Differential regulation and substrate preferences in two peptide transporters of Saccharomyces cerevisiae

Dal5p has been shown previously to act as an allantoate/ureidosuccinate permease and to play a role in the utilization of certain dipeptides as a nitrogen source in Saccharomyces cerevisiae. Here, we provide direct evidence that dipeptides are transported by Dal5p, although the affinity of Dal5p for allantoate and ureidosuccinate is higher than that for dipeptides. Allantoate, ureidosuccinate, and to a lesser extent allantoin competed with dipeptide transport by reducing the toxicity of the peptide Ala-Eth and decreasing the accumulation of [(14)C]Gly-Leu. In contrast to the well-studied di/tripeptide transporter Ptr2p, whose substrate specificity is very broad, Dal5p preferred to transport non-N-end rule dipeptides. S. cerevisiae W303 was sensitive to the toxic peptide Ala-Eth (non-N-end rule peptide) but not Leu-Eth (N-end rule peptide). Non-N-end rule dipeptides showed better competition with the uptake of [(14)C]Gly-Leu than N-end rule dipeptides. Similar to the regulation of PTR2, DAL5 expression was influenced by the addition of Leu and by the CUP9 gene. However, DAL5 expression was downregulated in the presence of leucine and the absence of CUP9, whereas PTR2 was upregulated. Toxic dipeptide and uptake assays indicated that either Ptr2p or Dal5p was predominantly used for dipeptide transport in the common laboratory strains S288c and W303, respectively. These studies highlight the complementary activities of two dipeptide transport systems under different regulatory controls in common laboratory yeast strains, suggesting that dipeptide transport pathways evolved to respond to different environmental conditions.     

Genomewide screen reveals a wide regulatory network for di/tripeptide utilization in Saccharomyces cerevisiae

Small peptides of two to six residues serve as important sources of amino acids and nitrogen required for growth by a variety of organisms. In the yeast Saccharomyces cerevisiae, the membrane transport protein Ptr2p, encoded by PTR2, mediates the uptake of di/tripeptides. To identify genes involved in regulation of dipeptide utilization, we performed a systematic, functional examination of this process in a haploid, nonessential, single-gene deletion mutant library. We have identified 103 candidate genes: 57 genes whose deletion decreased dipeptide utilization and 46 genes whose deletion enhanced dipeptide utilization. On the basis of Ptr2p-GFP expression studies, together with PTR2 expression analysis and dipeptide uptake assays, 42 genes were ascribed to the regulation of PTR2 expression, 37 genes were involved in Ptr2p localization, and 24 genes did not apparently affect Ptr2p-GFP expression or localization. The 103 genes regulating dipeptide utilization were distributed among most of the Gene Ontology functional categories, indicating a very wide regulatory network involved in transport and utilization of dipeptides in yeast. It is anticipated that further characterization of how these genes affect peptide utilization should add new insights into the global mechanisms of regulation of transport systems in general and peptide utilization in particular.     

High-Throughput Screening of Dipeptide Utilization Mediated by the ABC Transporter DppBCDF and Its Substrate-Binding Proteins DppA1-A5 in Pseudomonas aeruginosa

In this study, we show that the dppBCDF operon of Pseudomonas aeruginosa PA14 encodes an ABC transporter responsible for the utilization of di/tripeptides. The substrate specificity of ABC transporters is determined by its associated substrate-binding proteins (SBPs). Whereas in E. coli only one protein, DppA, determines the specificity of the transporter, five orthologous SBPs, DppA1–A5 are present in P. aeruginosa. Multiple SBPs might broaden the substrate specificity by increasing the transporter capacity. We utilized the Biolog phenotype MicroArray technology to investigate utilization of di/tripeptides in mutants lacking either the transport machinery or all of the five SBPs. This high-throughput method enabled us to screen hundreds of dipeptides with various side-chains, and subsequently, to determine the substrate profile of the dipeptide permease. The substrate spectrum of the SBPs was elucidated by complementation of a penta mutant, deficient of all five SBPs, with plasmids carrying individual SBPs. It became apparent that some dipeptides were utilized with different affinity for each SBP. We found that DppA2 shows the highest flexibility on substrate recognition and that DppA2 and DppA4 have a higher tendency to utilize tripeptides. DppA5 was not able to complement the penta mutant under our screening conditions. Phaseolotoxin, a toxic tripeptide inhibiting the enzyme ornithine carbamoyltransferase, is also transported into P. aeruginosa via the DppBCDF permease. The SBP DppA1, and with much greater extend DppA3, are responsible for delivering the toxin to the permease. Our results provide a first overview of the substrate pattern of the ABC dipeptide transport machinery in P. aeruginosa.     

Peptide utilization by group N streptococci.

The rate of glycylleucine uptake by Group N streptococci varied widely. One strain of Streptococcus cremoris did not transport the dipeptide or utilize tripeptides. In peptide-utilizing strains, amino acid, dipeptide and tripeptide transport were distinct, although dipeptides inhibited tripeptide utilization. Specificity determinants for peptide transport and utilization were similar to those reported in Gram-negative bacteria. Peptide utilization in S. lactis was not completely dependent on the transport of intact peptides.     

Probing the Putative Active Site of YjdL: An Unusual Proton-Coupled Oligopeptide Transporter from E. coli

YjdL from E. coli is an unusual proton-coupled oligopeptide transporter (POT). Unlike prototypical POTs, dipeptides are preferred over tripeptides, in particular dipeptides with a positively charged C-terminal residue. To further understand this difference in peptide specificity, the sequences of YjdL and YdgR, a prototypical E. coli POT, were compared in light of the crystal structure of a POT from Shewanella oneidensis. Several residues found in the putative active site were mutated and the activities of the mutated variants were assessed in terms of substrate uptake assays, and changes in specificity in terms of uptake inhibition. Most strikingly, changing the YjdL specific Asp392 to the conserved Ser in YjdL obliterated the preference for a positively charged C-terminal residue. Based on this unique finding and previously published results indicating that the dipeptide N-terminus may interact with Glu388, a preliminary orientation model of a dipeptide in the YjdL cavity is presented. Single site mutations of particularly Ala281 and Trp278 support the presented orientation. A dipeptide bound in the cavity of YjdL appears to be oriented such that the N-terminal side chain protrudes into a sub pocket that opens towards the extracellular space. The C-terminal side chain faces in the opposite direction into a sub pocket that faces the cytoplasm. These data indicated a stabilizing effect on a bulky N-terminal residue by an Ala281Phe variant and on the dipeptide backbone by Trp278. In the presented orientation model, Tyr25 and Tyr58 both appear to be in proximity of the dipeptide backbone while Lys117 appears to be in proximity of the peptide C-terminus. Mutational studies of these conserved residues highlight their functional importance.     

The benefits and risks of expressing the POT and FOT family of oligopeptide transporters in Saccharomyces cerevisiae

In the yeast Saccharomyces cerevisiae, all strains possess a gene for the evolutionarily conserved POT family peptide transporter, Ptr2; however, the genes for a novel FOT family transporter were found only in some wine brewing strains. The substrate specificity of the POT and FOT family of transporters was compared. Among the naturally occurring oligopeptides that were tested, Lys-Leu and Arg-Phe were Ptr2-specific substrates. Artificial dipeptide aspartame was imported specifically through the FOT transporter, but the structurally similar Asp-Phe was a substrate of both FOT and Ptr2 transporters. Furthermore, only the FOT transporter was important for high sensitivity to an antibiotic puromycin. These results demonstrate that the POT and FOT family of transporters have distinct substrate preferences although both transporters import overlapping dipeptide substrates. Having POT and FOT transporters is advantageous for cells to acquire nutrients, but also detrimental when these cells are exposed to the toxic molecules of their substrates.     

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.     

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.     

Transport of [14C]Gly-Pro in a proline peptidase mutant of Salmonella typhimurium.

The transport of [14C]Gly-Pro was examined using a mutant of Salmonella typhimurium (strain TN87) deficient in an X-Pro dipeptidase and an X-Pro-Y iminopeptidase. The dipeptide was taken up by one saturable transport system having a Km of 5.3-10(-7)M and a V of 1.4 nmol/mg dry wt cell per min. The uptake of Gly-Pro was not inhibited by amino acids or tripeptides and the transport system exhibited a rather broad side chain specificity for dipeptides. Dipeptides containing hydrophobic residues were the most potent inhibitors of this dipeptide transport system exhibiting Ki values between 10(-8) and 10(-7) M. In contrast, dipeptides containing glycine residues were particularly weak inhibitors. Finally, Gly-Pro was found to be in the intact form inside the cell and was concentrated more than 1000-fold.     

The Saccharomyces cerevisiae GATA Factors Dal80p and Deh1p Can Form Homo- and Heterodimeric Complexes

GATA family proteins Gln3p, Gat1p, Dal80p, and Deh1p mediate the regulation of nitrogen catabolite repression (NCR)-sensitive gene expression in Saccharomyces cerevisiae. Thus far, Gln3p, Dal80p, and Deh1p have been shown to bind to GATA sequences in NCR-sensitive promoters, in some cases to exactly the same GATA sequences. A minimal Gln3p binding site consists of a single GATA sequence, whereas a Dal80p binding site consists of two GATA sequences in specific orientation, 15 to 35 bp apart, suggesting that Dal80p may bind to DNA as a dimer. Additionally, both Dal80p and Deh1p are predicted to contain a leucine zipper motif near their C termini. Therefore, we tested whether they could form homo- and/or heterodimers in two-hybrid assays. We show that Dal80p-Dal80p, Dal80p-Dal80pLZ (leucine zipper), Dal80pLZ-Dal80pLZ, Dal80p-Deh1pLZ, Dal80pLZ-Deh1pLZ, and Deh1pLZ-Deh1pLZ complexes can form. Dal80p-Dal80p and Dal80pLZ-Dal80pLZ complexes yield 5- to 10-fold stronger signals than the other possible dimers. If Dal80p and Deh1p bind to DNA only after dimerization, then the difference in ability to form complexes could significantly affect their affinity for binding DNA and thus the degree of regulation exerted by each of the two factors.     

Transport and metabolism of bacilysin and other peptides by suspensions of Staphylococcus aureus

L-Alanyl-L-tyrosine and glycyl-L-phenylalanine labelled with 14C competed with each other and with the dipeptide antibiotic bacilysin for transport into Staphylococcus aureus NCTC 6571 in a medium which did not support growth. They also competed with other dipeptides and several tripeptides. The fast initial transport ofthe two labelled peptides appeared to show Michaelis-Menten kinetics. Neither was transported into a bacilysin-resistant mutant of S. aureus NCTC 6571, although tyrosine was taken up by the mutant as readily as it was by the parent strain. Uptake of alanyltyrosine or glycylphenylalanine was followed by rapid hydrolysis of the peptide and the excretion of tyrosine or phenylalanine. Glycine liberated from glycylphenylalanine was partly degraded and partly incorporated into the bacterial wall. The behaviour of these dipeptides paralleled the inactivation of bacilysin by suspensions of S. aureus and the appearance of its C-terminal amino acid, anticapsin, in the extracellular fluid.     

Peptide Utilization by Amino Acid Auxotrophs of Neurospora crassa

The ability of auxotrophs of Neurospora crassa to grow on certain tripeptides, despite the presence of excess competing amino acids, suggests it has an oligopeptide transport system. In general, dipeptides did not support growth except in those instances where extracellular hydrolysis occurred, or where the dipeptide appeared to be accumulated by an uptake system which is sensitive to inhibition by free amino acids. Considerable intracellular peptidase activity toward a large number of peptides was demonstrated, including a number of peptides which could not be utilized for growth. The intracellular peptidase activity was shown to be selective for amino acid composition and sequence (N-terminal or C-terminal) within the peptide; glycine-containing peptides were particularly poor substrates for peptidase activity. Only a small amount of extracellular peptidase activity could be detected.     

Isolation and characterization Salmonella typhimurium mutants lacking a tripeptidase (peptidase T).

Salmonella typhimurium contains an enzyme, peptidase T, that hydrolyzes a variety of tripeptides. Specificity studies with a peptidase activity stain after gel electrophoresis of crude cell extracts showed that peptidase T hydrolyzes tripeptides containing N-terminal methionine, leucine, or phenylalanine. Little or no activity could be detected against dipeptides, N-blocked or C-blocked tripeptides, and tetrapeptides. Analysis of reaction products by high-pressure liquid chromatography showed that peptidase T removes the N-terminal amino acid from tripeptides. Mutants lacking peptidase T were isolated by screening microcultures grown in the wells of plastic microtitration plates for hydrolysis of Met-Ala-Ser or Met-Gly-Gly. Mutations (pepT) that eliminate this enzyme were found to be phage P22 cotransducible with purB at approximately 25 map units on the S. typhimurium map. Comparison of the growth properties of mutant and wild-type strains suggests that peptidase T does not function in utilization of tripeptides to provide amino acids during growth.     

Ligand binding analyses of the putative peptide transporter YjdL from E. coli display a significant selectivity towards dipeptides

Proton-dependent oligopeptide transporters (POTs) are secondary active transporters that couple the inwards translocation of di- and tripeptides to inwards proton translocation. Escherichia coli contains four genes encoding the putative POT proteins YhiP, YdgR, YjdL and YbgH. We have over-expressed the previously uncharacterized YjdL and investigated the peptide specificity by means of uptake inhibition. The IC₅₀ value for the dipeptide Ala-Ala was measured to 22 mM while Ala-Ala-Ala was not able to inhibit uptake. In addition, IC₅₀ values of 0.3 mM and 1.5 mM were observed for Ala-Lys and Tyr-Ala, respectively, while the alanyl-extended tripeptides Ala-Lys-Ala, Ala-Ala-Lys, Ala-Tyr-Ala and Tyr-Ala-Ala displayed values of 8, >50, 31 and 31 mM, respectively. These results clearly indicate that unlike most POT members characterized to date, including YdgR and YhiP, YjdL shows significantly higher specificity towards dipeptides.     

Separation of N-TFA-Glycyl and N-TFA-l-Prolyl Dipeptide Methyl Esters by Gas-chromatography

We have prepared a series of N-TFA-glycyl and N-TFA-l-prolyl dipeptide methyl ester from the corresponding dipeptide methyl esters by treating them with (TFA-Gly)₂O and TFA-l-Pro-Cl, respectively. Separation of these tripeptide derivatives by G.L.C. was studied and a relationship between the amino acid compositions of the tripeptides and their q_i-values (relative retention values) was observed, analogous to the case of the dipeptides previously reported.