Isolation of a peptide transport-deficient mutant of yeast.

A peptide transport mutant of a leucine-lysine auxotroph of Saccharomyces cerevisiae (strain Z1-2D) was isolated on the basis of its resistance to L-ethionyl-L-alanine. The mutant, designated Z1-2D Etar, did not utilize di- and tripeptides containing leucine or lysine although it contained peptidases which released the required amino acids from these substrates. S. cerevisiae Z1-2D Etar did not accumulate radioactivity from [14C]glycyl-L-leucine under conditions identical to those in which the parent took up the label from this dipeptide. These results indicate that the mutant lacks the cellular mechanism to transport peptides to the site of the peptidase activity and that di- and tripeptides share a common mode of entry into yeast.     

AtPTR1 and AtPTR5 transport dipeptides in planta

Transporters for di- and tripeptides belong to the large and poorly characterized PTR/NRT1 (peptide transporter/nitrate transporter 1) family. A new member of this gene family, AtPTR5, was isolated from Arabidopsis (Arabidopsis thaliana). Expression of AtPTR5 was analyzed and compared with tissue specificity of the closely related AtPTR1 to discern their roles in planta. Both transporters facilitate transport of dipeptides with high affinity and are localized at the plasma membrane. Mutants, double mutants, and overexpressing lines were exposed to several dipeptides, including toxic peptides, to analyze how the modified transporter expression affects pollen germination, growth of pollen tubes, root, and shoot. Analysis of atptr5 mutants and AtPTR5-overexpressing lines showed that AtPTR5 facilitates peptide transport into germinating pollen and possibly into maturating pollen, ovules, and seeds. In contrast, AtPTR1 plays a role in uptake of peptides by roots indicated by reduced nitrogen (N) levels and reduced growth of atptr1 mutants on medium with dipeptides as the sole N source. Furthermore, overexpression of AtPTR5 resulted in enhanced shoot growth and increased N content. The function in peptide uptake was further confirmed with toxic peptides, which inhibited growth. The results show that closely related members of the PTR/NRT1 family have different functions in planta. This study also provides evidence that the use of organic N is not restricted to amino acids, but that dipeptides should be considered as a N source and transport form in plants.     

Cloning of a second Arabidopsis peptide transport gene.

Previously, we reported the isolation of a peptide transport gene designated AtPTR2 from Arabidopsis thaliana by functional complementation of a yeast peptide transport mutant. We now report the isolation of a second peptide transport gene (AtPTR2-B) from Arabidopsis using the same approach. Similar to the effects of transferring AtPTR2-A (previously called AtPTR2), transfer of AtPTR2-B to yeast peptide transport mutants restored the ability to grow on di- and tripeptides but not peptides four residues or longer. However, unlike yeast mutants complemented with either the yeast PTR2 gene or the AtPTR2-A gene, transformants expressing AtPTR2-B were only partially sensitive to toxic peptides. Northern analysis showed that AtPTR2-B was constitutively expressed in all plant organs. Studies of the kinetics indicated that AtPTR2-A and AtPTR2-B have Km values of 47 and 14 microM, respectively, with Vmax values of 0.061 and 0.013 nmol mg-1 cell dry weight s-1, respectively, when dileucine was used as a substrate. AtPTR2-B is encoded on a 2.0-kb cDNA corresponding to a 585-amino acid protein (64.4 kD). Hydropathy analysis indicates that the protein is highly hydrophobic and suggests that there are 12 putative transmembrane segments. AtPTR2-B, like AtPTR2-A, shares significant similarity to a number of other proteins involved in transport of peptides into cells.     

Toxicity of oxalysine and oxalysine-containing peptides against Candida albicans: regulation of peptide transport by amino acids.

A lysine antimetabolite, L-4-oxalysine [H2NCH2CH2OCH2CH(NH2)COOH], and oxalysine-containing di-, tri-, tetra- and pentapeptides inhibited growth of Candida albicans H317. Micromolar amounts of amino acids were found to overcome ammonium repression of the di- and tripeptide transport system(s) in strain H317. Several amino acids increased the toxicity of oxalysine-containing di- and tripeptides for C. albicans with little or no increase in toxicity of oxalysine or oxalysine-containing tetra- and pentapeptides. L-Lysine completely reversed the toxicity of oxalysine by competing with the transport of oxalysine into the cells. In contrast, L-lysine increased the toxicity of oxalysine-containing di- and tripeptides, but had no effect on the toxicity of oxalysine-containing tetra- and pentapeptides. Incubation of cells with L-lysine for 4 h resulted in a 15-fold increase in the rate of transport of radiolabelled dileucine, indicating that increased sensitivity of C. albicans to some toxic peptides in the presence of L-lysine may be attributed to an increased rate of transport of these peptides. Our results indicate that the dipeptide and tripeptide transport system(s) of C. albicans are regulated by micromolar amounts of amino acids in a similar fashion to the regulation of peptide transport in Saccharomyces cerevisiae and that multiple peptide transport systems differentially regulated by various nitrogen sources and amino acids exist in C. albicans.     

AtPTR4 and AtPTR6 are differentially expressed,tonoplast-localized members of the peptide transporter/nitrate transporter 1 (PTR/NRT1) family

Members of the peptide transporter/nitrate transporter 1 (PTR/NRT1) family in plants transport a variety of substrates like nitrate, di- and tripepetides, auxin and carboxylates. We isolated two members of this family from Arabidopsis, AtPTR4 and AtPTR6, which are highly homologous to the characterized di- and tripeptide transporters AtPTR1, AtPTR2 and AtPTR5. All known substrates of members of the PTR/NRT1 family were tested using heterologous expression in Saccharomyces cerevisiae mutants and oocytes of Xenopus laevis, but none could be identified as substrate of AtPTR4 or AtPTR6. AtPTR4 and AtPTR6 show distinct expression patterns, while AtPTR4 is expressed in the vasculature of the plants, AtPTR6 is highly expressed in pollen and during senescence. Phylogenetic analyses revealed that AtPTR2, 4 and 6 belong to one clade of subgoup II, whereas AtPTR1 and 5 are found in a second clade. Like AtPTR2, AtPTR4-GFP and AtPTR6-GFP fusion proteins are localized at the tonoplast. Vacuolar localization was corroborated by co-localization of AtPTR2-YFP with the tonoplast marker protein GFP-AtTIP2;1 and AtTIP1;1-GFP. This indicates that the two clades reflect different intracellular localization at the tonoplast (AtPTR2, 4, 6) and plasma membrane (AtPTR1, 5), respectively.     

A di- and tripeptide transport system can supply Listeria monocytogenes Scott A with amino acids essential for growth.

Listeria monocytogenes takes up di- and tripeptides via a proton motive force-dependent carrier protein. This peptide transport system resembles the recently cloned and sequenced secondary di- and tripeptide transport system of Lactococcus lactis (A. Hagting, E. R. S. Kunji, K. J. Leenhouts, B. Poolman, and W. N. Konings, J. Biol. Chem. 269:11391-11399, 1994). The peptide permease of L. monocytogenes has a broad substrate specificity and allows transport of the nonpeptide substrate 5-aminolevulinic acid, the toxic di- and tripeptide analogs, alanyl-beta-chloroalanine and alanyl-alanyl-beta-chloroalanine, and various di- and tripeptides. No extracellular peptide hydrolysis was detected, indicating that peptides are hydrolyzed after being transported into the cell. Indeed, peptidase activities in response to various synthetic substrates were detected in cell extracts obtained from L. monocytogenes cells grown in brain heart infusion broth or defined medium. The di- and tripeptide permease can supply L. monocytogenes with essential amino acids for growth and might contribute to growth of this pathogen in various foods where peptides are supplied by proteolytic activity of other microorganisms present in these foods. Possible roles of this di- and tripeptide transport system in the osmoregulation and virulence of L. monocytogenes are discussed.     

Peptide transport in yeast: utilization of leucine- and lysine-containing peptides by Saccharomyces cerevisiae.

A variety of leucine-containing di- and tripeptides and two lysine-containing dipeptides supported the growth of strain Z1-2D, a leucine, lysine auxotroph of Saccharomyces cerevisiae. However, (Lys)2, (Lys)3, (Lys)4, and (Lys)5 as well as Gly-Leu-Gly, three tetra- and one pentapeptide containing leucine were not utilized by the mutant. Cellular peptidases released leucine or lysine from all of these non-growth-supporting peptides, suggesting that the failure of strain Z1-2D to utilize these compounds reflects their failure to enter the yeast. Competition studies employing phenylalanine or non-leucine-containing peptides showed that the uptake of peptides into S. cerevisiae Z1-2D is distinct from that of amino acids and that di- and oligopeptides may share a common transport system. The failure of strain Z1-2D to utilize any peptide larger than (Leu)3 may indicate a transport size limit. Such a size limit would influence the construction of models that explain the action of yeast mating factors.     

Specificity of peptide transport systems in Lactococcus lactis: evidence for a third system which transports hydrophobic di- and tripeptides.

A proton motive force-driven di-tripeptide carrier protein (DtpT) and an ATP-dependent oligopeptide transport system (Opp) have been described for Lactococcus lactis MG1363. Using genetically well-defined mutants in which dtpT and/or opp were inactivated, we have now established the presence of a third peptide transport system (DtpP) in L. lactis. The specificity of DtpP partially overlaps that of DtpT. DtpP transports preferentially di- and tripeptides that are composed of hydrophobic (branched-chain amino acid) residues, whereas DtpT has a higher specificity for more-hydrophilic and charged peptides. The toxic dipeptide L-phenylalanyl-beta-chloro-L-alanine has been used to select for a di-tripeptide transport-negative mutant with the delta dtpT strain as a genetic background. This mutant is unable to transport di- and tripeptides but still shows uptake of amino acids and oligopeptides. The DtpP system is induced in the presence of di- and tripeptides containing branched-chain amino acids. The use of ionophores and metabolic inhibitors suggests that, similar to Opp, DtpP-mediated peptide transport is driven by ATP or a related energy-rich phosphorylated intermediate.     

Multiplicity and regulation of genes encoding peptide transporters in Saccharomyces cerevisiae

The model eukaryote Saccharomyces cerevisiae has two distinct peptide transport mechanisms, one for di-/tripeptides (the PTR system) and another for tetra-/pentapeptides (the OPT system). The PTR system consists of three genes, PTR1, PTR2 and PTR3. The transporter (Ptr2p), encoded by the gene PTR2, is a 12 transmembrane domain (TMD) integral membrane protein that translocates di-/tripeptides. Homologues to Ptr2p have been identified in virtually all organisms examined to date and comprise the PTR family of transport proteins. In S. cerevisiae, the expression of PTR2 is highly regulated at the cellular level by complex interactions of many genes, including PTR1, PTR3, CUP9 and SSY1. Oligopeptides, consisting of four to five amino acids, are transported by the 12-14 TMD integral membrane protein Opt1p. Unlike Ptr2p, distribution of this protein appears limited to fungi and plants, and there appears to be three paralogues in S. cerevisiae. This transporter has an affinity for enkephalin, an endogenous mammalian pentapeptide, as well as for glutathione. Although it is known that OPT1 is normally expressed only during sporulation, to date little is known about the genes and proteins involved in the regulation of OPT1 expression.     

Kinetics and substrate specificity of membrane-reconstituted peptide transporter DtpT of Lactococcus lactis

The peptide transport protein DtpT of Lactococcus lactis was purified and reconstituted into detergent-destabilized liposomes. The kinetics and substrate specificity of the transporter in the proteoliposomal system were determined, using Pro-[(14)C]Ala as a reporter peptide in the presence of various peptides or peptide mimetics. The DtpT protein appears to be specific for di- and tripeptides, with the highest affinities for peptides with at least one hydrophobic residue. The effect of the hydrophobicity, size, or charge of the amino acid was different for the amino- and carboxyl-terminal positions of dipeptides. Free amino acids, omega-amino fatty acid compounds, or peptides with more than three amino acid residues do not interact with DtpT. For high-affinity interaction with DtpT, the peptides need to have free amino and carboxyl termini, amino acids in the L configuration, and trans-peptide bonds. Comparison of the specificity of DtpT with that of the eukaryotic homologues PepT(1) and PepT(2) shows that the bacterial transporter is more restrictive in its substrate recognition.     

Stimulatory effects of peptides on growth of the free-living nematode Caenorhabditis briggsae.

Evidence is presented that peptide products of hydrolysis of casein, including some di- and tripeptides, but not the constituent amino acids, can stimulate growth of C. briggsae in defined basal medium supplemented with cytochrome C and B-sitosterol. Peptide activity may raise from amino acid imbalances in the medium which causes competitive inhibition of uptake of essential amino acids. Such activity precludes facilitation of heme transport as the sole function of growth factor in C. briggsae. This is the first report in a nutritional role of peptides in invertebrate metazoa.     

Peptidase activities in Saccharomyces cerevisiae.

At least four distinct aminopeptidase activities and a single dipeptidase activity were found in cell extracts of a leucine-lysine auxotroph of Saccharomyces cerevisiae. The assay for peptidase activity involved polyacrylamide gel electrophoresis followed by an enzyme-coupled activity staining procedure. The aminopeptidases had largely overlapping specificities but could be distinguished from one another by their electrophoretic mobilities and activities toward different peptide substrates. Substrates tested included both free and blocked di- and tripeptides and amino acid derivatives.     

Evidence for two different broad-specificity oligopeptide transporters in intestinal cell line Caco-2 and colonic cell line CCD841

Recently the existence of two different Na(+)-coupled oligopeptide transport systems has been described in mammalian cells. These transport systems are distinct from the previously known H(+)/peptide cotransporters PEPT1 and PEPT2, which transport only dipeptides and tripeptides. To date, the only peptide transport system known to exist in the intestine is PEPT1. Here we investigated the expression of the Na(+)-coupled oligopeptide transporters in intestinal cell lines, using the hydrolysis-resistant synthetic oligopeptides deltorphin II and [d-Ala(2),d-Leu(5)]enkephalin (DADLE) as model substrates. Caco-2 cells and CCD841 cells, both representing epithelial cells from human intestinal tract, were able to take up these oligopeptides. Uptake of deltorphin II was mostly Na(+) dependent, with more than 2 Na(+) involved in the uptake process. In contrast, DADLE uptake was only partially Na(+) dependent. The uptake of both peptides was also influenced by H(+) and Cl(-), although to a varying degree. The processes responsible for the uptake of deltorphin II and DADLE could be differentiated not only by their Na(+) dependence but also by their modulation by small peptides. Several dipeptides and tripeptides stimulated deltorphin II uptake but inhibited DADLE uptake. These modulating small peptides were, however, not transportable substrates for the transport systems that mediate deltorphin II or DADLE uptake. These two oligopeptide transport systems were also able to take up several nonopioid oligopeptides, consisting of 9-17 amino acids. This represents the first report on the existence of transport systems in intestinal cells that are distinct from PEPT1 and capable of transporting oligopeptides consisting of five or more amino acids.     

植物寡肽运输与硝酸根运输基因家族的研究进展

氮素是植物生长发育的重要营养元素,也是限制植物生物量尤其是经济产量的关键营养元素之一.植物不仅能从外界获取无机氮素(硝酸根、铵根和尿素等),还能以氨基酸、寡肽等形式获取有机氮素.植物已进化出复杂的运输系统来吸收与运输这些含氮化合物.硝酸根运输基因家族分为低亲和力硝酸根运输基因(low-affmity nitrate t...     

Synthesis of cyclic tryptathionine peptides

The helicity of the tryptathionine moiety of the phallotoxins has been recognized by comparison with cyclic tryptathionine tripeptides. In order to investigate the influence of the configuration of the component amino acids on the conformation of the cyclic peptides, six analogue thioether tripeptides containing L- and D-alanine and L- and D-cysteine, respectively, have been synthesized. The CD spectra of the peptides are very similar to each other, showing mirror images of the CD of phalloidin and, therefore, negative helicity. The spectra of the D-cysteine containing compounds differ from the L-cysteine containing compounds by their weakly positive ellipticity values around 270 nm. The cyclization reaction of Boc-Hpi-D-Ala-D-Cys(STrt)OCH3, along with the cyclic tripeptide, afforded a cyclic hexapeptide by dimerization. The CD spectrum of the dimer is very similar to that of phalloidin, thus pointing to a positive helicity of its two tryptathionine moieties. The dimeric thioether peptide forms a rather strong complex with Cu2+ ions.     

Substrate specificity of peptide permeases in Candida albicans

Abstract Specificity of peptide transport systems in Candida albicans was studied using as an experimental tool novel anticandidal peptides, containing the N³-4-methoxyfumaroyl- l -2,3-diamino-propanoic acid residue. Studies on cross-resistance and on peptide uptake by spontaneous mutants resistant to toxic peptides, confirmed the multiplicity of peptide permeases in Candida albicans . At least two peptide permeases exist in this microorganism; the first one, specific for di- and tripeptides and the second, for oligopeptides containing 3–6 amino acids. The rate of the tritetra tetra-, penta- and hexapeptide transport in the mycelial form of Candida albicans is about 2-times higher than in the yeast form, while that of dipeptides is markedly reduced.
Tripeptides are proposed as the most efficient carriers for the delivery of 'warhead' amino acids into Candida albicans cells.     

Trifluoroacetylated peptides as substrates and inhibitors of elastase: a nuclear magnetic resonance study.

Trifluoroacetyl di- and tripeptides have been synthesized in order to investigate their interactions with elastase by proton and fluorine magnetic resonance. These substituted peptides behave as substrates or inhibitors of the enzyme, depending upon their length. They are hydrolyzed with production of trifluoracetic acid and unsubstituted parent peptides exclusively. The amino acid specificity observed and the absence of hydrolysis in the presence of an enzyme substituted at the serine residue of the active site indicate that the trifluoracetic hydrolysis occurs at this site. It requires the fixation of the C-terminal amino acids at the two S' subsites, as does the peptidic hydrolysis of unsubstituted or acetylated oligoalanines. Trifluoracetyl tripeptides exhibit a much higher affinity for the protein, as compared with the unsubstituted or acetylated peptides as well as compared with the trifluoroacetyl dipeptides, and they act as powerful inhibitors of the enzyme. The inhibitory binding mode has been shown to involve the fixation of the trifluoroacetyl group at subsite S4 or in its vicinity, allowing for the cooperative fixation of the C-terminal alanine at S1 and the accommodation of a transproline at S2.     

Mechanisms and functional properties of two peptide transporters, AtPTR2 and fPTR2

The Arabidopsis AtPTR2 and fungal fPTR2 genes, which encode H+/dipeptide cotransporters, belong to two different subgroups of the peptide transporter (PTR) (NRT1) family. In this study, the kinetics, substrate specificity, stoichiometry, and voltage dependence of these two transporters expressed in Xenopus oocytes were investigated using the two-microelectrode voltage-clamp method. The results showed that: 1) although AtPTR2 belongs to the same PTR family subgroup as certain H+/nitrate cotransporters, neither AtPTR2 nor fPTR2 exhibited any nitrate transporting activity; 2) AtPTR2 and fPTR2 transported a wide spectrum of dipeptides with apparent affinity constants in the range of 30 microM to 3 mM, the affinity being dependent on the side chain structure of both the N- and C-terminal amino acids; 3) larger maximal currents (Imax) were evoked by positively charged dipeptides in AtPTR2- or fPTR2-injected oocytes; 4) a major difference between AtPTR2 and fPTR2 was that, whereas fPTR2 exhibited low Ala-Asp- transporting activity, AtPTR2 transported Ala-Asp- as efficiently as some of the positively charged dipeptides; 5) kinetic analysis suggested that both fPTR2 and AtPTR2 transported by a random binding, simultaneous transport mechanism. The results also showed that AtPTR2 and fPTR2 were quite distinct from PepT1 and PepT2, two well characterized animal PTR transporters in terms of order of binding of substrate and proton(s), pH sensitivity, and voltage dependence.     

Peptide utilization by Pseudomonas putida and Pseudomonas maltophilia.

Pseudomonas putida assimilates peptides and hydrolyses them with intracellular peptidases. Amino acid auxotrophs (his, trp, thr or met) grew on a variety of di- and tripeptides up to twice as slowly as with free amino acids. Pseudomonas putida has separate uptake systems for both dipeptides and oligopeptides (three or more residues). Although the dipeptide system transported a variety of structurally diverse dipeptides it did not transport peptides having either unprotonatable N-terminal amino groups, blocked C-terminal carboxyl groups, D-residues, three or more residues, N-methylated peptide bonds, or beta-amino acids. Oligopeptide uptake lacked amino acid side-chain specificity, required a free N-terminal L-residue and had an upper size limit. Glycylglycyl-D,L-p-fluorophenylalanine inhibited growth of P. putida. Uptake of glycylglycyl[I-14C]alanine was rapid and inhibited by 2,4-dinitrophenol. Both dipeptide and oligopeptide uptake were constitutive. Dipeptides competed with oligopeptides for oligopeptide uptake, but oligopeptides did not compete in the dipeptide system. Final bacterial yields were 5 to 10 times greater when P. putida his was grown on histidyl di- or tripeptides rather than on free histidine because the histidyl residue was protected from catabolism by L-histidine ammonia-lyase. Methionine peptides could satisfy the methionine requirements of P. maltophilia. Generation times on glycylmethionine and glycylmethionylglycine were equal to those obtained with free methionine. Methionylglycylmethionylmethionine gave a generation time twice that of free methionine. Growth of P. maltophilia was inhibited by glycylglycyl-D,L-p-fluorophenylalanine.