2.A.17 The Proton-dependent Oligopeptide Transporter (POT/PTR) Family
Proteins of the POT family (also called the PTR (peptide transport) family) consist of proteins 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).
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).
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.
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).
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 generalized transport reaction catalyzed by the proteins of the POT family is:
Substrate (out) nH (out) → substrate (in) nH+ (in)