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Solute carrier spinster homolog 2 (SPNS2), one of only four known major facilitator superfamily (MFS) lysolipid 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 mechanism. 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 conformational dynamics of SPNS2, we show how synergistic binding of PI(4,5)P₂ and S1P facilitates transport, increases dynamics of the extracellular gate, and stabilizes the intracellular …