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Eukaryotic Cell, October 2007, p. 1805-1813, Vol. 6, No. 10
1535-9778/07/$08.00+0     doi:10.1128/EC.00257-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Differential Regulation and Substrate Preferences in Two Peptide Transporters of Saccharomyces cerevisiae{triangledown}

Houjian Cai,1 Melinda Hauser,1 Fred Naider,2,3 and Jeffrey M. Becker1*

Department of Microbiology, University of Tennessee, Knoxville, Tennessee 37996-0845,1 Department of Chemistry and Macromolecular Assemblies Institute, College of Staten Island, CUNY, New York 10314,2 Graduate School and University Center, CUNY, New York 103143

Received 11 August 2006/ Accepted 9 July 2007


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ABSTRACT
 
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 [14C]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 [14C]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.


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INTRODUCTION
 
Utilization of di- and tripeptides (di/tripeptides) as nitrogen and carbon sources and for protein synthesis is an important cellular process documented in all organisms from archaea to bacteria, plants, and animals. Di/tripeptides are not only important nutrient sources but also participate in a variety of physiological functions. Several groups of naturally occurring di/tripeptides or synthesized peptide compounds show antitumor growth, neuroprotection activity, brain hormone activity, and stimulation of the immune system (25), and di/tri-peptides regulate a variety of cellular processes such as gene transcription, protein translation, and enzyme activity (22, 26, 31, 39).

The influx of di/tripeptides is mediated by two types of transport systems, ATP-binding cassette peptide transporters and proton-driven peptide transporters (38). In eukaryotic organisms, two distinct proton-coupled peptide transport systems have been reported in a variety of organisms (18): the peptide transport (PTR) system transports di/tripeptides (38), and the oligopeptide transport system highly favors the transport of peptides of four to five amino acid residues and also glutathione (27, 29). Both the PTR transport and the oligopeptide transport systems are predicted to contain 12 transmembrane domains and have specific signature sequences distinguishing them from one another, as well as from all other proteins in the database (18). Ptr2p, encoded by the PTR2 gene, is the only member of the PTR family that transports di/tripeptides in Saccharomyces cerevisiae.

In yeast, peptide transport is upregulated by growth media containing poor nitrogen sources, such as allantoin, isoleucine, or proline (21), due to the high upregulation of PTR2 resulting in efficient peptide transport. In media containing ammonium, a rich nitrogen source, PTR2 expression is downregulated via nitrogen catabolite repression (28). Peptide utilization is also regulated by the addition of micromolar amounts of certain amino acids, most notably leucine and tryptophan, to the growth medium (20), which results also in upregulation of the expression of PTR2 (32). Amino acids regulate PTR2 expression through the SPS (Ssy1p-Ptr3p-Ssy5p) signal transduction pathway (1, 13-15). In the SPS complex, Ssy1p is a transmembrane receptor that senses extracellular amino acids and results in the induction of the di/tripeptide transporter PTR2 and several amino acid permeases, such as the broad specificity amino acid permease (AGP1) and the branched-chain amino acid permeases (BAP2 and BAP3). Ssy1p also downregulates the expression level of the arginine permease gene (CAN1), the high-affinity proline permease (PUT4), and the general amino acid permease gene (GAP1) (2, 11, 13, 23, 24).

A variety of genes is involved in the regulation of PTR2 expression and ultimately influence dipeptide utilization (5). Cup9p has been identified as a repressor of PTR2 expression. In a cup9-null mutant, PTR2 is overexpressed leading to a high level of Ptr2p in the membrane and results in a marked increase in the uptake of dipeptides (4, 5). Cup9p is destabilized by the protein complex of Ptr1p, Ubc2p, and Ubc4p via the ubiquitination pathway. In this pathway, Ptr1p acts as a scaffolding protein or ubiquitin ligase (E3) for Ubc2p and Ubc4p, which serve as ubiquitin-conjugating (E2) enzymes in the Cup9p degradation process (44). Dipeptides containing N-terminal basic (Arg, Lys, and His) or bulky hydrophobic (Phe, Leu, Tyr, Trp, and Ile) amino acids, also called the N-end rule residues, bind directly to two distinct binding sites on Ptr1p and accelerate the Ptr1p-dependent degradation of Cup9p (39).

By investigating dipeptide utilization in yeast strains with different genetic backgrounds, Dal5p, previously defined as an allantoate/ureidosuccinate permease, was identified as playing a role in utilizing dipeptides as a nitrogen source when present at high (millimolar) concentrations (19). In the W303 strain background, a ptr2 deletion mutant could grow in medium supplemented with millimolar concentrations of Ala-Leu as the sole nitrogen source; however, the dal5 deletion mutant did not grow on the same medium (19). Similar to PTR2, the expression of DAL5 was highly upregulated when yeast cells were grown under poor nitrogen conditions, such as when proline, allantoin, or ornithine were supplied as the sole nitrogen source (34), and DAL5 was subjected to nitrogen catabolite repression as rich nitrogen sources such as asparagine, glutamine, or ammonium suppressed the allantoate transporter (6). In addition, several gene products, such as Dal80p, Gln3p, Ure2p, Mks1p, Rtg2p, Vid30p, and Tor1/2p, have been reported to be involved in the direct or indirect regulation of DAL5 expression (7-10, 12, 16, 33, 36, 42, 43).

We examine here the role of Dal5p in dipeptide transport. We found that allantoate, ureidosuccinate, and dipeptides are all substrates for Dal5p; however, dipeptides have a much lower affinity than either allantoate or ureidosuccinate. In addition, we show that Dal5p favors the transport of some non-N-end rule dipeptides but not N-end rule dipeptides. We found that the regulation of DAL5 is dependent on leucine and Cup9p. In response to leucine, DAL5 expression is downregulated, whereas Cup9p upregulates expression. These effects are opposite to those observed for leucine and Cup9p on the expression of PTR2. We show that Ptr2p activity is limited in the W303 wild-type strain of yeast but is predominant in the S288c background and that Dal5p provides an alternative pathway for dipeptide transport in W303, indicating that different routes for dipeptide transport may be shaped by natural selection in genetically diverse yeast strains.


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MATERIALS AND METHODS
 
Strains and media. We used S. cerevisiae W303-x (MATa ura3-1 can1-100 ho), a derivative of W303 in which auxotrophies for adenine, histidine, leucine, and tryptophan were eliminated (19), and S. cerevisiae FY3 (MATa ura3-1) in the present study. The FY3 strain belongs to the S288c lineage (3), while the W303 strain was derived from a different lineage as described previously (19). S288c and W303 strains are two commonly used laboratory strains. S288c was the strain chosen for the first eukaryotic genome sequencing (37). Deletions of PTR2, DAL5, PTR2 DAL5, and CUP9 in the W303 strain background were created to study the impact of the deleted genes on dipeptide utilization (19). Strains were inoculated into minimal proline medium [MP contained (per liter) 20 g of dextrose, 1.7 g of yeast nitrogen base (Difco, Sparks, MD) without (NH4)2SO4 and amino acids, and 1 g of proline as a nitrogen source] supplemented with uracil (20 µg/ml) to satisfy the auxotrophic requirement. In cases when the strain carried plasmid pRS426-DAL5 (19), uracil was not added to the medium.

Toxic halo assays. For the toxic dipeptide halo assay, the sensitivity of deletion mutants to the toxic dipeptide Ala-Eth or Leu-Eth was measured as previously described (20). Ethionine (Eth) is an analog of methionine, and utilization of Eth results in cell death. Yeast cells expressing a functional dipeptide transport system will take up and hydrolyze the toxic dipeptide and die (indicated by a clear halo of growth inhibition on plates spotted with Eth-containing peptides). Cells with a defective dipeptide uptake system will not take up the dipeptide efficiently and will survive (as indicated by a small halo or no halo). Cells were grown overnight in MP+Ura or MP medium for strains carrying p426-DAL5 and then harvested and washed three times with sterile, distilled water. Yeast cells were counted and adjusted to 5 x 106 cells/ml. One milliliter of the cell suspension was added to 0.8% noble agar (3 ml), and this top agar was spread onto solid MP+Ura medium (MP+Ura plus 2% agar). Two 6-mm sterile paper disks containing either 0.4 or 0.2 µmol of Ala-Eth or Leu-Eth were placed on the top agar, the plates were incubated at 30°C for 2 days, and the halo size was measured. For measuring the effect of leucine on the sensitivity to toxic dipeptide, top agar lawns were plated onto MP+Ura+Leu (MP+Ura containing Leu [30 µg/ml]). Each experiment was repeated at least three times, with similar or identical results obtained in each experiment.

For competition toxicity assays, the procedure and medium were the same as used for the toxic dipeptide halo assay, except that in addition to a disk containing 0.4 µmol of Ala-Eth another disk containing 0.4 µmol of allantoate, ureidosuccinate, or 0.65 µmol of allantoin was placed on the same plate, separated by a distance of 15 mm from the Ala-Eth disk. In addition, a third disk containing a mixture of Ala-Eth (0.4µmol) and either allantoate (0.4 µmol), ureidosuccinate (0.4 µmol), or allantoin (0.65 µmol) was also placed in the same plate. Photographs of the halo images were taken after 2 days incubation at 30°C. The data were analyzed statistically by a nonpaired Student t test.

Uptake assays. The strains were grown overnight in MP+Ura and then subcultured into 30 ml of fresh medium. Cells were harvested in log phase at a cell density of 5 x 106 cells/ml, washed with 2% glucose, and adjusted to a final concentration of 108 cells/ml in 2% glucose. The uptake assay was initiated by combining equal volumes of prewarmed (30°C) cells and 2x uptake medium composed of 2% glucose, 20 mM sodium citrate-potassium phosphate (pH 5.5), and 0.8 µCi of [14C]Gly-Leu/ml (20) supplemented with cold Gly-Leu (Sigma, St. Louis, MO) at the concentrations indicated below for each experiment. To compare Gly-Leu accumulation in various mutant strains, cells were incubated with peptide (110 µM final concentration) for 10 min. Cells were collected on a membrane filter (HAWP; Millipore, Billerica, MA) by vacuum filtration and washed four times with 1 ml of ice-cold water. The radioactivity retained on the filter was determined by liquid scintillation spectrometry. The results for each deletion mutant were normalized to the accumulation of [14C]Gly-Leu by the wild-type strain and are expressed as a percentage of the control. To measure the inhibition of [14C]Gly-Leu transport by allantoate, ureidosuccinate, or allantoin, 110 µM Gly-Leu and 220 µM concentrations of the competitors were added to the uptake medium, and the accumulation of Gly-Leu was measured after 10 min. To determine the Km of Dal5p for Gly-Leu, cells were incubated with the peptide at final concentrations of 1, 2, 5, 10, or 40 mM for 2 min, an interval in which accumulation versus time was increasing linearly. Cells were harvested and washed, the level of radioactivity was determined as described above, and the results are expressed as Gly-Leu uptake/108 cells/min. To correct for non-Dal5p-dependent accumulation, the uptake of Gly-Leu in the dal5 strain was subtracted from that measured for the wild-type strain. To measure the competition of representatives of non-N-end rule dipeptides (Ala-His, Met-Leu, Ser-Leu, and Val-Ala) and N-end rule dipeptides (Trp-Ala, Arg-Ala, Lys-Ala, His-Gly, and Leu-Leu) with Gly-Leu transport, the various dipeptides were added to the uptake medium at 10-fold excess (15 mM). All uptake experiments were repeated at least three times with similar or identical results obtained.

Real-time reverse transcription-PCR (RT-PCR). Approximately 3 x 108 cells of the tested deletion mutant strains were harvested after overnight growth in MP+Ura medium. Total RNA was isolated by using a RiboPure-Yeast extraction kit (Ambion, Austin, TX) and then treated with the TURBO DNA-free kit to eliminate any genomic DNA contamination (Ambion). The amount of total RNA was quantified by monitoring absorbance at 260 nm. To ensure that genomic DNA had been eliminated, the obtained total RNA was used as a template for PCR; no amplification of target genes was obtained. cDNA was synthesized by using M-MLV RT (Invitrogen, Carlsbad, CA) in a reaction mixture containing 1 µg of oligo(dT), 1 mM deooxynucleoside triphosphates, 14 U of anti-RNase, and 1 µg of total RNA at 42°C for 45 min, and the reaction was stopped after 5 min by incubation at 72°C.

Real-time PCR analysis was performed by using the DNA Engine Opticon (MJ Research, Boston, MA). QuantiTect SYBR green PCR kit (QIAGEN, Valencia, CA) and the primers PTR2(F) (5'-CAGTGACCGTTGATCCTAAAT-3'), PTR2(R) (5'-CTGAAGCACAACCAGAACAAA-3'), ACT1(F) (5'-CCACCATGTTCCCAGGTATT-3'), ACT1(R) (5'-CCAATCCAGACGGAGTACTT-3'), DAL5(F) (5'-CATCTCGCCCGTCTCATTTA-3'), DAL5(R) (5'-CTGCCAAGTTAGCTGCAGCAT-3'), GAP1(F) (5'-CCTTCCCACTTGTTATGGTTAT-3'), GAP1(R) (5'-CTTCTCTTCTACCCGTATCAAT-3'), AGP1(F) (5'-CTGCCTACGCTTGCATTATGA-3'), AGP1(R) (5'-CATCCAGCTTACCTTCACCAA-3'), BAP3(F) (5'-CTCCACAGGAAGAGCAAGCAT-3'), and BAP3(R) (5'-CCATATACCGGTATTGGAATTA-3'), resulting in an ~100-bp amplicon, were used for PCR. The reaction contained 12.5 µl of 2x qPCR reaction mix and 15 pmol of primers and was run with a cycle of 50°C for 2 min and 95°C for 15 min, followed by 40 cycles of 95°C for 15 s, 54°C for 30 s, and 72°C for 15 s. A standard curve for each primer set was performed with 1, 1:10, 1:100, 1:1,000, 1:10,000, and 1:100,000 dilutions of the wild-type cDNA. The CT value, the cycle when sample fluorescence exceeds a chosen threshold above background fluorescence, was determined by using the default program of the DNA Engine Opticon. The copy number of PTR2, DAL5, and ACT1 transcripts in each deletion mutant strain was calculated based on the standard curve. The ratio of the fold change of target transcripts (PTR2 and DAL5) versus the fold change of the internal control transcripts (ACT1) in the tested mutant strains was calculated to show upregulation or downregulation. The ratio of the fold change in the wild type was standardized as 1.0.

Structure comparison. The chemical structures of allantoate, ureidosuccinate, and Gly-Leu dipeptide were drawn by using the ChemSketch software (version 8.0; Advanced Chemistry Development, Inc., Toronto, Ontario, Canada). The chemical structures are automatically generated in the most favorable tautomeric form.


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RESULTS
 
Dal5p functions as a dipeptide transporter in W303. Using high-throughput phenotyping and several genetically diverse strains, we previously reported evidence that the Dal5p allantoate/ureidosuccinate permease is also capable of facilitating di/tripeptide utilization (19). In order to more thoroughly investigate the potential of Dal5p as a peptide transporter, we performed toxic dipeptide (Ala-Eth) "halo" assays in the W303 background as an indirect measure of dipeptide transport. A ptr2 deletion mutant showed about the same sensitivity to Ala-Eth as that shown by the wild type (Fig. 1A and B, halo diameter = 18.6 ± 0.7 mm). In contrast, both a dal5 deletion mutant (Fig. 1C) and a ptr2/dal5 double deletion strain (Fig. 1D) had reduced sensitivity to Ala-Eth compared to the wild type (halo size = 12.5 ± 0.6 mm). The experiments were performed at least three times, and the differences between the halo sizes of the wild-type W303 strain and either the dal5 or the ptr2/dal5 deletion strains were significantly different (P < 0.0002). The strains that overexpressed DAL5 (p426-DAL5) in a dal5 deletion and ptr2/dal5 deletion backgrounds (Fig. 1E and F) were more sensitive to Ala-Eth than the wild-type strain. These data indicate that Ala-Eth is a substrate of Dal5p. In contrast, Ala-Eth toxicity is greater in the S288c background and is dependent on Ptr2p. (Fig. 1G and H). In addition, the data in Fig. 1 demonstrate that Ala-Eth has the ability to enter W303 cells through an additional, as-yet-unidentified, transporter, as shown by the remaining sensitivity to Ala-Eth in the ptr2/dal5 double deletion strain.


Figure 1
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  		FIG. 1. Toxic dipeptide assay (halo assay). (A to F) The non-N-end rule toxic dipeptide (Ala-Eth) was placed on a disk (white ring, 0.4 µmol/disk,) on top of cell lawns of the "wild type" (W303) and the ptr2, dal5, ptr2 dal5, dal5 p426-DAL5, and ptr2 dal5 p426-DAL5 strains in the W303 background. (G and H) Ala-Eth (0.2 µmol/disk was added to the "wild type" (S288c) and the strain with ptr2 deleted in the S288c background. Plates were incubated for 2 days at 30°C, and photographs were taken.

In order to directly assess dipeptide transport, we used [14C]Gly-Leu as a substrate and measured its uptake in the ptr2, dal5, ptr2/dal5, dal5 (p426-DAL5), dal5/ptr2 (p426-DAL5) strains and the wild-type W303 background (Fig. 2). The ptr2 deletion mutant showed no significant difference in the accumulation of [14C]Gly-Leu compared to wild type. However, in the dal5 deletion and dal5/ptr2 double deletion mutants the accumulations of radiolabeled peptide were only 11 and 10% compared to the wild type, respectively. When expressing plasmid-borne DAL5, the accumulation of [14C]Gly-Leu was restored to the wild-type level in both the dal5-deletion mutant and the dal5/ptr2 double deletion mutant. The increased toxicity of Ala-Eth in the Dal5p-overexpressing strain (Fig. 1E) does not correspond to the similar rates of Gly-Leu accumulation in the overexpressing strain compared to the wild type (Fig. 2). This difference is due inherent differences in the assays; toxicity was measured over a 48-h time period, whereas accumulation was determined over the course of 10 min. These data indicate that Gly-Leu is a substrate of Dal5p and demonstrate that Gly-Leu, like Ala-Eth, may enter W303 cells through another, as-yet-unidentified, transporter, as shown by the residual 10% accumulation in the dal5/ptr2 double deletion strain. By measuring the initial rate of uptake at various concentrations of the Gly-Leu substrate (see Materials and Methods) the Km for Gly-Leu transport in W303 cells was determined to be 7.5 ± 1.3 mM. The affinity for this dipeptide was 150- to 300-fold lower than the reported Km for allantoate (50 µM) and ureidosuccinate (27 µM) (40, 41).


Figure 2
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  		FIG. 2. Accumulation of [14C]Gly-Leu in the wild type (W303) and th ptr2, dal5, ptr2 dal5, dal5 p426-DAL5, ptr2 dal5 p426-DAL5e deletion mutant strains. The accumulation of [14C]Gly-Leu in the wild type was 1.3 nmol/108cells at 10 min and used as the 100% accumulation in the calculation of the percent accumulation by different strains.

Allantoate, ureidosuccinate, and allantoin compete with Dal5p-dependent dipeptide uptake. Allantoate, urediosuccinate, and to a lesser extent allantoin have all been reported as substrates for Dal5p (6, 35, 41). If dipeptides are also substrates for Dal5p, allantoate, ureidosuccinate, or allantoin should be able to compete with dipeptide uptake. Therefore, the effect of allantoate, ureidosuccinate, or allantoin on the sensitivity to Ala-Eth in the wild type, ptr2 deletion mutant, and dal5 deletion mutant was tested. Allantoate (Fig. 3A, disk b) and ureidosuccinate (Fig. 3D, disk b) suppressed the sensitivity of the W303 strain to the toxic dipeptide Ala-Eth (panels A and D, disk a) since the halo around the Ala-Eth "disk a" was not fully formed on the side facing the disk containing the competitors. In addition, the halo size around the disk containing a mixture of Ala-Eth with allantoate (panel A, disk c) and ureidosuccinate (panel D, disk c) was reduced compared to the halo where only Ala-Eth was added. In contrast, allantoin (panel G, disk c) had limited effect on the sensitivity of W303 to Ala-Eth. Allantoate, ureidosuccinate and allantoin had effects similar to those seen in the wild type on Ala-Eth sensitivity in a ptr2 deletion mutant (Fig. 3B, E, and H). However, the dal5 deletion mutant showed less sensitivity to toxic dipeptide Ala-Eth and no suppression of toxicity by allantoate, ureidosuccinate or allantoin (Fig. 3C, F, and I).


Figure 3
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  		FIG. 3. Effect of allantoate (plates A, B, and C), ureidosuccinate (plate D, E, and F), and allantoin (plate G, H, and I) on the sensitivity of Ala-Eth to the wild-type (W303), ptr2, and dal5 strains. In each plate, 0.4 µmol of Ala-Eth was spotted onto the upper left disk (disk a); 0.4 µmol of allantoate, or 0.4 µmol of ureidosuccinate, or 0.65 µmol of allantoin was spotted into the upper right disk (disk b). In addition, the mixture of 0.4 µmol of Ala-Eth and 0.4 µmol of allantoate, 0.4 µmol of ureidosuccinate, or 0.65 µmol of allantoin was added into the bottom disk (disk c) in each plate.

To further evaluate the competition of allantoate, ureidosuccinate, or allantoin with dipeptide transport, the accumulation of [14C]Gly-Leu was measured in the presence of a twofold excess of competitor (Fig. 4). The presence of allantoate or ureidosuccinate (220 µM) resulted in decreased accumulation of [14C]Gly-Leu to 58 and 55% of the W303 wild-type level, respectively. However, the presence of allantoin (530 µM) at an ~5-fold excess resulted was not as effective as the other competitors, reducing accumulation of [14C]Gly-Leu to 82% of the W303 wild-type level. These results reflect those of the toxicity assay in which allantoin was less effective than either allantoate or ureidosuccinate in competing for peptide accumulation. When the concentration of allantoate was increased, the accumulation of Gly-Leu decreased (data not shown). Similar to the wild-type W303, the addition of allantoate, ureidosuccinate, and allantoin reduced the accumulation of [14C]Gly-Leu to 58, 44, and 71%, respectively, in the ptr2 deletion mutant strain. The dal5 deletion mutant did not accumulate [14C]Gly-Leu much above background levels, and therefore the effect of allantoate, ureidosuccinate, or allantoin on Ptr2p/Dal5p-independent transport could not be determined.


Figure 4
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  		FIG. 4. Accumulation of [14C]Gly-Leu in the presence of allantoate, ureidosuccinate, or allantoin. Wild type (W303) and the ptr2 and dal5 deletion mutant strains were grown in the MP+Ura medium. Allantoate (220 µM), ureidosuccinate (220 µM), and allantoin (530 µM) were added into the uptake medium solution containing 110 µM [14C]Gly-Leu. The accumulation of [14C]Gly-Leu in the wild type was 1.3 nmol/108cells at 10 min and used as the 100% rate of uptake.

Non-N-end rule dipeptides are preferred by Dal5p. After we examined 284 of the 400 possible dipeptides composed of naturally occurring amino acids, our previous research demonstrated that Dal5p preferred non-N-end rule dipeptides over N-end rule dipeptides (dipeptides with basic or bulky hydrophobic amino acid residues at the N terminus) as substrates when the ability of peptides to serve as a nitrogen source was assayed (19). In order to measure whether Dal5p can transport either non-N-end rule or N-end rule dipeptides, we used Ala-Eth as a representative non-N-end rule toxic dipeptide and Leu-Eth as a representative N-end rule toxic dipeptide. Wild-type, ptr2, dal5, ptr2/dal5, and dal5 (p426-DAL5) strains showed sensitivity to the non-N-end rule toxic dipeptide Ala-Eth (Fig. 1); however, none of the tested strains showed any sensitivity to the N-end rule toxic dipeptide Leu-Eth (data not shown). In addition, the direct assay of the uptake of [14C]Gly-Leu was tested in W303 in the presence of a 10-fold excess of selected representatives of non-N-end rule dipeptides (Ala-His, Met-Leu, Ser-Leu, and Val-Ala) and N-end rule dipeptides (Trp-Ala, Arg-Ala, Lys-Ala, His-Gly, and Leu-Leu) (Table 1). The presence of a 10-fold excess of N-end rule dipeptides only slightly reduced the accumulation of [14C]Gly-Leu compared to the control (no dipeptide competitor). In contrast, the addition of 10-fold excess of non-N-end rule dipeptides Ser-Leu, Ala-His, Met-Leu, and Val-Ala resulted in the accumulation of 54, 61, 75, and 84% [14C]Gly-Leu uptake, respectively, in comparison to the control uptake rates (Table 1). These data demonstrate that within the sampling of peptides substrates tested in the present study, Dal5p has a preference for non-N-end rule peptide substrates.


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  		TABLE 1. Effect of non-N-end rule dipeptides (Ala-His, Met-Leu, Ser-Leu, and Val-Ala) and N-end rule dipeptides (Trp-Ala, Arg-Ala, Lys-Ala, His-Gly, and Leu-Leu) on the rate of uptake of [14C]Gly-Leua

Leucine effects PTR2 and DAL5 expression and peptide toxicity. Microarray analyses (11, 13, 24) in S288c and close derivative strain backgrounds (see reference 30) indicated that the expression of PTR2 and DAL5 was regulated by the addition of leucine in an Ssy1p-dependent manner. In these studies PTR2 expression was upregulated by leucine, whereas DAL5 expression was downregulated. To further address the expression of PTR2 and DAL5 under the conditions used in our study using W303, real-time RT-PCR analysis was performed (Fig. 5). In contrast to the microarray data using S288c backgrounds, PTR2 expression in the W303 strain did not change upon addition of leucine, although DAL5 expression decreased to 30% of control levels (Fig. 5A). The decrease in DAL5 expression is similar to that observed in the S288c strains. The reduction of DAL5 expression was also reflected by the sensitivity to toxic dipeptide when measured in medium containing leucine. Both the W303 strain and the ptr2 deletion mutant were less sensitive to Ala-Eth when grown in medium containing Leu compared to cells grown in the absence of Leu (Fig. 5C). Furthermore, the accumulation of [14C]Gly-Leu by W303 grown in the presence of leucine was 63% of that measured in cells grown in medium without leucine (data not shown). Taken together, these data indicate that leucine addition results in the downregulation of DAL5 in W303 but that PTR2 in W303 is not regulated by leucine.


Figure 5
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  		FIG. 5. Effect of leucine on PTR2 and DAL5 transcription and toxic peptide response. (A) Real-time RT-PCR of PTR2 and DAL5 transcription is shown as a ratio calculated by the fold change of the target gene (PTR2 or DAL5) in the wild-type strain (W303) with or without leucine addition (30 µg/ml) divided by the fold change of ACT1 in the wild type with or without Leu. The ratio of the fold change in the wild type without Leu was standardized to 100%. (B) Real-time RT-PCR of AGP1, BAP3, and GAP1 transcription in the wild-type strain (W303) with or without leucine addition (30 µg/ml). The ratio of the fold change in the wild type without Leu was standardized to 100%. (C) Effect of leucine addition on the response to Ala-Eth for wild-type W303 and the W303 strain with ptr2 deleted. Portions, 0.4 and 0.2 µmol, of Ala-Eth were added to the left and right disks, respectively, in each plate.

To further test whether the SPS pathway is indeed inactive in the W303 strain, we monitored the expression of AGP1, BAP3, and GAP1 by real-time RT-PCR. The expression of these genes was previously reported to be under the regulation of the SPS sensor (11, 22, 23). The expression of AGP1, BAP3, and GAP1 under leucine induction showed less than a twofold change compared to cells grown with no leucine addition (Fig. 5B). This indicated that leucine did not regulate the expression of these genes in the W303 strain under the conditions tested.

CUP9 deletion effects PTR2 and DAL5 expression and peptide toxicity. An important aspect of PTR2 regulation in the S288c background is its control by the Cup9p repressor. In S288c, PTR2 is induced by leucine through the SPS pathway involving Ssy1p and derepressed by removal of the Cup9p repressor from the PTR2 promoter (18). To determine the role of Cup9p on PTR2 and DAL5 expression in W303, real-time RT-PCR was used (Fig. 6A). The expression level of PTR2 was ~7-fold higher in a W303 cup9 deletion mutant compared to that for the wild-type strain. Compared to wild-type W303, the cup9 deletion mutant in the W303 background showed increased sensitivity to both Ala-Eth and Leu-Eth (Fig. 6B, compare panels a and c and panels b and d). However, the expression level of DAL5 in the cup9 deletion mutant was reduced fourfold compared to the gene expression level in the W303 wild type. These results suggest PTR2 and DAL5 are regulated by Cup9p in an opposite manner in W303; Cup9p is a repressor of PTR2 as shown previously in S288c strains (39), but Cup9p appears to be a positive regulator for DAL5 in W303 based on RT-RCR results. The overall increase in sensitivity in the W303 cup9 deletion strain indicates that PTR2 is dominating the uptake under these conditions. Ptr2p has a much "higher capacity" for peptide uptake than Dal5p, so upregulation of PTR2 masks the downregulation of DAL5.


Figure 6
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  		FIG. 6. Effect of deletion of CUP9 on PTR2 and DAL5 transcription and peptide toxicity. (A) Real-time RT-PCR of PTR2 and DAL5 transcription is shown as a ratio calculated by the fold change of the target gene (PTR2 or DAL5) in the wild-type strain (W303) and W303 cup9 deletion strain divided by the fold change of ACT1. The ratio of the fold change in the wild type was standardized to 100%. (B) Effect of wild type (panels a and b) and cup9 deletion (panels c and d) on the response to Ala-Eth (panels a and c) and Leu-Eth (panels b and d) is shown. We added 0.4 and 0.2 µmol of Ala-Eth or 0.2 and 0.1 µmol of Leu-Eth were added to the left and right disks in each plate, respectively.


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DISCUSSION
 
Ptr2p has been reported many times as the sole dipeptide transporter in S. cerevisiae strains of the S288c background. However, we have recently reported that Dal5p played an important role in using many, but not all, dipeptides as the sole nitrogen source at a high peptide concentration (2 mM) in the W303 strain (19). Since Dal5p was characterized as an allantoate/ureidosuccinate permease (35, 41), we hypothesized that the role played by Dal5p in utilization in W303 was that of a dipeptide transporter that recognized not only allantoate/ureidosuccinate but also a subset of the 400 naturally occurring dipeptides. In the present study, we further characterize Dal5p as the route for dipeptide transport that predominates in wild-type strain W303, whereas Ptr2p is the dominant di/tripeptide transporter in S. cerevisiae 288c backgrounds (18). Furthermore, we found that the peptide transporters Ptr2p and Dal5p are regulated differently.

Dal5p is an alternative route in transporting dipeptides. Dal5p was important for transporting the toxic dipeptide Ala-Eth and [14C]Gly-Leu in W303; deletion of DAL5 strongly reduced both the sensitivity to toxic Ala-Eth and the ability to accumulate radiolabeled dipeptide. Furthermore, plasmid-based overexpression of DAL5 in the dal5 deletion mutant background led to the recovery of sensitivity to Ala-Eth and the accumulation of [14C]Gly-Leu (Fig. 1 and 2). In the W303 strain, Ptr2p was limited in activity since deletion of this gene did not change the sensitivity to the toxic dipeptide Ala-Eth, and the strain was still able to accumulate [14C]Gly-Leu at near wild-type levels. These results suggest that Dal5p, not Ptr2p, plays the major role in dipeptide transport in W303. These observations agree with previous results that showed that expression of Ptr2p-green fluorescent protein was not observed in W303 (19). Double deletion of PTR2 and DAL5 did not completely abolish the sensitivity to Ala-Eth, suggesting there might be an additional unknown protein involved in transporting Ala-Eth into the W303 strain.

Both W303 and S288c strains have been commonly used in genetic studies and have a close lineage relationship since W303 was derived in part from crosses with S288c (19). An S288c strain was the "standard" S. cerevisiae wild-type used for genome sequencing. However, S288c and W303 strains showed either Ptr2p-dependent or Dal5p-dependent dipeptide transport, respectively. Ptr2p and Dal5p share 21% identity in amino acid sequence. The highly conserved regions in the PTR family such as the consensus sequences in TM1, TM5, and TM10 of Ptr2p (18) were not identified in Dal5p. Given the close pedigrees of these two strains, it remains unsolved how or why these strains developed two dipeptide utilization pathways.

Substrates of Dal5p transporter. By testing 284 of 400 possible naturally occurring dipeptides, the W303 ptr2{Delta} mutant showed similar growth patterns on a subset of non-N-end rule dipeptides compared to the W303 strain (19). In addition, overexpression of DAL5 increased non-N-end rule dipeptide utilization, particularly for dipeptides with Ala, Ser, and Gly in the N terminus. In the present study, we examined whether Dal5p transported Gly-Leu, Ala-Eth, Ala-His, Met-Leu, Ser-Leu, and Val-Ala as examples of non-N-end rule dipeptides and Trp-Ala, Arg-Ala, Lys-Ala, His-Gly, Leu-Leu, and Leu-Eth as examples of N-end rule dipeptide. W303, which uses predominantly Dal5p for peptide transport, showed no sensitivity to Leu-Eth, while Ptr2p transport-dependent S288c is very sensitive to the same toxic peptide (20). Furthermore, high concentrations of Trp-Ala, Arg-Ala, Lys-Ala, His-Gly, and Leu-Leu (10-fold that of Gly-Leu) showed limited or no competition for the accumulation of [14C]Gly-Leu in the W303 background compared to the non-N-end rule dipeptides (Fig. 5). These results extend our previous observation that Dal5p-deficient strains were unable to utilize N-end rule dipeptides (19) and indicate that the block is at the peptide transport level.

Together with non-N-end rule dipeptides such as Gly-Leu or Ala-Eth, allantoate and ureidosuccinate are substrates for Dal5p (40, 41). The addition of allantoate and ureidosuccinate decreased the sensitivity of W303 to Ala-Eth (Fig. 3) and reduced the accumulation of [14C]Gly-Leu (Fig. 4). The Km values for Dal5p transport of allantoate and ureidosuccinate were determined to be 50 and 27 µM, respectively (40, 41). In contrast, we measured the Km for Gly-Leu transport to be 7.5 mM. The affinity of Dal5p for Gly-Leu was more than 150 times lower than that for allantoate or ureidosuccinate. Together, these data demonstrate that Dal5p strongly prefers allantoate/ureidosuccinate as its substrates. Upon alignment of allantoate, ureidosuccinate, and Gly-Leu, a common chemical moiety is observed (Fig. 7). The conserved grouping includes an amide proximal to a carboxylic acid and may be essential for substrate recognition by Dal5p. For allantoate and ureidosuccinate, the -NH2 group is directly connected to a common chemical group; however, for dipeptides such as Gly-Leu, an additional carbon is located between the conserved structure and -NH2. The addition of the -CH2 group may reduce Gly-Leu affinity for Dal5p. The larger chemical groups between the conserved chemical moiety and the -NH2 in N-end rule substrates apparently lowers the affinity as a Dal5p substrate (Table 1). Alanine, glycine, and serine residues have been shown to be particularly favorable to Dal5p-mediated growth (19) and better competitors to inhibit the uptake of [14C]Gly-Leu (Table 1). These amino acids contain a relatively small chemical group next to the -NH2 group.


Figure 7
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  		FIG. 7. Structure of Dal5p substrates. The common structure of allantoate, ureidosuccinate, and dipeptide is circled.

PTR2 regulation in the W303 and S288c backgrounds. In the present study we showed that Cup9p repressed the expression of PTR2 in W303, similar to the repression of PTR2 in the S288c background as the deletion of CUP9 leads to overexpression of PTR2 (1, 39). The cup9 deletion mutant showed a considerable increase in sensitivity to both the non-N-end rule dipeptide Ala-Eth and the N-end rule dipeptide Leu-Eth (Fig. 6B), indicating that PTR2 expression was upregulated in W303. We have previously sequenced the promoter region of PTR2 in the W303 strain and showed that the PTR2 promoter regions of the W303 and S288c strains had almost identical sequences (19). These results suggest that the difference in Ptr2p activity between the W303 and S288c strains is likely upstream of Cup9p in the signal transduction pathway for the regulation of PTR2 expression. However, in contrast to the strong induction of PTR2 by leucine in the S288c background (1) through the Ssy1p-Ptr3p-Ssy5p (SPS) protein complex, leucine did not affect PTR2 expression in W303. The PTR2 expression level remained constant in both the presence and the absence of leucine (Fig. 5A). The limited Ptr2p activity in W303 is possibly due to the inability to induce the SPS pathway in W303. Our results indicated that the expression of AGP1, BAP3, and GAP1 showed less than a twofold change in response to leucine induction in W303 as measured by RT-PCR (Fig. 5B). However, AGP1 and BAP3 have been reported to be upregulated, whereas GAP1 is downregulated by leucine induction via the SPS signal pathway in the S288c strain and its derivative strains such as FY2 (11, 22, 23). These results indicate that there are strain-dependent differences in the regulation of permease expression in response to leucine induction.

DAL5 regulation. The expression of DAL5 is regulated by Cup9p, but this regulation is in the opposite direction to that found for the expression of PTR2. The deletion of CUP9 downregulated the expression of DAL5 (Fig. 6A), whereas PTR2 expression was upregulated in the cup9 deletion strain. Either Cup9p somehow acts as an inducer of DAL5 or Cup9p regulates DAL5 expression indirectly via its regulation of another regulatory protein.

DAL5 expression is also downregulated by the addition of leucine in W303 (Fig. 5A), which is opposite to the PTR2 upregulation by leucine addition in the S288c background. Downregulation of DAL5 expression by the addition of Leu in an Ssy1p-dependent manner has also been observed in several microarray analyses in the S288c backgrounds (11, 24). To further define the role of Ssy1p on the regulation of DAL5 expression in the W303 background, we attempted to make a knockout strain using kanamycin as a dominant selectable marker (17). After repeated attempts we were not able to isolate kanamycin-resistant strains with the disruption cassette integrated into the SSY1 locus, suggesting that deletion of SSY1 is lethal in this background (data not shown). Therefore, the direct role of Ssy1p in regulation of expression of DAL5 expression is still unclear.

Other transcription factors are known to regulate DAL5 expression such as Gln3p, Ure2p, (9), and Tor1/2p (16). How these proteins affected peptide transport in direct assays was not investigated in the present study.

Preferences for dipeptide transporters in different strains. As shown in the present study of peptide transport and indicated previously when measuring peptides as sources for nitrogen (19), the activity of Dal5p for peptide utilization was dominant in W303, with little apparent activity by Ptr2p. In contrast, S288c strains showed low activity for Dal5p and high activity for Ptr2p. The strains have adapted to selective pressure influencing nutrient utilization from the extracellular environment resulting in preferences for one of the dipeptide transporters. Variation in the relative strengths of the activities contributing to dipeptide import appears to impact the range of dipeptide substrates utilized by the cell. For example, Ptr2p has a very broad range of dipeptide substrates, whereas the optimal substrates for Dal5p are limited to a smaller subset of naturally occurring dipeptides, mostly non-N-end rule dipeptides. While di/tripeptides can serve as a valuable source of nutrients, evolutionary pressures opposing indiscriminate import may reinforce mechanisms promoting utilization or expression of a specific transport system.


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ACKNOWLEDGMENTS
 
F.N. is the Leonard and Esther Kurtz Term Professor at the College of Staten Island.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Microbiology, University of Tennessee, Knoxville, TN 37996-0845. Phone: (865) 974-3006. Fax: (865) 974-0361. E-mail: jbecker{at}utk.edu Back

{triangledown} Published ahead of print on 10 August 2007. Back


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Eukaryotic Cell, October 2007, p. 1805-1813, Vol. 6, No. 10
1535-9778/07/$08.00+0     doi:10.1128/EC.00257-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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