High-affinity Proline Research Articles

The yeast α-arrestin Art3 is a key regulator for arginine-induced endocytosis of the high-affinity proline transporter Put4

Proline is one of the abundant amino acids in grape must, but in winemaking processes it is poorly assimilated by the yeast Saccharomyces cerevisiae. This often causes a nitrogen deficiency during fermentation and proline accumulation in wine. Our previous study showed that arginine inhibits proline utilization by specifically inducing the endocytosis of the high-affinity proline transporter Put4. However, the detailed mechanisms underlying this induction are still unclear. Here, we propose a possible mechanism mediated by the ubiquitin ligase Rsp5 and its adaptor protein, Art3. First, we found that the ubiquitination activity of Rsp5 was essential for the arginine-induced endocytosis of Put4. Because Put4 contains no Rsp5-binding motif, we next screened an adaptor protein that plays a role in the arginine-induced endocytosis of Put4. Our genetic and biochemical analyses clearly revealed that the ART3 gene-disrupted cells were defective in Put4 endocytosis, indicating that Art3 is a key regulator for Put4 endocytosis. More importantly, we discovered that deletion of ART3 remarkably canceled the inhibitory effects of arginine on proline utilization. The present results could hold promise for the development of wine yeast strains that can efficiently assimilate the abundant proline in grape must during the fermentation processes.

Proline Utilization by Bacillus subtilis: Uptake and Catabolism

L-Proline can be used by Bacillus subtilis as a sole source of carbon or nitrogen. We traced L-proline utilization genetically to the putBCP (ycgMNO) locus. The putBCP gene cluster encodes a high-affinity proline transporter (PutP) and two enzymes, the proline dehydrogenase PutB and the Δ(1)-pyrroline-5-carboxylate dehydrogenase PutC, which jointly catabolize L-proline to L-glutamate. Northern blotting, primer extension, and putB-treA reporter gene fusion analysis showed that the putBCP locus is transcribed as an L-proline-inducible operon. Its expression was mediated by a SigA-type promoter and was dependent on the proline-responsive PutR activator protein. Induction of putBCP expression was triggered by the presence of submillimolar concentrations of L-proline in the growth medium. However, the very large quantities of L-proline (up to several hundred millimolar) synthesized by B. subtilis as a stress protectant against high osmolarity did not induce putBCP transcription. Induction of putBCP transcription by external L-proline was not dependent on L-proline uptake via the substrate-inducible PutP or the osmotically inducible OpuE transporter. It was also not dependent on the chemoreceptor protein McpC required for chemotaxis toward L-proline. Our findings imply that B. subtilis can distinguish externally supplied L-proline from internal L-proline pools generated through de novo synthesis. The molecular basis of this regulatory phenomenon is not understood. However, it provides the B. subtilis cell with a means to avoid a futile cycle of de novo L-proline synthesis and consumption by not triggering the expression of the putBCP L-proline catabolic genes in response to the osmoadaptive production of the compatible solute L-proline.

L-Proline transport into renal OK epithelial cells: a second renal proline transport system is induced by amino acid deprivation

Influx of [(3)H]-L-proline into renal OK cells revealed that basal transport was mediated by the transporter SIT1. When cells were submitted for 8 h to amino acid deprivation, uptake of L-proline was now dominated by a low-affinity system with an apparent K (m) of 4.4 +/- 0.6 mM and a V (max) of 10.2 +/- 0.6 nmol/mg of protein/min operating in addition to the high-affinity SIT1 system with a K (m) of 0.12 +/- 0.01 mM and a V (max) of 0.28 +/- 0.04 nmol/mg of protein/min. The low- and high-affinity proline transporting systems were sensitive to inhibitors of JNK and PI-3 kinases, whereas a GSK-3 inhibitor affected only the upregulated transport system. Ion-replacement studies and experiments assessing substrate specificities for both systems provided strong evidence that SNAT2, that showed two- to threefold increased mRNA levels, is the responsible transporter mediating the increased proline influx under conditions of amino acid deprivation.

Discovery and characterization of potent small molecule inhibitors of the high affinity proline transporter

The mammalian proline transporter (PROT) is a high affinity Na +/Cl −-dependent transporter expressed in specific regions of the brain. It is homologous to other neurotransmitter transporters such as glycine, norepinephrine, serotonin, and dopamine transporters. PROT is enriched in glutamatergic synaptic terminals and may play an important role in the regulation of excitatory neurotransmission. No non-peptide small molecule inhibitors have been described for this transporter. To study its physiological role in the central nervous system and evaluate its potential as a therapeutic target, we established cell lines that stably express recombinant hPROT and characterized its kinetic properties for proline uptake. We then screened for inhibitors and identified a series of compounds that inhibit hPROT-mediated proline uptake. A known compound, benztropine, was found to inhibit hPROT with an IC 50 of 0.75 μM. A series of novel compounds were also found, one of which, LP-403812, showed an IC 50 of approximately 0.1 μM on both recombinant human and mouse PROT without significant inhibition of glycine and dopamine transporters at concentrations up to 10 μM. This compound also inhibited proline transporter activity of mouse brain synaptosomes with the same potency. These inhibitors provide important tools for the understanding of PROT functions in the brain and may lead to the development of therapeutic agents for certain neurological disorders.

Transcriptional activation of the Staphylococcus aureus putP gene by low-proline-high osmotic conditions and during infection of murine and human tissues.

Staphylococcus aureus can grow virtually anywhere in the human body but needs to import proline through low- and high-affinity proline transporters to survive. This study examined the regulation of the S. aureus putP gene, which encodes a high-affinity proline permease. putP::lacZ and putP::lux transcriptional fusions were constructed and integrated into the genomes of several S. aureus strains. Enzyme activity was measured after growth in media with various osmolyte concentrations. As osmolarity rose, putP expression increased, with a plateau at 2 M for NaCl in strain LL3-1. Proline concentrations as low as 17.4 muM activated expression of the putP gene. The putP::lux fusion was also integrated into the genomes of S. aureus strains that were either SigB inactive (LL3-1, 8325-4, and SH1003) or SigB active (Newman and SH1000). SigB inactive strains showed increased putP gene expression as NaCl concentrations rose, whereas SigB active strains displayed a dramatic decrease in putP expression, suggesting that the alternative sigma factor B plays a negative role in putP regulation. Mice inoculated with S. aureus strains containing the putP::lux fusion exhibited up to a 715-fold increase in putP expression, although levels in the various murine organs differed. Moreover, urine from human patients infected with S. aureus showed elevated putP levels by use of a PCR procedure, whereas blood and some abscess material had no significant increase. Thus, putP is transcriptionally activated by a low-proline and high osmotic environment both in growth media and in murine or human clinical specimens.

Low-proline environments impair growth, proline transport and in vivo survival of Staphylococcus aureus strain-specific putP mutants

Staphylococcus aureus is a common cause of disease in humans, particularly in hospitalized patients. This species needs to import several amino acids to survive, including proline. Previously, it was shown that an insertion mutation in the high-affinity proline uptake gene putP in strain RN6390 affected proline uptake by the bacteria as well as reducing their ability to survive in vivo. To further delineate the effect of the putP mutation on growth of S. aureus strain RN6390, a proline uptake assay that spanned less than 1 min was done to measure transport. An eightfold difference in proline levels was observed between the wild-type strain and the high-affinity proline transport mutant strain after 15 s, indicating that the defect was only in proline transport and not a combination of proline transport, metabolism and accumulation that would have been assessed with longer assays. A putP mutant of S. aureus strain RN4220 was then grown in minimal medium with different concentrations of proline. When compared to the wild-type strain, the putP mutant strain was significantly growth impaired when the level of proline was decreased to 1.74 microM. An assessment of proline concentrations in mouse livers and spleens showed proline concentrations of 7.5 micromol per spleen and 88.4 micromol per liver. To verify that the effects on proline transport and bacterial survival were indeed caused solely by a mutation in putP, the putP mutation was complemented by cloning a full-length putP gene on a plasmid that replicates in S. aureus. Complementation of the putP mutant strains restored proline transport, in vitro growth in low-proline medium, and in vivo survival within mice. These results show that the mutation in putP led to attenuated growth in low-proline media and by corollary low-proline murine organ tissues due to less efficient transport of proline into the bacteria.

Identification and characterization of the PutP proline permease that contributes to in vivo survival of Staphylococcus aureus in animal models.

Staphylococcus aureus is an important pathogen of humans and other animals, causing bacteremia, abscesses, endocarditis, and other infectious syndromes. A signature-tagged mutagenesis (STM) system was adapted for use in studying the genes required for in vivo survival of S. aureus. An STM library was ultimately created in S. aureus RN6390, with Tn917 being used to create the transposon mutations. Pools of S. aureus RN6390 mutants were screened in mouse abscess, bacteremia, and wound infection models for growth attenuation after in vivo passage. One of the mutants that was identified displayed marked attenuation following large-pool screening in all three animal models, which was confirmed in bacteremia and endocarditis models of infection with a smaller pool of mutants. Sequence analysis of the entire open reading frame showed a 99% identity to the high-affinity proline permease (putP) gene characterized in another strain of S. aureus. In wound and murine abscess infection models, the putP mutant was approximately 10-fold more attenuated than was wild-type strain RN6390. Another S. aureus strain transduced with the putP mutation also displayed an attenuated phenotype after passage in the wound model. A [3H]proline uptake assay showed that less proline was specifically transported into the putP mutant than into strain RN6390. The reduced viability of the bacteria possessing the mutation in the S. aureus high-affinity proline permease suggests that proline scavenging by the bacteria is important for in vivo growth and proliferation and that analogs of proline may serve as potential antistaphylococcal therapeutic agents.

A cryptic proline permease in Salmonella typhimurium.

Wild-type Salmonella typhimurium expresses three proline transport systems: a high-affinity proline transport system encoded by the putP gene, and two glycine betaine transport systems with a lower affinity for proline encoded by the proP and proU genes. Although proline uptake by the ProP and ProU transport systems is sufficient to supplement a proline auxotroph, it is not efficient enough to allow proline utilization as a sole source of carbon or nitrogen. Thus, the PutP transport system is required for utilization of proline as a carbon or nitrogen source. In this study, an overexpression suppressor, designated proY, which allows proline utilization in a putP genetic background and does not require the function of any of the known proline transport systems, was cloned and characterized. The suppressor gene, designated proY, maps at 8 min on the S. typhimurium linkage map, distant from any of the other characterized proline transport genes. The DNA sequence of the proY gene predicts that it encodes a hydrophobic integral membrane protein, with strong similarity to a family of amino acid transporters. The suppressor phenotype requires either a multicopy done of the proY+ gene or both a single copy of the proY+ gene and a proZ mutation. These results indicate that the proY gene is the structural gene for a cryptic proline transporter that is silent unless overexpressed on a multicopy plasmid or activated by a proZ mutation.

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.

Proline-induced potentiation of glutamate transmission

The amino acid proline has long been suspected to serve as a modulator of synaptic transmission in the mammalian brain, but no such function has been identified. The selective expression of high affinity proline transport by a subset of glutamate pathways suggested that proline might play a role in synaptic transmission at these sites. This idea was tested with use of one such pathway, the Schaffer collateral-commissural projection to CA1 pyramidal cells of the rat hippocampus. Proline enhanced the initial slope of the field EPSP without affecting axonal excitability or the magnitude of paired-pulse facilitation. Proline-induced potentiation far outlasted the period of proline application and required the activation of NMDA receptors. Proline enhanced Schaffer collateral-commissural synaptic transmission even when the connections between areas CA1 and CA3 had been interrupted. Potentiation was observed with a proline concentration normally present in human CSF (3 μM). A concentration typical of CSF in persons with the genetic disorder hyperprolinemia type II (30 μM) produced a somewhat greater effect. Occlusion experiments suggested that proline-induced potentiation and tetanus-induced long-term potentiation utilize largely distinct transduction mechanisms. Proline-induced potentiation could be blocked by a prior high frequency stimulus, whether or not the stimulus evoked long-term potentiation. These results suggest that endogenous extracellular proline regulates the basal function of some glutamate synapses by maintaining them in a partially potentiated state. They may also facilitate understanding of the seizures and/or mental retardation associated with genetic disorders of proline metabolism.

Sodium-dependent proline and glutamate uptake by hippocampal synaptosomes during postnatal development

Na +-dependent uptake of proline and glutamate by hippocampal synaptosomes was studied during postnatal development. At all ages from 9 days to adulthood, hippocampal synaptosomes transported proline by both a high-affinity and a low-affinity process, whereas glutamate was always transported predominantly by a high-affinity process. During the period of rapid synaptogenesis, the K T for high-affinity proline transport overshot the adult value, whereas the K T for glutamate transport increased steadily toward the adult value. The ratio of K T values for proline and glutamate was 2–3 times the adult value between 12 and 24 days of age. Although high-affinity transporters for proline and glutamate are expressed by nearly the same hippocampal pathways, they are differentially regulated during postnatal development.

Asparaginase II of Saccharomyces cerevisiae. GLN3/URE2 regulation of a periplasmic enzyme.

The production of some extracellular enzymes is known to be negatively affected by readily metabolized nitrogen sources such as NH4+ although there is no consensus regarding the involved mechanisms. Asparaginase II is a periplasmic enzyme of Saccharomyces cerevisiae encoded by the ASP3 gene. The enzyme activity is not found in cells grown in either ammonia, glutamine, or glutamate, but it is found in cells that have been subjected to nitrogen starvation or have been grown on a poor source of nitrogen such as proline. In this report it is shown that the formation of this enzyme is dependent upon the functional GLN3 gene and that the response to nitrogen availability is under the control of the URE2 gene product. In this respect the expression of ASP3 is similar to the system that regulates the GLN1, GDH2, GAP1, and PUT4 genes that codes for glutamine synthetase, NAD-linked glutamate dehydrogenase, general amino-acid permease, and high affinity proline permease, respectively.

Ser57 in the Na+/proline permease of Escherichia coli is critical for high-affinity proline uptake.

Ser57 in the Na+/proline permease of Escherichia coli has been replaced with alanine, cysteine, glycine, or threonine, and properties of the corresponding putP mutants have been analyzed. Although Ser57 is not essential for activity, the amino acid side chain at this position is critical for proline uptake. Thus, alanine, cysteine, glycine, or threonine in place of Ser57 reduces the initial rate of proline transport under standard conditions to less than 10% of the wild-type value. In addition, substitution of Ser57 in the Na+/proline permease reduces the sensitivity of E. coli cells to the toxic proline analogs L-azetidine-2-carboxylate and 3.4-dehydro-D.L-proline. Replacement of Ser57 with alanine or cysteine results in apparent affinities for proline that are reduced by more than two orders of magnitude, and permeases with threonine and glycine in place of Ser57 yield apparent affinities reduced by a factor of 60 and 18 respectively, relative to wild-type. In contrast, all of the Ser57 replacements analyzed cause only small changes in Vmax values. All permease molecules containing Ser57 substitutions are inserted into the membrane in amounts comparable to the wild-type protein as shown by immunoblot analysis. These results indicate that alterations of proline transport and sensitivity to toxic proline analogs have to be attributed primarily to defects in substrate binding. It is suggested that the serine residue at position 57 of the permease is located within the substrate-binding domain of the protein.

Identification of a PutP proline permease gene homolog from Staphylococcus aureus by expression cloning of the high-affinity proline transport system in Escherichia coli

The important food-borne pathogen Staphylococcus aureus is distinguished by its ability to grow at low water activity values. Previous work in our laboratory and by others has revealed that proline accumulation via transport is an important osmoregulatory strategy employed by this bacterium. Furthermore, proline uptake by this bacterium has been shown to be mediated by two distinct transport systems: a high-affinity system and a low-affinity system (J.-H. Bae, and K. J. Miller, Appl. Environ. Microbiol. 58:471-475, 1992; D. E. Townsend and B. J. Wilkinson, J. Bacteriol. 174:2702-2710, 1992). In the present study, we report the cloning of the high-affinity proline transport system of S. aureus by functional expression in an Escherichia coli host. The sequence of the staphylococcal proline permease gene was predicted to encode a protein of 497 amino acids which shares 49% identity with the PutP high-affinity proline permease of E. coli. Analysis of hydropathy also indicated a common overall structure for these proteins.

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.

Nucleotide sequence of the Saccharomyces cerevisiae PUT4 proline-permease-encoding gene: similarities between CAN1, HIP1 and PUT4 permeases

The complete nucleotide (nt) sequence of the PUT4 gene, whose product is required for high-affinity proline active transport in the yeast Saccharomyces cerevisiae, is presented. The sequence contains a single long open reading frame of 1881 nt, encoding a polypeptide with a calculated M r of 68795. The predicted protein is strongly hydrophobic and exhibits six potential glycosylation sites. Its hydropathy profile suggests the presence of twelve membrane-spanning regions flanked by hydrophilic N- and C-terminal domains. The N terminus does not resemble signal sequences found in secreted proteins. These features are characteristic of integral membrane proteins catalyzing translocation of ligands across cellular membranes. Protein sequence comparisons indicate strong resemblance to the arginine and histidine permeases of S. cerevisiae, but no marked sequence similarity to the proline permease of Escherichia coli or to other known prokaryotic or eukaryotic transport proteins. The strong similarity between the three yeast amino acid permeases suggests a common ancestor for the three proteins.

Genetic and physical characterization of putP, the proline carrier gene of Escherichia coli K12.

Two new mutants of E. coli K12, strains PT9 and PT32 were isolated, that were defective in proline transport. They had no high affinity proline transport activity, but their cytoplasmic membranes retained proline binding activity with altered sensitivity to inhibition by p-chloromercuribenzoate (pCMB). The lesion was mapped at the putP gene, which is located at min 23 on the revised E. coli genetic map (Bachmann 1983) as a composite gene in the proline utilization gene cluster, putP, putC, and putA, arranged in this order. The putC gene was shown to regulate the synthesis of proline dehydrogenase (putA gene product). Hybrid plasmids carrying the put region (Motojima et al. 1979; Wood et al. 1979) were used to construct the physical map of the put region. The possible location of the putP gene in the DNA segment was determined by subcloning the putP gene, genetic complementation, and recombination analyses using several proline transport mutants.

High affinity proline uptake in rat brain synaptosomes

The uptake of L-proline by synaptosomes isolated from different regions of the brain was investigated. The highest rates of transport and the largest accumulation ratios were found in synaptosomes from the midbrain, striatum, hippocampus and hypothalamus, with lower activity in the cortex and medulla (+ pons) and lowest in the cerebellum. The high affinity, Na + -dependent proline uptake had a K m of 12 μM, a V max of 0.6 nmol.min −1.mg protein −1 and the maximum accumulation ratio ([proline] i/[proline] o) at 2 μM added radioactive amino acid was 40–50. Our results suggest that: (1) only 5–10% of the synaptosomal population accumulates proline through the high-affinity uptake; (2) the characteristics of the proline uptake system into the prolinergic nerve endings are remarkably similar to those of other amino acid neurotransmitters (GABA, aspartate and glutamate); (3) the behavior of proline is consistent with this amino acid being a neurotransmitter in the central nervous system.

Proline transport in Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae is capable of utilizing proline as the sole source of nitrogen. Mutants of S. cerevisiae with defective proline transport were isolated by selecting for resistance to either of the toxic proline analogs L-azetidine-2-carboxylate or 3,4-dehydro-DL-proline. Strains carrying the put4 mutation are defective in the high-affinity proline transport system. These mutants could still grow when given high concentrations of proline, due to the operation of low-affinity systems whose existence as confirmed by kinetic studies. Both systems were repressed by ammonium ions, and either was induce by proline. Low-affinity transport was inhibited by histidine, so put4 mutants were unable to grow on a medium containing high concentrations of proline to which histidine has been added.