Distinct Cation Gradients Power Cholesterol Transport at Different Key Points in the Hedgehog Signaling Pathway

Cholesterol plays two critical roles in Hedgehog signaling, a fundamental pathway in animal development and cancer: it covalently modifies the Sonic hedgehog (SHH) ligand, restricting its release from producing cells, and directly activates Smoothened in responding cells. In both contexts, a membrane protein related to bacterial RND transporters regulates cholesterol: Dispatched1 controls release of cholesterylated SHH, and Patched1 antagonizes Smoothened activation by cholesterol. The mechanism and driving force for eukaryotic RND proteins, including Dispatched1 and Patched1, are unknown. Here, we show that Dispatched1 acts enzymatically to catalyze SHH release. Dispatched1 uses the energy of the plasma membrane Na⁺ gradient, thus functioning as an SHH/Na⁺ antiporter. In contrast, Patched1 repression of Smoothened requires the opposing K⁺ gradient. Our results clarify the transporter activity of essential eukaryotic RND proteins and demonstrate that the two main cation gradients of animal cells differentially power cholesterol transport at two crucial steps in the Hedgehog pathway.