The cell membrane encases cells and functions as a protective barrier. Although this has the benefit of preventing harmful substances from entering a cell, it also keeps beneficial molecules out. The cell membrane therefore contains a system of different ‘gates’, called transporters, through which selected supplies can pass.

One large family of transporters, found in bacteria, mammals, and plants, is the ‘proton coupled oligopeptide transporter’ family, called POTs for short. These transport over 8000 types of small peptide molecule, each of which is made up of two or three smaller molecules called amino acids. The energy for this transport process is gained by simultaneously transporting charged ions called protons with the peptides. Because these transporters also recognize and transport various drugs, they are currently being investigated to discover whether they could be manipulated to increase how much of a drug is taken up into cells.

It remains unknown how the POT family of transporters imports so many different small peptides across the cell membrane, or how many protons are needed to transport a peptide. A study published earlier in 2014 has nevertheless provided some hints: it appears that small peptides adopt different shapes when bound to a bacterial POT transporter depending on whether they consist of two or three amino acids. This suggests that two different transport mechanisms operate from the same binding site, which may account for the wide variety of molecules that can be transported.

In a follow up to this work, Parker et al., including some of the researchers involved in the earlier 2014 work, now look in detail at how many protons this bacterial transporter uses to import these small peptides. This reveals that while the transport of peptides made of three amino acids requires three protons to also be moved through the transporter, the transport of peptides containing two amino acids requires four, or possibly five, protons. This challenges previous findings that these transporters transport one peptide for every proton, and further supports the idea that a single transporter can use more than one method to bind to and transport molecules. Whether other membrane transporters, particularly the human versions of the POT family, share this ability remains an open question.

Secondary active transporters are integral membrane proteins that couple the energy stored in an ion gradient to drive the uptake of a solute against its concentration gradient (Nicholls and Ferguson, 2013). This can be accomplished through either a symport mechanism, with the solute being moved in the direction of the driving ion, or antiport, where the solute movement is counter to that of the driving ion (Shi, 2013). The ion gradients utilised by secondary active transporters include proton (ΔμH⁺), sodium (ΔμNa⁺), or chloride (ΔμCl⁻) gradients, which are in turn established through the action of the primary ATP driven P-, F-, V-, and A-type ion pumps (Voet and Voet, 2011). A fundamental characteristic of these systems is that, in general, transport is strictly coupled; the movement of solutes and ions is obligatory and one cannot be transported without the other. If these systems were to operate in a decoupled manner, they would act as leaks and dissipate the ion gradients across the membrane, quickly leading to cell death. Given the strict requirement for coupling solute binding and transport to ion movement, the stoichiometry of these mechanisms is normally a fixed ratio. Examples include the Escherichia coli lactose transporter, LacY, which transports lactose in a symport mechanism with one proton (Kaback et al., 2011) and EmrE, the small multidrug extrusion transporter, which moves both monovalent and divalent substrates in a 1:2 drug:proton stoichiometry (Rotem and Schuldiner, 2004).

A number of membrane proteins have been identified that recognise multiple structurally and chemically diverse solutes (Koepsell, 2013; Pelis and Wright, 2014). Prominent among these are the proton coupled oligopeptide transporters or POTs (Hillgren et al., 2013; Smith et al., 2013). POT family transporters are widely distributed within bacterial, fungal, and plant genomes where they are responsible for the uptake of di- and tri-peptides from the external environment (Daniel et al., 2006). Mammals contain four POT family transporters, PepT1 (SLC15A1), PepT2 (SLC15A2), PHT1 (SLC15A4), and PHT2 (SLC15A3). PepT1 and PepT2 are expressed at the plasma membrane, whereas PHT1 and PHT2 are found in lysosomal membranes (Daniel and Kottra, 2004). Throughout the POT family the transport mechanism and peptide binding site are highly conserved, with bacterial counterparts sharing ∼80% identity to human PepT1 and PepT2 within their peptide binding sites (Terada and Inui, 2012; Newstead, 2014). All POT family members studied to date transport their substrates into the cell in a coupled symport mechanism, driven by the proton electrochemical gradient. While a number of mutational studies on the mammalian PepT1 and PepT2 transporters address peptide recognition (Terada et al., 1996; Fei et al., 1997, 1998; Yeung et al., 1998; Uchiyama et al., 2003; Luckner and Brandsch, 2005; Kulkarni et al., 2007; Pieri et al., 2009), the question of how many protons are coupled to peptide transport remains unresolved; early studies using Caco-2 cell lines derives a ratio of greater than two protons per peptide (Thwaites et al., 1993). However due to experimental design, narrowing this figure to a more precise stoichiometry was not possible (Kottra et al., 2002). Electrophysiological studies using two electrode voltage clamping (TEVC) in Xenopus oocytes in tandem with radio ligand transport assays on non hydrolysable peptide (D-Phe-L-Gln/Glu/Lys or Gly-Sar) have reported stoichiometry ratios of 1:1 and 2:1 proton:peptide for neutral/basic and acidic di-peptides respectively for PepT1 (Fei et al., 1994; Steel et al., 1997; Chen et al., 1999). Similar experiments on PepT2 have given different ratios either D-Phe-L-ala of 2:1 and for D-Phe-L-Glu 3:1 (Chen JBC 1999) or 1:1 for D-Phe-L-Gln/Glu or Lys (Fei et al., 1999).

Recently, we reported two crystal structures of a bacterial POT family transporter, PepT_St, from Streptoccocus thermophilus, which revealed di- and tri-peptides interacting differently within the binding site (Lyons et al., 2014). Whereas the di-peptide L-Ala-L-Phe binds in a horizontal position with respect to the plane of the membrane, whilst the tri-peptide L-Ala-L-Ala-L-Ala resides in a vertical orientation and makes subtly different interactions within the binding site. This raised the interesting and to our knowledge unique proposition, that two different transport mechanisms may have evolved within the same binding site as a way to accommodate a diverse library of peptide ligands, >8000 (Ito et al., 2013). To address whether PepT_St could indeed operate using distinct mechanisms to drive di- and tri-peptides, we explored the coupling mechanism between protons and peptide in a reconstituted system, determining the coupling stoichiometries of protons and peptides. We show that whilst tri-peptide import is coupled to three protons, the mechanism for di-peptide import requires at least four and possibly five protons. These results provide further biochemical evidence that POT family transporters do operate via multiple mechanisms for coupling peptide transport to the proton gradient. The ability to couple different numbers of protons to structurally and chemically diverse ligands may explain how the POT family is able to accommodate and transport such a large library of di- and tri-peptides.

To address the question of stoichiometry, we developed a sensitive and robust assay to follow the proton movement during the transport cycle. Previously published peptide transport assays tend to follow peptide uptake using a radiolabeled peptide substrate. To instead follow proton movement, we monitored the internal pH with the ratiometric pH sensitive fluorophore, pyranine (Figure 1A and Figure 1—figure supplement 1). We first performed a number of control experiments to see whether our system could indeed follow proton coupled peptide transport into a liposome. Acidification of the lumen was only observed in the presence of peptide and a large hyperpolarized (negative inside) membrane potential imposed by adding the potassium ionophore valinomycin in the presence of a K⁺ gradient (Figure 1B,C and Figure 1—figure supplement 2). We did not see such acidification either in the absence of valinomycin or in the presence of amino acid (alanine) or tetra peptide (Ala-Ala-Ala-Ala), confirming that this transporter is indeed specific for di- and tri-peptide substrates (Figure 1C). PepT_St has been shown previously to transport Ala–Ala with a 10-fold higher activity than Ala-Ala-Ala as judged by IC₅₀ values using tritiated Ala–Ala as a reporter (Solcan et al., 2012). We confirmed this 10-fold difference in uptake by using our proton-based assay, which can now report the direct uptake of any transported substrate rather than inferring substrate specificity through inhibition of Ala–Ala (Figure 1—figure supplement 3). This assay is also useful to study poor competitors of di-alanine for example, di-lysine where no competition could be observed previously (Solcan et al., 2012). Using this assay, we can now observe uptake of this substrate indirectly by monitoring the coupled proton movement (Figure 1C).

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Since PepT_St is an electrogenic transporter, we predict that imposition of a membrane potential in the presence of a pH gradient should drive uphill substrate transport. By loading the liposomes with high concentration of peptide and imposing a large hyperpolarising membrane potential (negative inside), we observe acidification of the lumen, indicating that the voltage can drive PepT_St-mediated transport against a 100-fold peptide gradient (Figure 2A). Importantly, we can also manipulate this system to see protons leaving the liposomal lumen as would be expected under an oppositely orientated membrane potential (positive inside). With an assay system set up where we could drive transport in predicted directions, we were now in a position to assess proton:peptide stoichiometry by measuring the equilibrium potential for proton flux using pyranine at a series of membrane voltages, set at the start of the assay with the appropriate potassium ion concentration gradient and the addition of valinomycin. This type of assay was used previously to address the stoichiometry of a lysosomal Cl−/H+ antiporter, CLC-7 (Graves et al., 2008).

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We assume that PepT_St operates via the coupled mechanism, nH_out + mPep_out ⇔nH_in + mPep_in, where the relative stoichiometry of protons:peptide is n/m. For this coupled system, the equilibrium potential is defined as the voltage at which there is no net substrate flux. This voltage, also known as the reversal potential ($\Delta \Psi$), is independent of the reaction mechanism. Rather, it depends only on the concentrations of protons and substrate and on the coupling stoichiometry with the form: $\Delta \Psi$ = 60{[pH_in − pH_out] − m/n log ([Pep]_in/[Pep]_out)}, where m and n are the stoichiometric coefficients in the chemical reaction above and $\Delta \Psi$ is in mV (see derivation in Figure 2—figure supplement 1). For a given combination of pH and peptide gradients this equation predicts a voltage at which no net pH change will occur; voltages above and below that value should produce inward or outward proton flux, depending on the voltage. Conversely, if at a series of voltages (set using K⁺/valinomycin), we observe acidification/no flux/alkalinzation, we can derive the relative stoichiometry of PepT_St for protons and peptide. We performed such experiments for the neutral peptide, Ala-Ala-Ala with no net pH difference between the inside and outside of the liposome and a 100-fold peptide gradient (higher concentration inside) and observed an absence of proton flux at a membrane potential of−40 mV (inside negative) which corresponds to a 3:1 proton:peptide stoichiometry (Figure 3A and Figure 3—source data 1). In contrast, voltages corresponding to reversal potentials for stoichiometries of 2:1 and 4:1 produced clearly distinguishable inward and outward fluxes respectively, strongly pointing to a 3:1 stoichiometry for the transporter. A very different combination of proton and peptide gradients, where we now included a proton gradient, also (pH more acidic outside) gave the same stoichiometry of 3:1 for the same tri-alanine peptide (Figure 3B). We also obtained this three proton:peptide stoichiometry for a different tri-peptide substrate, Ala-Leu-Ala (Figure 3C).

Figure 3

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Figure 3—source data 1

Table showing the conditions used to calculate the stoichiometry of transport.

https://doi.org/10.7554/eLife.04273.010

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We went on to determine the proton:peptide stoichiometry of PepT_St when transporting di-peptide substrates. However, when we performed experiments with the same gradients as our initial tri-peptide measurements only now with the neutral peptide Ala–Ala, we still observed transport at a voltage of −40 mV, where before we saw no net proton flux for tri-alanine. Transport is still also occurring at −30 mV which under these conditions would correspond to a proton:peptide stoichiometry of 4:1 and where we saw proton influx for tri-alanine (Figure 4A). Therefore, surprisingly, the stoichiometry of protons to peptides in PepT_St appears to be different for tri-Ala as compared with di-Ala. Further experiments to try to pin down the number of protons being co-transported with di-alanine lead to slightly ambiguous results, as increasing proton:peptide stoichiometries predict decreasing increments in reversal potential. These are, in turn, harder to generate reproducibly with our valinomycin/K⁺ system. We performed experiments with voltages set at reversal potentials predicted for symport ratios of 5 and 6 protons:peptide and proton flux was minimal at −20 mV (6 protons) but it is hard to be able to fully distinguish. Importantly, the system can still be driven in the opposite direction with higher voltages (0 mV, Figure 4B). Regardless of whether the actual stoichiometry is 5 or 6 protons:peptide, these experiments strongly suggest that the stoichiometry is higher for di-peptides than for tri-peptides. We confirmed this higher ratio for di-peptide transport using the substrate Ala–Phe (Figure 4C). Again transport is clearly observed at a membrane potential at −30 mV, so the stoichiometry for di-peptide transport by PepT_St is greater than four protons, clearly different from that of tri-peptides.

Figure 4

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The reversal potential equation above predicts that if $\Delta \Psi$ = 0, then [Pep_in]/[Pep_out] = ([H⁺_out]/[H⁺_in])ⁿ, where n is the number of protons transported per peptide. Therefore, if our results are truly indicative of higher coupling ratios for di-peptides than for tri-peptides, the same proton electrochemical gradient should accumulate di-peptides to a higher steady-state concentration than tri-peptides. We tested this prediction by measuring the uptake of radiolabeled di- and tri-peptides in the presence of a fixed, 1-unit pH gradient (acid outside). As shown in Figure 5, we find dramatically higher uptake of the di-peptide in this gradient, conclusively supporting the idea that different length peptides couple to the proton gradient with differing stoichiometries.

Figure 5

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A different coupling stoichiometry for di- vs tri-peptides raises an interesting question of whether different amino-acid side chains within the transporter are required for di-peptide vs tri-peptide transport. PepT_St contains six-protonatable side chains within its binding site (Glu 22, 25, 299, 300, 400, and K126, Figure 6A). All of these with the exception of Glu299 are conserved across the PTR family from bacteria through to mammalian PepT1 and PepT2. Previous biochemical studies have shown that in PepT_St this non-conserved residue is likely to be involved in structural and/or stability features specific to this protein as mutation of this residue results in no expression of the protein. Glu400 and Lys126 are likely to form a salt bridge that stabilises the outward open confirmation of the transporter, a feature that would be conserved regardless of the substrate. Glu300 has been shown to interact with both a di-peptide and a tri-peptide substrate and therefore likely to be involved in the transport mechanism for both di- and tri-peptides and has been shown to be important for di-alanine transport previously (Solcan et al., 2012; Doki et al., 2013). This leaves Glu22 and Glu25 as candidates for differential effects on di- and tri-peptide transport. Previously these residues have been shown to be important for proton coupling for di-alanine, however, here we also found that mutating either of these residues to alanine yielded proteins unable to catalyse proton coupled tri-alanine transport (Figure 6B). Therefore, despite different proton:peptide stoichiometries are apparently required for di- and tri-alanine transport, all five protonatable side chains within the binding site of PepT_St are likely to be important for the transport mechanism.

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Author details

1. Joanne L Parker

   Department of Biochemistry, University of Oxford, Oxford, United Kingdom

   Contribution

   JLP, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

2. Joseph A Mindell

   Membrane Transport Biophysics Unit, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, United States

   Contribution

   JAM, Conception and design, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

3. Simon Newstead

   Department of Biochemistry, University of Oxford, Oxford, United Kingdom

   Contribution

   SN, Conception and design, Analysis and interpretation of data, Drafting or revising the article

   For correspondence

   simon.newstead@bioch.ox.ac.uk

   Competing interests

   The authors declare that no competing interests exist.

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