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Physiology 21: 93-102, 2006; doi:10.1152/physiol.00054.2005
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Physiology, Vol. 21, No. 2, 93-102, April 2006
© 2006 Int. Union Physiol. Sci./Am. Physiol. Soc.

REVIEW

From Bacteria to Man: Archaic Proton-Dependent Peptide Transporters at Work

Hannelore Daniel, Britta Spanier, Gabor Kottra and Dietmar Weitz

Department of Food and Nutrition, Molecular Nutrition Unit, Technical University of Munich, Freising-Weihenstephan, Germany, daniel{at}wzw.tum.de


   Abstract
 
Uptake of nutrients into cells is essential to life and occurs in all organisms at the expense of energy. Whereas in most prokaryotic and simple eukaryotic cells electrochemical transmembrane proton gradients provide the central driving force for nutrient uptake, in higher eukaryotes it is more frequently coupled to sodium movement along the transmembrane sodium gradient, occurs via uniport mechanisms driven by the substrate gradient only, or is linked to the countertransport of a similar organic solute. With the cloning of a large number of mammalian nutrient transport proteins, it became obvious that a few "archaic" transporters that utilize a transmembrane proton gradient for nutrient transport into cells can still be found in mammals. The present review focuses on the electrogenic peptide transporters as the best studied examples of proton-dependent nutrient transporters in mammals and summarizes the most recent findings on their physiological importance. Taking peptide transport as a general phenomenon found in nature, we also include peptide transport mechanisms in bacteria, yeast, invertebrates, and lower vertebrates, which are not that often addressed in physiology journals.


   Introduction
Top
 Introduction
Basic Characteristics of...
 The Unique Substrate Specificity...
 Comparative Physiology of the...
 References
 
Nutrient transport across the plasma membrane of cells is a critical step in nutrient homeostasis since the cell membrane compartmentalizes metabolic processes and serves as a selective barrier for permeation of nutrients and xenobiotics. The maintenance of an intracellular environment that is distinctly different from the extracellular milieu is essential to life, and therefore a large spectrum of membrane proteins with highly specialized functions has emerged during evolution. Whereas the transport pathways mediating nutrient uptake in bacteria, yeast, and plants are mainly energized by a transmembrane electrochemical proton gradient, the systems mediating nutrient influx into mammalian cells are more frequently energized by the electrochemical Na+ gradient or occur via uniport or antiport processes and thus are driven solely by the substrate gradients. However, some of the mammalian nutrient transporter proteins utilize existing, although mostly shallow, transmembrane proton gradients that, in combination with the inside negative membrane potential, provide a powerful driving force for transport, allowing nutrients to be accumulated above extracellular concentration. Representative proton-dependent carrier systems are those for di- and tripeptides (SLC15 family), for selected free amino acids (PAT system, SLC36 family), for some vitamins (for example folate; SLC19 family), and for some trace elements (for example iron; SLC40), but also for a large number of organic acids from intermediate metabolism (for example, the monocarboxylate transporter MCT1-4; SLC16 family). We shall focus here exclusively on electrogenic peptide transporters as a paradigm for mammalian proton-coupled nutrient carriers and review the current knowledge on their structure and physiological functions in various organisms.


   Basic Characteristics of Electrogenic Proton-Dependent Peptide Transporters
Top
 Introduction
 Basic Characteristics of...
The Unique Substrate Specificity...
 Comparative Physiology of the...
 References
 
Proton-dependent transmembrane transport of short-chain peptides occurs in all living organisms and provides an efficient and energy-saving route for uptake of bulk quantities of amino acids in peptide form. When peptide transport in mammals was first described, most studies revealed that peptide uptake was Na+ dependent. Later, it was proven that peptide transport in mammalian intestine and kidney is energized, like in prokaryotes and lower eukaryotes, by an inwardly directed electrochemical proton gradient existing across brush-border membranes. The apparent sodium dependence found in tissues is essentially based on the requirement of Na+/H+ exchanger activity for recovery from cellular acid loading by recycling protons entering via peptide transporters back into the gut and tubular lumen. The transporter proteins from numerous species were identified by expression cloning in the mid-1990s and later by homology screening and cloning. In mammals, the SLC15 family is composed of four members: two proteins of the PEPT series that transport exclusively di- and tripeptides and two proteins of the PHT series that seem to transport histidine as well as selected peptides but that remain obscure in terms of functional importance. The mammalian intestinal transporter form was designated as PEPT1 (SLC15A1) and the renal isoform as PEPT2 (SLC15A2). Functional analysis of the transporters in Xenopus oocytes and in mammalian cells established PEPT1 to represent the low-affinity, high-capacity variant and PEPT2 the high-affinity, low-capacity variant, although both proteins essentially transport the same substrates. Information on the transporter genes in the different species, their putative structure, and other relevant features are provided in Table 1Go and have also been reviewed elsewhere (11, 14–16, 68).


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  		Table 1. Molecular characteristics and additional information of POT family members from higher and lower vertebrates, worm, and bacteria
 
The amino acid sequences for PEPT1 and PEPT2 and the related proteins in other species predict 12 membrane-spanning domains with both amino and carboxy termini localized in the cytosol (FIGURE 1Go). In case of the prokaryotic peptide transporter DtpT, this 12-transmembrane domain (TMD) structure has experimentally been proven by a gene fusion approach (30). Functional analysis of chimeras of the mammalian PEPT1 and PEPT2 proteins or of transporters with single-point mutations suggests the amino-terminal regions with TMDs 2 to 5 and 7 to play a predominant role in determining substrate affinity and also other characteristic features. Recent studies addressing the functional consequences of single-nucleotide polymorphisms (SNPs) in the PEPT proteins identified in individuals revealed only minor differences in the kinetic behavior of the mutant proteins (76). In the public SNP database NCBI Gene Expression Omnibus (GEO) dbSNP (http://www.ncbi.nlm.nih.gov/SNP/), five cSNPs with nonsynonymous changes in hPEPT2 are reported, two selected SNPs resulting in the amino acid changes Arg57His (R57H) and Pro409Ser (P409S) have been functionally characterized, and the R57H mutant protein was shown to lack transport activity despite proper membrane targeting (69). A genetic and haplotype analysis of the gene encoding hPEPT2 revealed the presence of two distinct, common variants that are present at a high frequency in a variety of ethnic groups. A functional analysis of the two common haplotypes termed hPEPT2*1 and hPEPT2*2, which differ by three amino acid residues, revealed a twofold difference in apparent substrate affinity but otherwise no major changes in function (56).


Figure 1
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  		FIGURE 1. Conserved regions and protein domains in members of the PTR family
Based on the predicted protein structure of human PEPT1, the location of the three conserved domains is shown. Alignments were made by using MultAlin Software. Identical amino acid residues are indicated in bold letters. The NCBI Gene Expression Omnibus (GEO) accession numbers of all analyzed proteins are also listed.

 
Although peptide transporter homologs differ strongly in genetics, sequence as well as protein size (see Table 1Go), small protein stretches are highly conserved. Three such domains, from which two are known as PTR (Peptide TRansporter) motifs, are located in the first 180–250 amino acids of the various PEPT homologs proteins (FIGURE 1Go). The first motif (ExCERFxYYG) is located at the outward-facing end of the first TMD (TMD1) and stretches into the following extracellular loop (32). Between the COOH-terminal half of TMD2 and the first NH2-terminal amino acid of TMD3, the PTR 2-1 motif, which is less conserved, is found (GxxxADxxxGKxxTI). The third FYxxINxG motif (FYING-PTR-motif) in TMD5 shows absolute conservation among all members of the peptide transporter family from bacteria to mammals. Point mutations introduced into these regions cause a loss of function of the corresponding protein (22, 31, 75), indicating the essentiality of these domains for the formation of the transporter pore and the interaction with the di-and tripeptide substrates.


   The Unique Substrate Specificity of Peptide Transporters
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 Introduction
 Basic Characteristics of...
 The Unique Substrate Specificity...
Comparative Physiology of the...
 References
 
The di- and tripeptide transporters display a unique substrate specificity that one could call "nonspecifically specific" or "specifically nonspecific." They essentially transport all possible di- and tripeptides (with a few exceptions) composed of L-{alpha} amino acids as well as a large variety of derivatives. This means that a minimum of 400 different dipeptides and 8,000 different tripeptides serve as substrates. Taking peptides that contain D-enantiomers of amino acids into account, there is a seemingly endless number of potential substrates. However, specificity comes into play by quite impressive differences in substrate affinity depending on the particular sequence of the substrate, and transport always occurs in a stereoselective manner with a preference for L-{alpha} amino acids. Although peptides containing D-isomers of amino acids are accepted, their affinity for interaction depends on the location of the D-amino acid residue in the peptide backbone and the polarity of the amino acid side chain. Peptides containing solely D-amino acids do not bind with appreciable affinity to the substrate binding domain as do free amino acids, and peptides with four or more amino acids do not serve as substrates of the proteins of the PEPT series. In addition to the large number of di- and tripeptides, peptidomimetics such as amino beta-lactam antibiotics, selected angiotensin-converting enzyme inhibitors, and some antiviral nucleoside prodrugs as well as omega-amino fatty acids do serve as substrates (11, 14, 68). Detailed information on the structural requirements in substrates that determine affinity has been compiled over years, and recent QSAR studies have defined the template that allows affinities of new substrates to be predicted quite accurately in silico. Essential features for substrate binding to PEPT1 are a substrate donor and acceptor group separated by 2.5–3.4 Å and a region of high electronic density at a distance of 5.2–5.6 Å from the donor and 2.9–3.7 Å from the acceptor site that defines a three-point recognition model (4, 7, 25). Since protein crystals required for X-ray diffraction are still not available for any of the peptide transporters, the three-dimensional architecture of the substrate binding pocket is still unknown.

The proton-to-substrate stoichiometric ratios for co-transport of differently charged substrates vary, but transport produces positive inward currents under voltage clamp conditions in any case, regardless of the substrate charge. Neutral and most cationic dipeptides are co-transported with one proton in case of PEPT1, whereas anionic dipeptides are transported both in their neutral form with one proton and in their charged form with two protons (39, 42, 49, 67), in which one proton neutralizes the negative side chain charge of the substrate. Electrophysiological analysis of pre-steady-state currents suggested for hPEPT1 an ordered, simultaneous transport model in which H+ binds first (50) and the return of the unloaded carrier to the outside provides the rate-limiting step in the transport cycle. However, some observations such as different Vmax or Imax values for different substrates cannot be explained with this model. Giant patch-clamp experiments provided evidence that PEPT1 and PEPT2 are capable of transporting also in the reverse direction when membrane voltage and substrate gradients are reversed, and transport for inward and outward transporting modes appeared symmetric, whereas substrate binding sites are asymmetric with a considerably lower binding affinity for the same substrates on the cytosol-facing binding domain (42).


   Comparative Physiology of the Transporters: From Bacteria to Humans
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 Introduction
 Basic Characteristics of...
 The Unique Substrate Specificity...
 Comparative Physiology of the...
References
 
As shown in FIGURE 2Go, peptide transport processes have a distinct phylogeny and evolutionary conservation. Whereas prokaryotes possess different classes of membrane peptide transporters, including ATP-dependent oligopeptide carriers, such transporters could not be identified in cell membranes of eukaryotes. From yeast on toward mammals, only di- and tripeptide transporters are found, and those have diverged in multicellular species into two structurally related but kinetically different isoforms with tissue-specific expression patterns.


Figure 2
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  		FIGURE 2. Phylogeny of the peptide transporters
In prokaryotes, both oligopeptide and di- and tripeptide transporters are expressed, whereas in simple eukaryotes, as for example in Saccharomyces cerevisae, only the PEPT1-type transporters, specialized in proton-dependent uptake of di- and tripeptides, are found (gray box). The driving force, the proton gradient over the inner membrane, is indicated by the electron transport chain, allowing ATP synthesis as well as peptide transport (top left box). In higher eukaryotes, from Caenorhabditis elegans to Homo sapiens, the PEPT family has diverged into the low-affinity, high-capacity isoform PEPT1, with prominent expression in intestinal epithelial cells, and the high-affinity, low-capacity isoform PEPT2 found in a variety of different cells, including also epithelial cells (gray box). In polarized cells, both transporter forms are located in apical membranes only (bottom right box), with a functional coupling to the apical Na+/H+ antiporter (NHE-3) for pH recovery from the peptide-transport-induced intracellular acid load.

 
Peptide transport in bacteria and yeast
In bacteria, peptide transport plays an important role not only in providing amino acids for metabolic needs but also for signaling processes that regulate gene expression, sporulation, chemotaxis, and other processes (17). Bacterial peptide transport systems have been characterized, for example, in Salmonella typhimurium and Escherichia coli and gram-positive Lactococcus lactis and Bacillus subtilis strains. Three distinct systems with overlapping substrate specificity are known to mediate peptide transport in bacteria. Two of these transport systems, the oligopeptide per-mease (Opp) and dipeptide permeases (Dpp), are complex transport machineries consisting of several gene products organized in operons and code for the substrate-binding proteins OppA and DppA, which then directly interact with the translocator units OppBCDF and DppBCDF, respectively. The translocator units belong to the large family of ATP-binding cassette (ABC) transporters (18, 34, 36).

In mutants of S. typhimurium, a third peptide transport system was identified. Mutations in the locus encoding a tripeptide permease (tppB) provides resistance to the toxic phosphonopeptide alafosfalin (27). The TppB protein seems transcriptionally tightly regulated by leucine and anaerobiosis in an additive manner but via different pathways (36). By locus analysis, the YdgR protein in E. coli was identified as the corresponding tppB system, and YgdR shows a sequence homology to the proton-dependent di-/tripeptide transporter family that is found in mammals (28). YdgR has two PTR domains as well as the conserved additional CERFxYYG domain shown in FIGURE 1Go. Expression of YdgR is regulated by the EnzZ/OmpR two-component regulatory system for environmental stress response. The functions and substrate specificity of YdgR or TppB are not well-defined. Only a few number of transport experiments in S. typhimurium strains deficient in Opp and Dpp have been conducted and revealed that TppB prefers hydrophobic tripeptides and transports only some dipeptides (27, 35). Functional analysis of the overex-pressed YdgR protein in our laboratory revealed a proton-dependent di-/tripeptide transporter activity with a very broad substrate specificity resembling that of the mammalian PEPT1 (unpublished data).

In L. lactis, a similar member of the POT family, designated as DtpT has in extenso been characterized using native membrane vesicles or the reconstituted protein. DtpT is energized by a proton-motive force and transports di- and tripeptides with a substrate recognition pattern comparable with the mammalian peptide transporters, although in some aspects it appears a bit more restrictive (19, 29, 30, 43). Transport studies with DtpT suggest constitutive expression in L. lactis, and a recent DNA-array profiling study identified the transcript as repressed during heat shock and acidic stress (73).

In summary, bacteria possess three peptide transport systems, two (Opp and Dpp) belonging to the large family of ABC transporter and one for di- and tripeptides belonging to the PTR family. For peptide uptake, only the latter system is conserved in higher eukaryotic organisms starting with yeast.

From bacteria to yeast
Two peptide transport systems are known in Saccharomyces cerevisiae: one for di-/tripeptides (belonging to the PTR family) and a second for tetra-/pentapeptides belonging to the oligopeptide transporter (OPT) system (32). The OPT gene family is very small, is not related to the Opp system of bacteria, and has up to now only been identified in fungi and plants. The best characterized member, OPT1, represents a membrane protein with 12–14 transmembrane helices expressed only during sporulation. In overexpression studies, OPT1 was characterized as a proton-dependent transporter for tetra-/pentapeptides (48) that is also involved in glutathione transport (9). This carrier also showed a relatively high affinity for Leu-enkephalin (310 µM), a pentapeptide involved in analgesia control in the central nervous system of vertebrates. Interestingly, an enkephalin transporting system in the retinal pigment epithelium in mammalian cells was identified as a Na+- and Cl–-dependent but not proton-dependent system (38).

The PTR system of S. cerevisiae consists of three genes (40): two transcriptional regulators of the system and one integral membrane protein as the transporter unit. The PTR2 gene encodes the integral membrane protein Ptr2 that belongs to the POT family (55) and that mediates proton-dependent uptake of di-/tripeptides across the membrane with a transport optimum at pH 5.5 and a preference for dipeptides with hydrophobic amino acids similar to the mammalian PEPT proteins. PTR2 expression is highly regulated by the gene products PTR1 and PTR3. PTR1 encodes the protein Ubr1p (3), which was known as N-recognin before, and this protein is part of a ubiquitin-dependent protein degradation system (6). Cup9p, a homeodomain-containing protein, is a repressor of the PTR2 gene, and after ubiquitination by Ubr1p it is released from the promoters, which in turn induces PTR2 gene expression (13). This functional regulation explains why a loss of PTR1 causes a loss of the entire PTR system. The Ubr1p protein contains several functional domains, and in vitro studies show that binding of dipeptides to one of these domains leads to an increase in Ubr1p ubiquitination activity, which in turn by de-repression drives PTR2 expression (70). PTR3, although not essential for the PTR system, also mediates expression control of PTR2. PTR3 and the amino acid permease SSY1 are part of a nutrient-sensing system for amino acids and peptides in yeast (41). Null mutations in one of these genes delete the ability of yeast cells to sense and react to available extracellular amino acids and peptides with an increase in amino acid and peptide transporter expression.

In summary, despite low overall sequence similarities to the mammalian proteins, all PTR members of bacteria or yeast so far described do show very similar features as the mammalian proteins. An interesting question is why cell membrane transporters for peptides larger than three amino acids as found in a large number in bacteria, fungi, and plants cannot be found in vertebrates. It may be speculated that peptides with longer backbone length can represent epitopes and would be a constant challenge for the immune system in a higher developed organism of the animal kingdom. Similarly, they could serve as ligands and bind to peptidergic targets such as membrane receptors. To restrict the size to three amino acid residues may therefore be strategically important in allowing efficient delivery of amino acids while minimizing possible other interfering biological activities.

From yeast to worms and flies
Three peptide transporters from C. elegans [pep-1 (opt-1), pep-2 (opt-2), and opt-3] have been cloned and initially characterized by functional expression in Xenopus oocytes (20, 23). The kinetic analysis revealed PEP-1 as the high-affinity, low-capacity isoform, whereas PEP-2 is the low-affinity, high-capacity isoform in worms (20). OPT-3 appears to function predominantly as H+ channel in neurons of the nematode (23). For the sake of clarity, here we will use PEPT1 (for C. elegans PEP-2) and PEPT2 (for C. elegans PEP-1), as these proteins have essentially the same functions in the nematode as in mammals.

The expression of PEPT1 in C. elegans is restricted to intestinal cells (FIGURE 3Go) and suggests a role in absorption of peptides derived from luminal protein breakdown as in mammals (51). In contrast, PEPT2, as shown in FIGURE 3Go, is expressed in several neurons, in the ventral and dorsal nerve cord, and in the vulva and anal muscles. The early division into the intestinal PEPT1 isoform with a narrow expression pattern and the PEPT2 isoform with a much broader expression pattern points to the importance of a well-defined compartmentation of uptake and recycling systems for di- and tripep-tides in a complex organism during evolution.


Figure 3
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  		FIGURE 3. Expression pattern of the peptide transporters PEPT1 (PEP-2) and PEPT2 (PEP-1) in C. elegans
A: the pep-2promoter::GFP reporter construct indicates exclusive expression of the PEPT1 protein in intestinal cells. B: the pep-1promoter::GFP reporter construct is expressed in a variety of cells of the nematode, including some head neurons, the ventral and dorsal nerve cord, and the vulva and anal muscles (indicated by arrow heads), suggesting a widespread expression of the PEPT2 protein in the worm.

 
Uptake of amino acids in the gut in di- and tripep-tide-bound form seems to play an essential role in the development of C. elegans. PEPT1-deficient worms [pep-2(lg601)] that completely lack intestinal di- and tripeptide transport show a retarded postembryonic development (~80% compared with wild type), an ~28% reduced body size, and an ~64% decreased production of progeny (33, 51). Interestingly, in worms lacking proper Na+/H+ exchanger function in gut cells induced by silencing nhx-2, which also impairs intestinal peptide transport, a comparable but even stronger phenotype was found. Animals deficient for NHX-2 expression showed reduced development and body size, impaired pharyngeal pumping, and fat storage as well as reproduction defects, but a 40% increased life span (53).

Growth and viability of cells are strongly dependent on the nutritional status and two interconnected signaling routes; the TOR (target of rapamycin) and the insulin-signaling pathways are centrally involved in sensing of amino acids as well as the protein and energy status of the cell. TOR integrates as a main sensor for free amino acids and via AMP-kinase as a sensor for the energy status various cellular processes, including translation, protein degradation, and autophagy (1, 64). A link between proper PEPT1 function and the TOR signaling cascade was proposed based on the fact that a weak TOR (let-36) interference RNA phenotype in C. elegans was enhanced in all phenotypic alterations in a background of animals additionally lacking PEPT1 (46, 51).

The importance of the insulin/IGF signaling pathway in development, metabolism, stress response, and aging of C. elegans has been demonstrated in numerous studies (5, 10, 37). Despite the fact that, in mammals as well as in Drosophila, the availability of dietary proteins or amino acids is known to affect the insulin signaling cascade and affect growth and ageing processes (12, 26) in C. elegans, that link had not been demonstrated. Surprisingly, PEPT1-deficient animals, although growth-retarded by a limited amino acid and caloric supply, showed a wild-type-like aging phenotype. However, when the PEPT1-lacking animals were crossed with animals deficient in proper function of the insulin receptor [daf-2(e1370)], which is known to induce by its own a twofold increase in average life span, the double mutant worms [pep-2(lg601); daf-2(e1370)] displayed a further increase in life span by 60% (51). This suggested a close physiological link between the availability of dietary amino acids and the insulin/IGF signaling pathway also in C. elegans. The large life-extending effect was completely reversed when, additionally, a mutant deficient in DAF-16 function was crossed in. DAF-16 is a transcription factor, and the main target of the insulin/IGF signaling pathway in worms, and is homologous to the mammalian FOXO protein. These studies in the various C. elegans mutant lines demonstrated that the PEPT1-mediated transport of peptide-bound amino acids in the gut is important for delivery of bulk quantities of amino acids for growth and development. The impaired amino acid status is sensed most likely via the TOR signaling pathway in parallel with the insulin signaling pathway and transmitted into a reduced growth and reproduction phenotype. Along this line, studies in human intestinal Caco-2 cells have demonstrated that PEPT1 expression and function is regulated by nutrients as well as hormones and growth factors such as insulin, leptin, epidermal growth factor, and triiodothyronine (2), and these findings suggest that intestinal peptide uptake is embedded into a regulatory network in which transport function is hormonally linked to growth and development. In contrast to PEPT1, gene deletions of the pept2 gene in C. elegans did not reveal any obvious phenotypical alterations in the animals (unpublished observations).

From D. melanogaster, only one di- and tripeptide transporter homolog has been cloned and functionally characterized after expression in HeLa cells (57). The proton-dependent transporter (encoded by the gene opt1 = yin) is expressed in germinal and somatic tissues of both fly genders with highest expression in the nurse cells of the female ovary, in the fat body, in various neurons, as well as in the epithelium of the midgut and rectum. OPT1, which is most likely a PEPT1 type, displays a high affinity for di- and tripeptides as well as aminocephalosporins and aminopenicillins (57). Its expression in midgut correlates to PEPT1 in the small intestine in mammals. In Drosophila, as in most insects, the formation of primary urine by filtration and the active secretion of selected substances occurs within the Malpighian tubules with the fluid then deposited into the hindgut, where many filtered metabolites are reabsorbed by the rectum, and the presence of the peptide transporters here suggests a similar function as PEPT1 and PEPT2 in renal cells in mammals.

In lower vertebrates, the first cloned and characterized peptide transporter ortholog is zPEPT1 from the zebrafish Danio rerio (71). In zebrafish, PEPT1 is exclusively expressed in intestinal epithelial cells and shows essentially all characteristics of PEPT1 as a low-affinity, high-capacity transport, except for a unique pH dependence. Voltage clamp studies in ooyctes expressing zPEPT1 revealed that the Vmax of transport increases when extracellular pH is raised to neutral or alkaline pH values (71). This phenomenon has never been observed in any PEPT homolog and is currently unexplained. The zebrafish PEPT2 has very recently also been cloned, and when expressed in oocytes it displays all features of the mammalian PEPT2. It is expressed in the fish in an early stage in a variety of tissues and most interestingly also in inner ear-like structures (otis vesicle), which so far have not been analyzed for expression of PEPT2 in mammals (57a).

From fish to mammals
Although mammalian PEPT1 and PEPT2 proteins have been characterized extensively in a variety of heterologous expression systems, their functions in native organs and for overall body nitrogen homeostasis are still not well-defined. The studies in C. elegans suggest that PEPT1 in the gut epithelium contributes substantially to overall amino acid absorption. Studies in mammals addressing the quantitative importance of PEPT1 over that of the amino acid transporting systems are lacking, since a PEPT1-deficient mouse line has not yet been established. Hereditary disorders in humans linked to a malfunction or absence of PEPT1 are also not known. However, because PEPT1 has a prominent expression throughout the small intestine in the upper villus region and due to its high transport capacity and very high turnover number, it can also be anticipated to play an important role in the mammalian and human gut in overall absorption of dietary amino acids. Its central importance for delivery of peptidomimetic drugs to intestinal epithelial cells and systemic availability has been recognized. All drug substrates of PEPT1 do show an excellent oral availability, and for this reason PEPT1 has become a prime target for drug delivery via pro-drug approaches by rendering the pharmacologically active compound into a substrate for PEPT1.

PEPT2-deficient mice have been generated in two laboratories (59, 66). These mice have been studied for functional impairments in peptide transport in kidney and choroid plexus and for changes in overall clinical chemistry and physiology. In kidney, PEPT2 is mainly found in the S3 segment of the tubule, whereas PEPT1 is expressed in S1 and S2 segments, although with a much lower density. In PEPT2-null mice, transport of a radiolabeled dipeptide into kidney after intraperitoneal administration was reduced by almost 65%, and there was no change in the level of expression (mRNA and protein) of either PEPT1 or the PHT transporters (54) that could have compensated for the loss of PEPT2 function. In a very elegant study based on a 11C-labeled glycylsarcosine and PET scanning, efficient transport of the dipeptide into kidney tissues of wild-type mice was demonstrated, but essentially no renal uptake was found in the animals lacking PEPT2 (52). Together, these studies strongly suggest that PEPT2 is the functionally important peptide transporter in kidney, and PEPT1 may play a minor role in the clearance of filtered peptides. By use of a variety of model substrates (glycylsarcosine, carnosine, aminolevulinic acid, cefadroxil), it was also shown that PEPT2-null mice completely lack the CSF-to-brain peptide transport in the choroid plexus epithelium (65). Moreover, our laboratory recently demonstrated that PEPT2 is also expressed in glia cells in the enteric nervous system and that glia cells throughout the enteric nervous system can selectively be identified by ß-Ala-Lys-AMCA, a fluorescent high-affinity dipeptide substrate of PEPT2 with a complete lack of staining in animals deficient for PEPT2 (60). Recently, PEPT2 mRNA expression was also demonstrated in pituitary and reproductive organs (testis, prostate, ovary, and uterus) of rats and mice (47). However, despite its widespread expression in normal animals and the established functional loss of transport activity in various tissues, the PEPT2-null animals are healthy and fertile, and neither clinical chemistry data obtained from plasma and urine samples nor general physiological measures (growth and development) indicate any significant metabolic perturbation. The "real" physiological role of PEPT2 therefore remains hidden.

In summary, peptide transporters of the PEPT class with specificity for transport of di- and tripeptides are found in bacteria, yeast, invertebrates, and vertebrates. They are electrogenic and couple substrate movement to proton movement with variable substrate-to-proton coupling stoichometries and the driving force provided by the membrane voltage. Despite quite impressive differences in overall amino acid sequence, transport characteristics are almost identical when compared among species. In this respect, the transporters may be considered archaic with evolutionary alterations in sequence but less so in functions. Whereas the role of the PEPT1-like proteins in the various species appears to lie in the provision of bulk quantities of amino acids for growth and development at low cost (ATP requirement), the "true" physiological role of the PEPT2-type transporters in higher organisms remains to be determined.


   References
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 Introduction
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 The Unique Substrate Specificity...
 Comparative Physiology of the...
 References
 

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