The solute carrier SPNS2 recruits PI(4,5)P-2 to synergistically regulate transport of sphingosine-1-phosphate

Solute carrier spinster homolog 2 (SPNS2), one of only four known major facilitator superfamily (MFS) lyso-lipid transporters in humans, exports sphingosine-1-phosphate (S1P) across cell membranes. Here, we explore the synergistic effects of lipid binding and conformational dynamics on SPNS2's transport mecha-nism. Using mass spectrometry, we discovered that SPNS2 interacts preferentially with PI(4,5)P2. Together with functional studies and molecular dynamics (MD) simulations, we identified potential PI(4,5)P2 binding sites. Mutagenesis of proposed lipid binding sites and inhibition of PI(4,5)P2 synthesis reduce S1P transport, whereas the absence of the N terminus renders the transporter essentially inactive. Probing the conforma-tional dynamics of SPNS2, we show how synergistic binding of PI(4,5)P2 and S1P facilitates transport, in-creases dynamics of the extracellular gate, and stabilizes the intracellular gate. Given that SPNS2 transports a key signaling lipid, our results have implications for therapeutic targeting and also illustrate a regulatory mechanism for MFS transporters.