Organisms must accumulate scarce nutrients from their environments to survive. This often involves moving molecules up a concentration gradient, which is entropically unfavorable. Active transport proteins embedded in cell membranes harness electrochemical energy to catalyze this “uphill” movement of molecules. Symporters are active transporters that simultaneously transport in the same direction both a primary substrate (such as a desired nutrient) and one or more abundant ions (such as sodium) down its concentration gradient. A canonical symporter works by an alternating-access process, exposing its substrate binding sites to only one side of the membrane at a time. Importantly, a canonical symporter only interconverts between its outward-open and inward-open states when it is either empty or fully loaded with both substrates. The transport of the two substrates thus occurs at a fixed ratio and the substrates are said to be “coupled.” In this model all steps are readily reversible, and the concentration gradients of the substrates determine the net direction of transport (Figure, left panel).
We study transporters from the Nramp family, which import transition metal ions like iron and manganese into cells. These proteins also transport protons, which led researchers to propose that Nramps are symporters that use a favorable proton gradient to power uphill metal ion movements. We recently determined high-resolution structures of an Nramp transporter (Bozzi & Zimanyi et al., eLife, 2019) and identified the conserved amino acids that form the substrate binding sites. Strikingly, while protons and metal ions transit the same vestibule in the outward-open state to reach a binding site in the middle of the protein, they take separate parallel pathways through the inside half of the protein. These separate pathways may have evolved because both substrates are positively charged and therefore liable to repel each other if constrained to the same binding site during the transport process.
In this work we show that unlike canonical symporters the proton and metal transport are not tightly coupled in Nramps. Proton transport can occur readily without accompanying metals. In addition, protons can co-transport with some metals like iron and manganese, but not with others like cadmium (a toxic non-physiological substrate).
We find that Nramps have evolved unique features to favor metal import and prevent export regardless of the thermodynamic driving forces. They achieve that by being highly sensitive to pH and membrane potential such that metal import is most efficient under physiological conditions due to a conserved network of polar residues that prevents the deleterious back-transport of intracellular metals (Figure, right panel).
This work describes a mechanistic model for Nramp metal and proton transport that deviates from the canonical symport. While the mechanism may be specific to Nramps, it may be relevant to understanding other non-canonical transport proteins.