In the Hedgehog signaling pathway cholesterol is required to

In the Hedgehog signaling pathway, cholesterol is required to activate SMO (Cooper et al., 2003), the downstream target of PTCH1. A previous study (Bidet et al., 2011) reported that Patched expression in yeast AZD3514 increased extrusion of a fluorescent boron dipyromethanene sterol derivative (BODIPY-cholesterol) into the external medium by 2-fold. Interpretation of this study, however, is unclear because significant amounts of BODIPY-cholesterol were spontaneously released into the medium without PTCH1 expression, whereas spontaneous release of cholesterol into aqueous medium is implausible without an acceptor. Our direct measurements of endogenous cholesterol in the membrane and the presence in our PTCH1 structure of cholesterol-like densities within and at either end of a hydrophobic conduit together constitute strong evidence that PTCH1 may function in cholesterol transport. Nevertheless, some questions remain. Although PTCH1 appears to alter inner leaflet cholesterol in cells, the extra density present in the transmembrane domain is located at the level of the outer leaflet of the membrane. This observation may be accounted for by different dwell times of substrate along the transport path. Inner leaflet cholesterol may move outward along a shallow groove present between TM1 and TM2 but remain longer at the level of the outer leaflet because of higher affinity for that site prior to entry into the extracellular conduit. In addition, cholesterol is insoluble in water and would require a sink for export. HpnN delivers lipidic substrates directly to the outer membrane, and NPC1 apparently receives cholesterol from NPC2, a lipid-carrying partner. Because eukaryotic cells lack an outer membrane, PTCH1 export of a cholesterol-like lipid may require a lipid carrier as the sink, suggesting the possible existence of an unidentified lipid carrier protein that partners with PTCH1. Finally, it seems unlikely that PTCH1, under physiological conditions, would alter the cholesterol distribution of the entire plasma membrane because PTCH1 is normally localized to the primary cilium (Corbit et al., 2005, Kim et al., 2015, Rohatgi et al., 2007). Given the presence of a distinct ionic and membrane environment in the primary cilium (Chávez et al., 2015, DeCaen et al., 2013, Garcia-Gonzalo et al., 2015, Raleigh et al., 2018), PTCH1 activity may differ from that in the plasma membrane; more refined measurements might be possible with future availability of a sensor for ciliary membrane cholesterol. PTCH1 clearly affects IPM cholesterol activity, but we have not yet biochemically reconstituted cholesterol transport, which may require incorporation of an unidentified cholesterol acceptor. Nevertheless, our data limit the conceivable modes of indirect PTCH1 action on cholesterol, leaving direct action as the simplest explanation of our findings. Reversal of PTCH1-mediated reduction in IPM cholesterol activity begins immediately and is completed within minutes of ShhN addition. PTCH1-regulated transcriptional and translational events are unlikely to generate a sufficient effect to account for the observed change within this time frame. In addition, the NNQ and IVL residues altered in inactive mutated forms of PTCH1 are internal, making their participation in an interaction between PTCH1 and a protein partner unlikely. PTCH1 action on a non-cholesterol lipid, however, remains a possibility because the change in our sensor readout may arise from a change in availability of cholesterol through indirect effects on a lipid that forms a complex with cholesterol (Das et al., 2014). Given the sterol-like densities present in and near the essential hydrophobic conduit in our PTCH1 structure, however, the PTCH1 substrate seems likely to be a sterol, and cholesterol redistribution is the most parsimonious interpretation of our observations that links PTCH1 activity to SMO regulation.