2.A.17 The Proton-dependent Oligopeptide Transporter (POT/PTR) Family

Proteins of the POT family (also called the PTR (peptide transport) family) are from animals, plants, yeast, archaea and both Gram-negative and Gram-positive bacteria. Several of these organisms possess multiple POT family paralogues. The proteins are of about 450-600 amino acyl residues in length with the eukaryotic proteins in general being longer than the bacterial proteins. They exhibit 12 putative or established transmembrane α-helical spanners.  The plant homologues have been examined from phylogenetic standpoints (von Wittgenstein et al. 2014). This family is also called the solute carrier 15 family (Slc15). They utilize the proton gradient and negative membrane protential to drive the transmembrane transporter of di-/tripeptide and peptide-mimetic molecules, and they also play important roles in immunoreaction (Dong et al. 2020). Rehmannia glutinosa (Chinese foxglove) has 18 ntr1 genes encoding proteins of 419 to 601 aas with 7 - 12 TMSs. They are found primarily in the plasma membranes of various plant tissues (Gu et al. 2021). 109 members of the PTR family are encoded within the genome of tea plants (Camellia sinensis) (Wang et al. 2022).

Pairs of salt bridge interactions between transmembrane helices work in tandem to orchestrate alternating access transport within the PTR family (Newstead 2014). Key roles for residues conserved between bacterial and eukaryotic homologues suggest a conserved mechanism of peptide recognition and transport that in some cases has been subtly modified in individual species.  PepT1 and PepT2, mammalian members of this family, are responsible for the uptake of many pharmaceutically important drug molecules, including antibiotics and antiviral medications.  Thus, their promiscuity can be used for improving the oral bioavailability of poorly absorbed compounds (Newstead 2014). Signature peptides enabled the reliable quantification of human hepatic transporters (Mori et al. 2022). The QTAP protocol using these optimal signature peptides provides quantitative data on hepatic transporters, usable for integrated pharmacokinetic studies.

While most members of the POT family catalyze peptide transport, one is a nitrate permease and one can transport histidine as well as peptides. A nitrate permease of Arabidopsis, Chl1 (TC #2.A.17.3.1), exhibits dual affinity. When phosphorylated at threonine-101, it exhibits high affinity (50 μM) for nitrate, but when not phosphorylated, it exhibits low affinity (~5 mM) (Liu and Tsay, 2003). Some of the peptide transporters can also transport antibiotics. They function by proton symport, but the substrate:H⁺ stoichiometry is variable: the high affinity rat PepT2 carrier catalyzes uptake of 2 and 3H⁺ with neutral and anionic dipeptides, respectively, while the low affinity PepT1 carrier catalyzes uptake of one H⁺ per neutral peptide. In eukaryotes, some of these transporters may be in organellar membranes such as the lysosomes.  Candida tropicalis oligopeptide transporters assist in the transmembrane transport of the antimicrobial peptide, CGA-N9 (Wu et al. 2023).

Di- and tripeptide transporters of the POT/PTR/NRT1 family are localized either to the tonoplast (TP) or plasma membrane (PM). A 7 amino acid fragment of the hydrophilic N-terminal region of Arabidopsis PTR2, PTR4 and PTR6 is required for TP localization and sufficient to redirect not only PM-localized PTR1 or PTR5, but also sucrose transporter SUC2 to the tonopolast (Komarova et al., 2012). L(11) and I(12) of PTR2 are essential for TP targeting, while only one acidic amino acid at position 5, 6 or 7 is required, revealing a dileucine (LL or LI) motif with at least one upstream acidic residue. Similar dileucine motifs could be identified in other plant TP transporters. Targeting to the PM required the loop between transmembrane domains 6 and 7 of PTR1 or PTR5. Deletion of either PM or TP targeting signals resulted in retention in internal membranes, indicating that PTR trafficking to these destination membranes requires distinct signals and is in both cases not by default (Komarova et al., 2012). A comprehensive genome-wide systematic characterization of the PTR family led to the identification of 193 PTR genes in the genome of Brassica napus (Rapeseed) (Zhang et al. 2020). The  family exhibited high levels of genetic diversity among sub-families, and expression analyses identified a broad range of expression patterns for individual gene in response to multiple nutrient stresses, including nitrogen (N), phosphorus (P) and potassium (K⁺) deficiencies, as well as ammonium toxicity. Ten core genes induced in response to N stress (Zhang et al. 2020).

Both proton and ligand significantly change the conformational free-energy landscape of PepT (Batista et al. 2019). In the absence of ligand and protonation, only transitions involving inner facing (IF) and occluded (OC) states are allowed. After protonation of residue Glu300, the wider free-energy well  indicates a greater conformational variability relative to the apo system, and outward facing (OF) conformations become accessible. For the Glu300 protonated holo-PepT, the presence of a second free-energy minimum suggests that OF conformations are accessible and stable. Thus, the differences in the free-energy profiles suggest that transitions toward outward-facing conformations occur only after protonation, which is likely the first step in the mechanism of peptide transport. The transmembrane proton flux drives essential steps of the full functional cycle: 1) protonation of a glutamate in TMS 7 opens the extracellular gate, allowing ligand entry; 2) inward proton flow induces the cytosolic release of the ligand by varying the protonation state of a second conserved glutamate in TMS 10; 3) proton movement between TMS 7 and TMS 10 is thermodynamically driven and kinetically permissible via water proton shuttling without the participation of ligand (Li et al. 2022).

The generalized transport reaction catalyzed by the proteins of the POT family is:

Substrate (out) +  nH⁺ (out) → substrate (in) + nH⁺ (in)