in Escherichia coli membrane vesicles. Release of co-substrates is rate limiting for permease cycling

The mechanism of melibiose symport by the melibiose permease of Escherichia coli was studied by looking at the modifications of the facilitated diffusion properties of the permease which arise upon substitution of the coupled cations (H+, Na+, or Li+). Kinetic analysis of melibiose influx and efflux down a concentration gradient, exchange at equilibrium, and counterflow were examined in de-energized membrane vesicles resuspended in media allowing melibiose to be co-transported with either H+, Na+, or Li+. The data show that the maximal rates of melibiose efflux coupled to either H+, Na+, or Li+ are between 10 and 40 times faster than the corresponding influxes. This suggests that the permease functions asymmetrically. Cross-comparison between the rates of net [3H]melibiose entry during the influx reactions coupled to either cation and corresponding unidirectional sugar inflow during exchange and counterflow reactions leads to the conclusions that: 1) the step involving release of the co-substrates from the permease on the inner surface of the membrane is sequenced (sugar first and then coupled cation); 2) this step is rate determining for cycling of the permease. The Na+-melibiose passive flux data indicate in particular that release of Na+ ions rather than release of sugar into the intravesicular space is the slowest step during permease cycling. This property would hamper net passive Na+-melibiose influx but should allow exchange of sugar without concomitant exchange of the coupled cation. Finally, evidence is provided suggesting that the relative rates of release of the two co-substrates from the permease on the inner membrane surface varied considerably in relation to the identity of the coupled cation.     

Mutations of putP that alter the lithium sensitivity of Salmonella typhimurium

The putP gene encodes the major proline permease in Salmonella typhimurium that couples transport of proline to the sodium electrochemical gradient. To identify residues involved in the cation binding site, we have isolated putP mutants that confer resistance to lithium during growth on proline. Wild-type S. typhimurium can grow well on proline as the sole carbon source in media supplemented with NaCl, but grows poorly when LiCl is substituted for NaCl. In contrast to the growth phenotype, proline permease is capable of transporting proline via Na+/proline or Li+/proline symport. Therefore, we selected mutants that grow well on media containing proline as the sole carbon source in the presence of lithium ions. All of the mutants assayed exhibit decreased rates of Li+/proline and Na+/proline cotransport relative to wild type. The location of each mutation was determined by deletion mapping: the mutations cluster in two small deletion intervals at the 5' and 3' termini of the putP gene. The map positions of these lithium resistance mutations are different from the locations of the previously isolated substrate specificity mutations. These results suggest that Lir mutations may define domains of the protein that fold to form the cation binding site of proline permease.     

Regulation of amino acid uptake by phorbol esters and hypertonic solutions in rat thymocytes

Growth factors, mitogens, and malignant transformation can alter the rate of amino acid uptake in mammalian cells. It has been suggested that the effects of these stimuli on proliferation are mediated by activation of Na+/H+ exchange. In lymphocytes, Na+/H+ exchange can also be activated by phorbol esters and by hypertonic media. To determine the relationship between the cation antiport and amino acid transport, we tested the effects of these agents on the uptake of alpha-aminoisobutyric acid (AIB), methyl-AIB, proline, and leucine in rat thymocytes. Both 12-O-tetradecanoylphorbol-13-acetate (TPA) and hypertonicity stimulated amino acid uptake through system A (AIB, proline, and methyl-AIB). In addition, TPA, but not hypertonicity, also elevated leucine uptake. The stimulation of the Na+ -dependent system A was not due to an increased inward electrochemical Na+ gradient. The effects of TPA and hypertonic treatment were not identical: Stimulation of AIB uptake by TPA was observed within minutes, whereas at least 1 hr was required for the effect of hypertonicity to become noticeable. Moreover, stimulation by hypertonicity but not that by TPA, was partially inhibited by cycloheximide, suggesting a role of protein synthesis. That stimulation of Na+/H+ exchange does not mediate the effects on amino acid transport is suggested by two findings: 1) the stimulation of AIB uptake was not prevented by concentrations of amiloride or of 5-(N,N-disubstituted) amiloride analogs that completely inhibit the Na+/H+ antiport and 2) conditions that mimic the effect of the antiport, namely, increasing [Na+]i or raising pHi failed to stimulate amino acid uptake. Thus, in lymphocytes, activation of Na+/H+ exchange and stimulation of amino acid transport are not casually related.     

Energetics of alanine, lysine, and proline transport in cytoplasmic membranes of the polyphosphate-accumulating Acinetobacter johnsonii strain 210A.

Amino acid transport in right-side-out membrane vesicles of Acinetobacter johnsonii 210A was studied. L-Alanine, L-lysine, and L-proline were actively transported when a proton motive force of -76 mV was generated by the oxidation of glucose via the membrane-bound glucose dehydrogenase. Kinetic analysis of amino acid uptake at concentrations of up to 80 microM revealed the presence of a single transport system for each of these amino acids with a Kt of less than 4 microM. The mode of energy coupling to solute uptake was analyzed by imposition of artificial ion diffusion gradients. The uptake of alanine and lysine was driven by a membrane potential and a transmembrane pH gradient. In contrast, the uptake of proline was driven by a membrane potential and a transmembrane chemical gradient of sodium ions. The mechanistic stoichiometry for the solute and the coupling ion was close to unity for all three amino acids. The Na+ dependence of the proline carrier was studied in greater detail. Membrane potential-driven uptake of proline was stimulated by Na+, with a half-maximal Na+ concentration of 26 microM. At Na+ concentrations above 250 microM, proline uptake was strongly inhibited. Generation of a sodium motive force and maintenance of a low internal Na+ concentration are most likely mediated by a sodium/proton antiporter, the presence of which was suggested by the Na(+)-dependent alkalinization of the intravesicular pH in inside-out membrane vesicles. The results show that both H+ and Na+ can function as coupling ions in amino acid transport in Acinetobacter spp.     

How many Na+-dependent carriers for L-alanine and L-proline in the eel intestine? Studies with brush-border membrane vesicles

Using brush-border membrane (BBM) vesicles prepared from the intestine of the European eel, the specificity of L-alanine and L-proline Na+-dependent transport was investigated by measuring the uptake of isotopically labelled substrates. In the presence of Na+ ions, cross-inhibition between alanine and proline transports was observed; in addition alpha-(methylamino)isobutyric acid (MeAIB) inhibited proline but had no effect on alanine uptake. These results can be explained by the presence, in eel intestinal BBM vesicles, of at least two distinct agencies for Na+-dependent proline and alanine translocation. The first system is specific for alanine and short-chain neutral amino acids; the second system, specific for imino acids and the N-methylated analogues, is regulated by alanine concentration.     

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.     

Na+ -dependent proline transport in isolated membrane vesicles from the L6 muscle cell line. Stimulation of uptake by intravesicular proline

Membrane vesicles of L6 myoblasts were prepared in order to study the amino acid transport system A. The role of the membrane in the adaptive response of transport to amino acid-supplementation was assessed. The membranes, prepared by N2 cavitation, displayed Na+ (but not K+)-dependent L-proline uptake. An overshoot of L-[3H]proline uptake was observed after exposure of the vesicles to an inward Na+ gradient. Isolated membrane vesicles loaded with 50 microM proline displayed countertransport (stimulation of proline uptake). It is concluded that the adaptive decrease of proline uptake observed in amino acid-supplemented cells cannot be accounted for by trans-inhibition of transport.     

Evidence for a sodium-dependent proline and glycine-betaine uptake in the cyanobacterium Nostoc muscorum

The cyanobacterium Nostoc muscorum is able to utilized proline and glycine-betaine as a nitrogen source under unstressed growth conditions. This cyanobacterium when grow in modified Chu No. 10 medium (without Na+) unable to utilized proline and glycine-betaine as a nitrogen source. Spontaneously occurring mutant clones defective in Na+ transport (Na+-R) were isolated and analyzed for proline and glycine-betaine utilization. The mutant phenotype showed normal heterocyst frequency and nitrogenase activity even in the medium containing 1 mM proline or 1 mM glycine-betaine, indicates the role of Na+ for proline/glycine-betaine uptake. The Na+-R mutant showed 100% survival at pH 11 and was simultaneously able to uptake and utilize proline/glycine-betaine at higher alkaline pH. This indicates that proline and glycine-betaine uptake systems are more efficient at higher alkaline pH. Since, the hypersaline environments are rich in Na+ contents and have alkaline pH, therefore it is suggested that the origin and evolution of specific compatible solutes may not depend only on the osmoregulatory role they play, but also on the other ecological factors operating simultaneously in the organism's niche.     

Glutamate-induced uptake of proline by Streptomyces antibioticus.

Streptomyces antibioticus possesses an energy-dependent, carrier mediated transport system for the uptake of L-glutamate and L-proline. Amino acid transport was found to have a temperature optimum of 35 degrees C and a pH optimum from 7.0 to 8.0 for glutamate and 6.5 to 7.5 for proline uptake. Uptake did not depend upon Mg2+, Ca2+, Zn2+, Na+, or Fe2+ ions. Reversible p-hydroxymercuribenzoate inhibition of uptake indicated the involvement of an active sulfhydryl group. L-Glutamate uptake was mediated by a glutamate-inducible, nonspecific transport system, which was extremely stable and was not subject to substrate inhibition by L-proline. On the other hand, L-proline transport was mediated by at least two systems. The L-glutamate-inducible nonspecific system can account for uptake of proline by the mycelium grown in glutamate. In addition, a proline-specific, constitutive transport system was found to be present in the mycelium grown in organic and inorganic nitrogen sources other than L-glutamate. Shift experiments revealed that proline transport is not as stable as glutamate transport when the glutamate-inducible nonspecific system is utilized.     

Energetics of galactose,proline, and glutamine transport in a cytochrome-deficient mutant of salmonella typhimurium

The effect of inhibitors and uncouplers on the osmotic shock-sensitive transport systems for glutamine and galactose (by the β-methyl galactoside permease) was compared to their effect on the osmotic shock-resistant proline and galactose permease systems in cytochrome-deficient cells of Salmonella typhimurium SASY28. Both osmotic shock-sensitive and -resistant systems were sensitive to uncouplers and to inhibitors of the membrane-bound Ca²⁺, Mg²⁺-activated adenosine triphosphatase. This suggests that uptake by both types of systems is energized in these cells by an electrochemical gradient of protons formed by ATP hydrolysis through the ATPase.     

Characteristics of proline transport into R3230AC mammary tumor cells

Cells separated by enzyme treatment of the R3230AC mammary carcinoma were used to characterize the entry of proline. These cells showed minimal changes in cell viability and intracellular volume and were found to be suitable for transport studies, since the vi of proline was maintained for at least 4 h when cells were stored at 37 or 4 degrees C, or when transport was measured in the presence or absence of Na+. Proline was acitvely transported by these tumor cells, reaching a distribution ratio ([proline] intracellular/[proline] extracellular) of 20 after 2 h. Proline entry consisted of two processes, one saturable (carrier mediated) and the other, non-saturable. The carrier-mediated entry, Km - 0.83 mM and V = 151.10(-5) mumol/min per 5.10(6) cells, was Na+-dependent, sensitive to pH and metabolic inhibitors, and completely inhibited by alpha-(methylamino)-isobutyric acid (Ki = 0.34 mM). Proline entry in the absence of Na+ was 20% that in the presence of Na+ and was found to be due to a non-saturable process, since (a) vi of proline uptake in the absence of Na+ increases linearly with increasing proline concentration and (b) was not suppressed by either 20 mM alpha-(methyl-amino)-isobutyric acid, 50 mM glycine +20 mM phenylalanine, or 50 mM serine +20 mM phenylalanine when proline uptake was measured in the presence or absence of Na+. Therefore, under the conditions studied, we conclude that proline transport appears to be restricted to the A (alanine-preferring) system. Furthermore, these cells should provide a suitable model to study the effect of hormonal manipulations on the amino acid transport process.     

Ureidosuccinic Acid Uptake in Yeast and Some Aspects of Its Regulation

Ureidosuccinic acid (USA) is an intermediary product in pyrimidine biosynthesis. When proline was the sole nitrogen source, USA uptake occurred; however, when ammonium sulfate or glutamic acid was the nitrogen source, uptake was inhibited. Thus, a ura2 strain which does not synthesize USA would not grow when this substance was supplied on an ammonium sulfate or glutamic acid medium. Mutants are described in which uptake was constitutive on such a medium. Permeaseless mutants for USA have been found, and evidence is presented for permease specificity. It is shown that all constitutive mutants use the same transport system that is missing in the permeaseless mutant. These mutants are constitutive for two permeases: the specific USA permease and the general amino acid permease. The transport system studied here, like the general amino acid transport system, is regulated by nitrogen metabolism. These facts and others suggest that our permease constitutive mutants are impaired in nitrogen metabolism.     

Regulation of amino acid transport in L6 myoblasts. II. Different chemical properties of transport after amino acid deprivation

The mechanism of stimulation of amino acid transport system A caused by amino acid deprivation in L6 cells was investigated. In cells loaded with alpha-aminoisobutyric acid (AIB), amino acid deprivation increased the rate of proline uptake only after the intracellular [AIB] dropped below 7 mM. Efflux of proline was not sensitive to the presence of proline in the outer medium (with or without external Na+), suggesting that efflux through system A (and possibly uptake) is not susceptible to transinhibition. Transport (stimulated uptake) into amino acid-deprived cells and that into amino acid-supplemented cells differed in several chemical properties: 1) In the former group, transport was higher at lower pH values than in the latter, and the optimum pH values were 7.5 and 7.8, respectively. 2) Unlike proline uptake in supplemented cells, uptake in deprived cells was inhibited by 50% with N-ethylmaleimide (1 mM) or by 50 microM p-chloromercuribenzoate (PCMBS). Inhibition by PCMBS was not due to collapse of the Na+ gradient. The mercurial inhibited only the deprivation-induced stimulation of transport, bringing the rate of proline uptake to the "basal" uptake level observed in amino acid-supplemented cells. Proline uptake was not stimulated by a second deprivation following treatment with PCMBS and a supplementation-deprivation cycle. However, in untreated cells, or by reversing mercaptide formation with dithiotreitol, the second deprivation stimulated transport. Deprivation at 4 degrees C did not elicit stimulation of proline uptake. Cycloheximide prevented the stimulation and decreased the rate of proline uptake in deprived cells more efficiently than in supplemented cells. Actinomycin D prevented stimulation when added at the onset of deprivation. The above data indicate that stimulation of transport by deprivation is protein synthesis-dependent and that the stimulated transport had chemical properties distinct from the "basal" transport in supplemented cells. The evidence presented is consistent with a model of activation of a finite pool of transporters upon deprivation, the chemical characteristics of which differ from those of the "basal" transport system.     

In squid nerve fibers monovalent activating cations are not cotransported during Na+/Ca2+ exchange

Squid axons display a high activity of Na+/Ca2+ exchange which is largely increased by the presence of external K+, Li+, Rb+ and NH+4. In this work we have investigated whether this effect is associated with the cotransport of the monovalent cation along with Ca2+ ions. 86Rb+ influx and efflux have been measured in dialyzed squid axons during the activation (presence of Ca2+i) of Ca2+o/Na+i and Ca2+i/Ca2+o exchanges, while 86Rb+ uptake was determined in squid optic nerve membrane vesicles under equilibrium Ca2+/Ca2+ exchange conditions. Our results show that although K+o significantly increases Na+i-dependent Ca2+ influx (reverse Na+/Ca2+ exchange) and Rb+i stimulates Ca2+o-dependent Ca2+ efflux (Ca2+/Ca2+ exchange), no sizable transport of rubidium ions is coupled to calcium movement through the exchanger. Moreover, in the isolated membrane preparation no 86Rb+ uptake was associated with Ca2+/Ca2+ exchange. We conclude that in squid axons although monovalent cations activate the Na+/Ca2+ exchange they are not cotransported.     

Stimulatory effect of lithium ion on proline transport by whole cells of Escherichia coli.

The effect of monovalent cations on proline transport in whole cells of Escherichia coli K-12 has been examined. Lithium ion added to the uptake medium stimulated proline transport severalfold and K+ and Na+ were slightly effective, whereas Rb+, Cs+, and NH4+ were completely without effect. The stimulatory effect of Li+ on proline transport was not due to an increase in osmolarity of the uptake medium, and d 5 mM p-chloromercuribenzene sulfonic acid completely blocked this effect of Li+ without having any effect on the basal rate of proline transport. The Arrhenius plots for Li+-stimulated transport showed a clear transition point at 35 degrees C in addition to 20 degrees C which was also detectable in the basal transport. Lithium ion stimulated proline transport synergistically in the presence of glucose and succinate as a carbon source. The addition of 2.5 mM KCN or 0.5 mM arsenate did not inhibit this synergistic effect, although the presence of these inhibitors inhibited completely the stimulation of proline transport induced by the addition of carbon source. Carbonylcyanide m-chlorophenylhydrazone and 2,4-dinitrophenol blocked both the basal and Li+-stimulated proline transport. When membrane potential of E. coli cells was measured by the dibenzyldimethylammonium uptake method, the incubation of Li+ with the cells did not affect the preexisting membrane potential. These results suggest that Li+ stimulates proline transport by intact cells of E. coli in a manner somewhat affecting membrane component(s) different from the transport carrier of proline. It is uncertain whether the effect of Li+ is directly involved in the mechanisms of energy coupling of proline transport.     

Transport of Na+ inside the giant axon of squid

The transport mechanism of Na ions within the nerve cell was studied by measuring the radioactivity distribution profile of 22Na that had been intracellularly injected into the giant axon. Specifically, we tested whether or not the movement of Na ions is coupled with the process of "fast axonal transport." Results of our measurements indicate that the intracellular transport of Na+ and the fast axonal transport are two independent processes. Very few Na ions are irreversibly sequestered into the axoplasmic vesicles involved in axonal transport. The movement of Na+ inside the axon can be modeled by a one-dimension diffusion. The effective diffusion coefficient of the intracellular Na+ was determined in this study.     

Sodium-Dependent Proline Uptake in the Rat Hippocampal Formation: Association with Ipsilateral-Commissural Projections of CA3 Pyramidal

Na+-dependent uptake of L-[3H]proline was measured in a crude synaptosomal preparation from the entire rat hippocampal formation or from isolated hippocampal regions. Among hippocampal regions, Na+-dependent proline uptake was significantly greater in areas CA1 and CA2-CA3-CA4 than in the fascia dentata, whereas there was no marked regional difference in the distribution of Na+-dependent gamma-[14C]aminobutyric acid ([14C]GABA) uptake. A bilateral kainic acid lesion, which destroyed most of the CA3 hippocampal pyramidal cells, reduced Na+-dependent proline uptake by an average of 41% in area CA1 and 52% in area CA2-CA3-CA4, without affecting the Na+-dependent uptake of GABA. In the fascia dentata, neither proline nor GABA uptake was significantly altered. Kinetic studies suggested that hippocampal synaptosomes take up proline by both a high-affinity (KT = 6.7 microM) and a low-affinity (KT = 290 microM) Na+-dependent process, whereas L-[14C]glutamate is taken up predominantly by a high-affinity (KT = 6.1 microM) process. A bilateral kainic acid lesion reduced the Vmax of high-affinity proline uptake by an average of 72%, the Vmax of low-affinity proline uptake by 44%, and the Vmax of high affinity glutamate uptake by 43%, without significantly changing the affinity of the transport carriers for substrate. Ipsilateral-commissural projections of CA3 hippocampal pyramidal cells appear to possess nearly as great a capacity for taking up proline as for taking up glutamate, a probable transmitter of these pathways. Therefore proline may play an important role in transmission at synapses made by the CA3-derived Schaffer collateral, commissural, and ipsilateral associational fibers.