Dispatched uses Na.sup.+ flux to power release of lipid-modified Hedgehog

The Dispatched protein, which is related to the NPC1 and PTCH1 cholesterol transporters.sup.1,2 and to H.sup.+-driven transporters of the RND family.sup.3,4, enables tissue-patterning activity of the lipid-modified Hedgehog protein by releasing it from tightly -localized sites of embryonic expression.sup.5-10. Here we determine a cryo-electron microscopy structure of the mouse protein Dispatched homologue 1 (DISP1), revealing three Na.sup.+ ions coordinated within a channel that traverses its transmembrane domain. We find that the rate of Hedgehog export is dependent on the Na.sup.+ gradient across the plasma membrane. The transmembrane channel and Na.sup.+ binding are disrupted in DISP1-NNN, a variant with asparagine substitutions for three intramembrane aspartate residues that each coordinate and neutralize the charge of one of the three Na.sup.+ ions. DISP1-NNN and variants that disrupt single Na.sup.+ sites retain binding to, but are impaired in export of the lipid-modified Hedgehog protein to the SCUBE2 acceptor. Interaction of the amino-terminal signalling domain of the Sonic hedgehog protein (ShhN) with DISP1 occurs via an extensive buried surface area and contacts with an extended furin-cleaved DISP1 arm. Variability analysis reveals that ShhN binding is restricted to one extreme of a continuous series of DISP1 conformations. The bound and unbound DISP1 conformations display distinct Na.sup.+-site occupancies, which suggests a mechanism by which transmembrane Na.sup.+ flux may power extraction of the lipid-linked Hedgehog signal from the membrane. Na.sup.+-coordinating residues in DISP1 are conserved in PTCH1 and other metazoan RND family members, suggesting that Na.sup.+ flux powers their conformationally driven activities.