Oligopeptide Transporter Family Research Articles

An oligo peptide transporter family member, OsOPT7, mediates xylem unloading of Fe for its preferential distribution in rice.

Iron (Fe) needs to be delivered to different organs and tissues of above-ground parts for playing its multiple physiological functions once it is taken up by the roots. However, the mechanisms underlying Fe distribution are poorly understood. We functionally characterized OsOPT7, a member of oligo peptide transporter family in terms of expression patterns, localization, transport activity and phenotypic analysis of knockdown lines. OsOPT7 was highly expressed in the nodes, especially in the uppermost node I, and its expression was upregulated by Fe-deficiency. OsOPT7 transports ferrous iron into the cells coupled with proton. Immunostaining revealed that OsOPT7 is mainly localized in the xylem parenchyma cells of the enlarged vascular bundles in the nodes and vascular tissues in the leaves. Knockdown of OsOPT7 did not affect the Fe uptake, but altered Fe distribution; less Fe was distributed to the new leaf, upper nodes and developing panicle, but more Fe was distributed to the old leaves. Furthermore, knockdown of OsOPT7 also resulted in less Fe distribution to the leaf sheath, but more Fe to the leaf blade. Taken together, OsOPT7 is involved in the xylem unloading of Fe for both long-distance distribution to the developing organs and local distribution within the leaf in rice.

Proton-Coupled Oligopeptide Transport (Slc15) in the Brain: Past and Future Research.

This mini-review describes the role of the solute carrier (SLC)15 family of proton-coupled oligopeptide transporters (POTs) and particularly Pept2 (Slc15A2) and PhT1 (Slc15A4) in the brain. That family transports endogenous di- and tripeptides and peptidomimetics but also a number of drugs. The review focuses on the pioneering work of David E. Smith in the field in identifying the impact of PepT2 at the choroid plexus (the blood-CSF barrier) as well as PepT2 and PhT1 in brain parenchymal cells. It also discusses recent findings and future directions in relation to brain POTs including cellular and subcellular localization, regulatory pathways, transporter structure, species differences and disease states.

A Remarkable Expansion of Oligopeptide Transporter Genes in Rust Fungi (Pucciniales) Suggests a Specialization in Nutrient Acquisition for Obligate Biotrophy.

Nutrient acquisition by rust fungi during their biotrophic growth has been assigned to a few transporters expressed in haustorial infection structures. We performed a comparative genomic analysis of all transporter genes (hereafter termed transportome) classified according to the Transporter Classification Database, focusing specifically on rust fungi (order Pucciniales) versus other species in the Dikarya. We also surveyed expression of transporter genes in the poplar rust fungus for which transcriptomics data are available across the whole life cycle. Despite a significant increase in gene number, rust fungi presented a reduced transportome compared with most fungi in the Dikarya. However, a few transporter families in the subclass Porters showed significant expansions. Notably, three metal transport-related families involved in the import, export, and sequestration of metals were expanded in Pucciniales and expressed at various stages of the rust life cycle, suggesting a tight regulation of metal homeostasis. The most remarkable gene expansion in the Pucciniales was observed for the oligopeptide transporter (OPT) family, with 25 genes on average compared with seven to 14 genes in the other surveyed taxonomical ranks. A phylogenetic analysis showed several specific expansion events at the root of the order Pucciniales with subsequent expansions in rust taxonomical families. The OPT genes showed dynamic expression patterns along the rust life cycle and more particularly during infection of the poplar host tree, suggesting a possible specialization for the acquisition of nitrogen and sulfur through the transport of oligopeptides from the host during biotrophic growth.

Genome-wide analysis of PTR transporters in Candida species and their functional characterization in Candida auris.

The peptide transport (PTR) or proton-dependent oligopeptide transporter (POT) family exploits the inwardly directed proton motive force to facilitate the cellular uptake of di/tripeptides. Interestingly, some representatives are also shown to import peptide-based antifungals in certain Candida species. Thus, the identification and characterization of PTR transporters serve as an essential first step for their potential usage as antifungal peptide uptake systems. Herein, we present a genome-wide inventory of the PTR transporters in five prominent Candida species. Our study identifies 2 PTR transporters each in C. albicans and C. dubliniensis, 1 in C. glabrata, 4 in C. parapsilosis, and 3 in C. auris. Notably, despite all representatives retaining the conserved features seen in the PTR family, there exist two distinct classes of PTR transporters that differ in terms of their sequence identities and lengths of certain extracellular and intracellular segments. Further, we also evaluated the contribution of each PTR protein of the newly emerged multi-drug-resistant C. auris in di/tripeptide uptake. Notably, deletion of two PTR genes BNJ08_003830 and BNJ08_005124 led to a marked reduction in the transport capabilities of several tested di/tripeptides. However, all three genes could complement the role of native PTR2 gene of Saccharomyces cerevisiae, albeit to varied levels. Besides, BNJ08_005124 deletion also resulted in increased resistance toward the peptide-nucleoside drug Nikkomycin Z as well as the glucosamine-6-phosphate synthase inhibitor, L-norvalyl-N3-(4-methoxyfumaroyl)-L-2,3-diaminopropionoic acid (Nva-FMDP), pointing toward its predominant role in their uptake mechanism. Altogether, the study provides an important template for future structure-function investigations of PTR transporters in Candida species. KEY POINTS: • Candida genome encodes for two distinct classes of PTR transporters. • Candida auris encodes for 3 PTR transporters with different specificities. • BNJ08_005124 in C. auris is involved in the uptake of Nikkomycin Z and Nva-FMDP.

Genome-Wide Identification and Transcript Analysis Reveal Potential Roles of Oligopeptide Transporter Genes in Iron Deficiency Induced Cadmium Accumulation in Peanut.

The oligopeptide transporter (OPT) family is a group of proton-coupled symporters that play diverse roles, including metal homeostasis. However, little is known about this family of peanuts. To reveal the potential roles of AhOPT genes in Fe/Cd interactions, peanut AhOPT genes were genome-widely identified, and the relationships between gene expression and Cd accumulation were detected in two contrasting peanut cultivars (Fenghua 1 and Silihong) under Fe-sufficient or Fe-deficient conditions. A total of 40 AhOPT genes were identified in peanuts, which were divided into two subfamilies (PT and YS). Most AhOPT genes underwent gene duplication events predominated by whole-genome duplication. Clustered members generally have similar protein structures. However, gene structural divergences occurred in most of the duplicated genes. Transcription analysis revealed that AhOPT3.2/3.4 and AhYSL3.1/3.2 might be responsible for Fe deficiency tolerance, while AhOPT3.1/3.4, AhOPT7.1/7.2, and AhYSL1.1 be involved in Fe/Cd interactions. These genes might be regulated by transcription factors, including ATHB-12, ATHB-6, DIVARICATA, MYB30, NAC02, DOF3.4, IDD7, and LUX. Reduced expressions of AhYSL3.1/3.2 and higher expressions of AhOPT3.4 might contribute to higher Fe-deficiency tolerance in Silihong. Higher expression of AhOPT7.3 and AhOPT6.1 might be responsible for low Cd accumulation in Fenghua 1. Our results confirmed that AhOPT3/6/7 and AhYSL1/3 might be involved in the transport of Fe and/or Cd in peanuts and provided new clues to understanding potential mechanisms of Fe/Cd interactions.

Yeast Plasma Membrane Fungal Oligopeptide Transporters Display Distinct Substrate Preferences despite Their High Sequence Identity.

Fungal Oligopeptide Transporters (Fot) Fot1, Fot2 and Fot3 have been found in Saccharomyces cerevisiae wine strains, but not in strains from other environments. In the S. cerevisiae wine strain EC1118, Fot1 and Fot2 are responsible for a broader range of oligopeptide utilization in comparison with strains not containing any Fot. This leads to better fermentation efficiency and an increased production of desirable organoleptic compounds in wine. Despite the benefits associated with Fot activity in S. cerevisiae within the wine environment, little is known about this family of transporters in yeast. The presence of Fot1, Fot2 and Fot3 in S. cerevisiae wine strains is due to horizontal gene transfer from the yeast Torulaspora microellipsoides, which harbors Fot2Tm, FotX and FotY proteins. Sequence analyses revealed that Fot family members have a high sequence identity in these yeast species. In this work, we aimed to further characterize the different Fot family members in terms of subcellular localization, gene expression in enological fermentation and substrate specificity. Using CRISPR/Cas9, we constructed S. cerevisiae wine strains containing each different Fot as the sole oligopeptide transporter to analyze their oligopeptide preferences by phenotype microarrays. The results of oligopeptide consumption show that Fot counterparts have different di-/tripeptide specificities, suggesting that punctual sequence divergence between FOT genes can be crucial for substrate recognition, binding and transport activity. FOT gene expression levels in different S. cerevisiae wine strains during enological fermentation, together with predicted binding motifs for transcriptional regulators in nitrogen metabolism, indicate that these transporters may be under the control of the Nitrogen Catabolite Repression (NCR) system. Finally, we demonstrated that Fot1 is located in the yeast plasma membrane. This work contributes to a better understanding of this family of oligopeptide transporters, which have demonstrated a key role in the utilization of oligopeptides by S. cerevisiae in enological fermentation.

Identification and distinct regulation of three di/tripeptide transporters in Aspergillus oryzae.

The uptake of di/tripeptides is mediated by the proton-dependent oligopeptide transporter (POT) family. In this study, 3 POT family transporters, designated PotA, PotB, and PotC were identified in Aspergillus oryzae. Growth comparison of deletion mutants of these transporter genes suggested that PotB and PotC are responsible for di/tripeptide uptake. PotA, which had the highest sequence similarity to yeast POT (Ptr2), contributed little to the uptake. Nitrogen starvation induced potB and potC expression, but not potA expression. When 3 dipeptides were provided as nitrogen sources, the expression profiles of these genes were different. PrtR, a transcription factor that regulates proteolytic genes, was involved in regulation of potA and potB but not in potC expression. Only potC expression levels were dramatically reduced by disruption of ubrA, an orthologue of yeast ubiquitin ligase UBR1 responsible for PTR2 expression. Expression of individual POT genes is apparently controlled by different regulatory mechanisms.

Identification of Differentially Expressed Drought-Responsive Genes in Guar [Cyamopsis tetragonoloba (L.) Taub

Drought remains one of the most serious environmental stresses because of the continuous reduction in soil moisture, which requires the improvement of crops with features such as drought tolerance. Guar [Cyamopsis tetragonoloba (L.) Taub], a forage and industrial crop, is a nonthirsty plant. However, the information on the transcriptome changes that occur under drought stress in guar is very limited; therefore, a gene expression analysis is necessary in this context. Here, we studied the differentially expressed genes (DEGs) in response to drought stress and their metabolic pathways. RNA-Seq via an expectation-maximization algorithm was used to estimate gene abundance. Subsequently, an Empirical Analysis of Digital Gene Expression Data in the R Bioconductor package was used to identify DEGs. Blast2GO, InterProScan, and the Kyoto Encyclopedia of Genes and Genomes were used to explore functional annotation, protein analysis, enzymes, and metabolic pathways. Transcription factors were identified using the PlantTFDB database. Our study identified 499 upregulated and 191 downregulated genes in response to drought stress. Of those, 32 upregulated and six downregulated genes were deemed as novel genes exclusive to guar. An aggregate of 137 protein families, 306 domains, 12 repeats, and two sites were upregulated. The proton-dependent oligopeptide transporter family and transferase, aquaporin transporter, calcium/calmodulin-dependent/calcium-dependent protein kinase, aspartic peptidase A1 family, UDP-glucuronosyl/UDP-glucosyltransferase, and major intrinsic protein were the most upregulated protein families. The upregulated unigenes were associated with 88 enzymes and 77 KEGG pathways. Finally, the MYB-related, MYB, and ERF transcription factor families were upregulated. These data may be useful for understanding the plant molecular response to drought stress.

Comparative genomics of Sporothrix species and identification of putative pathogenic-gene determinants.

Aim: To understand the phylogenomics, pathogenic/virulence-associated genes and genomic evolution of pathogenic Sporothrix species. Materials & methods: We performed in silico comparative genome analysis of Sporothrix species using ab initio tools and in-house scripts. We predicted genes and repeats, compared genomes based on synteny, identified orthologous clusters, assessed genes family expansion/contraction, predicted secretory proteins and finally searched for similar sequences from various databases. Results: The phylogenomics revealed that Sporothrix species are closely related to Ophiostoma species. The gene family evolutionary analysis revealed the expansion of genes related to virulence (CFEM domain, iron acquisition genes, lysin motif domain), stress response (Su[var]3-9, Enhancer-of-zeste and Trithorax domain and Domain of unknown function 1996), proteases (aspartic protease, x-pro dipeptidyl-peptidase), cell wall composition associated genes (chitin deacetylase, chitinase) and transporters (major facilitator superfamily transporter, oligo-peptide transporter family) in Sporothrix species. Conclusion: The present study documents the putative pathogenic/virulence-associated genes in the Sporothrix species.

Membrane Chemistry Tunes the Structure of a Peptide Transporter.

Membrane proteins require lipid bilayers for function. While lipid compositions reach enormous complexities, high‐resolution structures are usually obtained in artificial detergents. To understand whether and how lipids guide membrane protein function, we use single‐molecule FRET to probe the dynamics of DtpA, a member of the proton‐coupled oligopeptide transporter (POT) family, in various lipid environments. We show that detergents trap DtpA in a dynamic ensemble with cytoplasmic opening. Only reconstitutions in more native environments restore cooperativity, allowing an opening to the extracellular side and a sampling of all relevant states. Bilayer compositions tune the abundance of these states. A novel state with an extreme cytoplasmic opening is accessible in bilayers with anionic head groups. Hence, chemical diversity of membranes translates into structural diversity, with the current POT structures only sampling a portion of the full structural space.

Membrane Chemistry Tunes the Structure of a Peptide Transporter

AbstractMembrane proteins require lipid bilayers for function. While lipid compositions reach enormous complexities, high‐resolution structures are usually obtained in artificial detergents. To understand whether and how lipids guide membrane protein function, we use single‐molecule FRET to probe the dynamics of DtpA, a member of the proton‐coupled oligopeptide transporter (POT) family, in various lipid environments. We show that detergents trap DtpA in a dynamic ensemble with cytoplasmic opening. Only reconstitutions in more native environments restore cooperativity, allowing an opening to the extracellular side and a sampling of all relevant states. Bilayer compositions tune the abundance of these states. A novel state with an extreme cytoplasmic opening is accessible in bilayers with anionic head groups. Hence, chemical diversity of membranes translates into structural diversity, with the current POT structures only sampling a portion of the full structural space.

Diversity of Oligopeptide Transport in Yeast and Its Impact on Adaptation to Winemaking Conditions.

Nitrogen is an essential nutrient for yeasts and its relative abundance is an important modulator of fermentation kinetics. The main sources of nitrogen in food are ammonium and free amino acids, however, secondary sources such as oligopeptides are also important contributors to the nitrogen supply. In yeast, oligopeptide uptake is driven by different families of proton–coupled transporters whose specificity depends on peptide length. Proton-dependent Oligopeptide Transporters (POT) are specific to di- and tri-peptides, whereas the Oligopeptide Transport (OPT) family members import tetra- and pentapeptides. Recently, the novel family of Fungal Oligopeptide Transporters (FOT) has been identified in Saccharomyces cerevisiae wine strains as a result of a horizontal gene transfer from Torulaspora microellipsoides. In natural grape must fermentations with S. cerevisiae, Fots have a broader range of oligopeptide utilization in comparison with non-Fot strains, leading to higher biomass production and better fermentation efficiency. In this review we present the current knowledge on the diversity of oligopeptide transporters in yeast, also discussing how the consumption of oligopeptides provides an adaptive advantage to yeasts within the wine environment.

Iron deficiency triggered transcriptome changes in bread wheat

A series of complex transport, storage and regulation mechanisms control iron metabolism and thereby maintain iron homeostasis in plants. Despite several studies on iron deficiency responses in different plant species, these mechanisms remain unclear in the allohexaploid wheat, which is the most widely cultivated commercial crop. We used RNA sequencing to reveal transcriptomic changes in the wheat flag leaves and roots, when subjected to iron limited conditions. We identified 5969 and 2591 differentially expressed genes (DEGs) in the flag leaves and roots, respectively. Genes involved in the synthesis of iron ligands i.e., nicotianamine (NA) and deoxymugineic acid (DMA) were significantly up-regulated during iron deficiency. In total, 337 and 635 genes encoding transporters exhibited altered expression in roots and flag leaves, respectively. Several genes related to MAJOR FACILITATOR SUPERFAMILY (MFS), ATP-BINDING CASSETTE (ABC) transporter superfamily, NATURAL RESISTANCE ASSOCIATED MACROPHAGE PROTEIN (NRAMP) family and OLIGOPEPTIDE TRANSPORTER (OPT) family were regulated, indicating their important roles in combating iron deficiency stress. Among the regulatory factors, the genes encoding for transcription factors of BASIC HELIX-LOOP-HELIX (bHLH) family were highly up-regulated in both roots and the flag leaves. The jasmonate biosynthesis pathway was significantly altered but with notable expression differences between roots and flag leaves. Homoeologs expression and induction bias analysis revealed subgenome specific differential expression. Our findings provide an integrated overview on regulated molecular processes in response to iron deficiency stress in wheat. This information could potentially serve as a guideline for breeding iron deficiency stress tolerant crops as well as for designing appropriate wheat iron biofortification strategies.

OsmiR528 regulates rice-pollen intine formation by targeting an uclacyanin to influence flavonoid metabolism

The intine, the inner layer of the pollen wall, is essential for the normal development and germination of pollen. However, the composition and developmental regulation of the intine in rice (Oryza sativa) remain largely unknown. Here, we identify a microRNA, OsmiR528, which regulates the formation of the pollen intine and thus male fertility in rice. The mir528 knockout mutant aborted pollen development at the late binucleate pollen stage, significantly decreasing the seed-setting rate. We further demonstrated that OsmiR528 affects pollen development by directly targeting the uclacyanin gene OsUCL23 (encoding a member of the plant-specific blue copper protein family of phytocyanins) and regulating intine deposition. OsUCL23 overexpression phenocopied the mir528 mutant. The OsUCL23 protein localized in the prevacuolar compartments (PVCs) and multivesicular bodies (MVBs). We further revealed that OsUCL23 interacts with a member of the proton-dependent oligopeptide transport (POT) family of transporters to regulate various metabolic components, especially flavonoids. We propose a model in which OsmiR528 regulates pollen intine formation by directly targeting OsUCL23 and in which OsUCL23 interacts with the POT protein on the PVCs and MVBs to regulate the production of metabolites during pollen development. The study thus reveals the functions of OsmiR528 and an uclacyanin during pollen development.

Exploring Conformational Transitions and Free-Energy Profiles of Proton-Coupled Oligopeptide Transporters.

Proteins involved in peptide uptake and transport belong to the proton-coupled oligopeptide transporter (POT) family. Crystal structures of POT family members reveal a common fold consisting of two domains of six transmembrane α helices that come together to form a "V" shaped transporter with a central substrate binding site. Proton-coupled oligopeptide transporters operate through an alternate access mechanism, where the membrane transporter undergoes global conformational changes, alternating between inward-facing (IF), outward-facing (OF), and occluded (OC) states. Conformational transitions are promoted by proton and ligand binding; however, due to the absence of crystallographic models of the outward-open state, the role of H+ and ligands is still not fully understood. To provide a comprehensive picture of the POT conformational equilibrium, conventional and enhanced sampling molecular dynamics simulations of PepTst in the presence or absence of ligand and protonation were performed. Free-energy profiles of the conformational variability of PepTst were obtained from microseconds of adaptive biasing force (ABF) simulations. Our results reveal that both proton and ligand significantly change the conformational free-energy landscape. In the absence of ligand and protonation, only transitions involving IF and OC states are allowed. After protonation of the residue Glu300, the wider free-energy well for Glu300 protonated PepTst indicates a greater conformational variability relative to the apo system, and OF conformations became accessible. For the Glu300 protonated Holo-PepTst, the presence of a second free-energy minimum suggests that OF conformations are not only accessible, but also stable. The differences in the free-energy profiles demonstrate that transitions toward outward-facing conformation occur only after protonation, which is likely the first step in the mechanism of peptide transport. Our extensive ABF simulations provide a fully atomic description of all states of the transport process, offering a model for the alternating access mechanism and how protonation and ligand control the conformational changes.

Substrate Transport Properties of the Human Peptide/Histidine Transporter PHT2 in Transfected MDCK Cells

PHT2, a member of the proton-coupled oligopeptide transporter family, participates in the transportation of small peptides and histidine from lysosomes to the cytosol. It facilitates maintenance of intracellular peptide homeostasis. However, it remains a challenge to elucidate the functional properties of PHT2 due to its localization in the lysosomal membrane. The aim of this study was to explore the transport function and substrate properties of human PHT2 (hPHT2) by transfecting Madin-Darby canine kidney cells with hPHT2 mutants to obtain stably expressed protein in the cell membrane. Using this cell model, we found that the transport activity of hPHT2 reached a maximum capacity when the extracellular pH was 5.5. hPHT2 showed relatively low affinity for Gly-Sar and relatively high affinity for d3-L-histidine, with Km values of 428 ± 88 μM and 66.9 ± 5.7 μM, respectively. Several typical substrates or inhibitors of PEPT1 and PEPT2, including valacyclovir, Gly-Gly-Gly, and cefadroxil but not 5-aminolevulinic acid or captopril, were proven to be substrates of hPHT2. However, hPHT2 showed low affinity for valacyclovir with a Km value of 5350 ± 1234 μM. In conclusion, this study established a suitable and efficient cell model to explore the function of hPHT2 in vitro and provided important information on the transport activity and substrate properties of hPHT2.

Genome Mining of Plant NPFs Reveals Varying Conservation of Signature Motifs Associated With the Mechanism of Transport.

Nitrogen is essential for all living species and may be taken up from the environment in different forms like nitrate or peptides. In plants, members of a transporter family named NPFs transport nitrate and peptides across biological membranes. NPFs are phylogenetically related to a family of peptide transporters (PTRs) or proton-coupled oligopeptide transporters (POTs) that are evolutionarily conserved in all organisms except in Archaea. POTs are present in low numbers in bacteria, algae and animals. NPFs have expanded in plants and evolved to transport a wide range of substrates including phytohormones and glucosinolates. Functional studies have shown that most NPFs, like POTs, operate as symporters with simultaneous inwardly directed movement of protons. Here we focus on four structural features of NPFs/POTs/PTRs that have been shown by structural and functional studies to be essential to proton-coupled symport transport. The first two features are implicated in proton binding and transport: a conserved motif named ExxER/K, located in the first transmembrane helix (TMH1) and a D/E residue in TMH7 that has been observed in some bacterial and algal transporters. The third and fourth features are two inter-helical salt bridges between residues on TMH1 and TMH7 or TMH4 and TMH10. To understand if the mechanism of transport is conserved in NPFs with the expansion to novel substrates, we collected NPFs sequences from 42 plant genomes. Sequence alignment revealed that the ExxER/K motif is not strictly conserved and its conservation level is different in the NPF subfamilies. The proton binding site on TMH7 is missing in all NPFs with the exception of two NPFs from moss. The two moss NPFs also have a positively charged amino acid on TMH1 that can form the salt bridge with the TMH7 negative residue. None of the other NPFs we examined harbor residues that can form the TMH1–TMH7 salt bridge. In contrast, the amino acids required to form the TMH4–TMH10 salt bridge are highly conserved in NPFs, with some exceptions. These results support the need for further biochemical and structural studies of individual NPFs for a better understanding of the transport mechanism in this family of transporters.

Function, Regulation, and Pathophysiological Relevance of the POT Superfamily, Specifically PepT1 in Inflammatory Bowel Disease.

Mammalian members of the proton-coupled oligopeptide transporter family are integral membrane proteins that mediate the cellular uptake of di/tripeptides and peptide-like drugs and couple substrate translocation to the movement of H+ , with the transmembrane electrochemical proton gradient providing the driving force. Peptide transporters are responsible for the (re)absorption of dietary and/or bacterial di- and tripeptides in the intestine and kidney and maintaining homeostasis of neuropeptides in the brain. These proteins additionally contribute to absorption of a number of pharmacologically important compounds. In this overview article, we have provided updated information on the structure, function, expression, localization, and activities of PepT1 (SLC15A1), PepT2 (SLC15A2), PhT1 (SLC15A4), and PhT2 (SLC15A3). Peptide transporters, in particular, PepT1 are discussed as drug-delivery systems in addition to their implications in health and disease. Particular emphasis has been placed on the involvement of PepT1 in the physiopathology of the gastrointestinal tract, specifically, its role in inflammatory bowel diseases. © 2018 American Physiological Society. Compr Physiol 8:731-760, 2018.

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.

Identification of residues critical for proton-coupled glutathione translocation in the yeast glutathione transporter, Hgt1p.

The proton gradient acts as the driving force for the transport of many metabolites across fungal and plant plasma membranes. Identifying the mechanism of proton relay is critical for understanding the mechanism of transport mediated by these transporters. We investigated two strategies for identifying residues critical for proton-dependent substrate transport in the yeast glutathione transporter, Hgt1p, a member of the poorly understood oligopeptide transporter family of transporters. In the first strategy, we tried to identify the pH-independent mutants that could grow at higher pH when dependant on glutathione transport. Screening a library of 269 alanine mutants of the transmembrane domains (TMDs) along with a random mutagenesis strategy yielded two residues (E135K on the cusp of TMD2 and N710S on TMD12) that permitted growth on glutathione at pH 8.0. Further analysis revealed that these residues were not involved in proton symport even though they conferred better transport at a higher pH. The second strategy involved a knowledge-driven approach, targeting 31 potential residues based on charge, conservation and location. Mutation of these residues followed by functional and biochemical characterization revealed E177A, Y193A, D335A, Y374A, H445A and R554A as being defective in proton transport. Further analysis enabled possible roles of these residues to be assigned in proton relay. The implications of these findings in relation to Hgt1p and the suitability of these strategic approaches for identifying such residues are discussed.