Extended Data Fig. 1: Structurally related transporters Dispatched and Patched in Hedgehog signalling.

a, b, Opposing functions of Dispatched and Patched in Hedgehog signalling. a, The HH protein signal, covalently modified by cholesterol and palmitate, requires the action of DISP1 and SCUBE for release from the membrane of producing cells. HH then uses its palmitoyl adduct to clog the sterol transport conduit and block the function of its receptor PTCH1 in responding cells. The loss of PTCH1 sterol transport activity permits accumulation of cholesterol within the inner leaflet to levels that activate SMO by binding within its seven transmembrane helix bundle, resulting in activation of the GLI transcriptional effector of Hedgehog signalling. b, As Ptch1 is a target for GLI activation, the X-Gal staining of a Ptch1^LacZ knock-in allele⁵³ provides an indication of Hedgehog pathway activity (leftmost embryo). Homozygous mutation of Disp1^6,7,8,9,10 causes a loss of nearly all embryonic Hedgehog pathway activity (2^nd embryo from left)⁵⁴, whereas homozygous disruption of Ptch1 leads to unregulated ectopic pathway activity, regardless of the functional status of Disp1 (rightmost two embryos)⁵³. c–f, Truncated DISP1 protein. As murine full-length DISP1 protein was poorly expressed in HEK293 cells, we tested a variety of constructs, and ultimately settled on an N-terminal truncation. Although its export activity was partially reduced, truncated DISP1-A protein nevertheless mediated efficient release of ShhNp (autoprocessed, lipid-modified Shh protein) into cell culture medium containing SCUBE2 (mouse SCUBE2, lacking amino acids 30-281) upon transfection into Disp^-/- mouse embryonic fibroblasts^6,20 (MEFs), indicating preservation of its function. c, DISP1-mEGFP and DISP1-A-mEGFP in HEK293T cells with magnified insets showing expression on the cell membrane. d, Western blot showing high level of DISP1-A expression relative to DISP1 (both proteins were SBP and HA-tagged at the C-terminus). e, Functional assay (Methods, ShhNp release endpoint assay) in Disp^-/- MEFs. Culture media and cell lysates from transiently transfected Disp^-/- MEFs were probed by immunoblotting for expression of DISP-mEGFP (SBP, HA and mEGFP tagged at the C-terminus, see panel c), ShhNp, and SCUBE2. DISP1 and DISP1-A both released ShhNp in the presence of SCUBE2. β-ACTIN, loading control. f, Size exclusion chromatography of purified DISP1-A, together with the SDS-PAGE of the indicated fraction, corresponding to monomeric DISP1-A. Panels c–f, show representative results (n = 4 biologically independent replicates). g, Structural comparison between DISP1-A and mouse PTCH1⁵⁵ (PDB ID: 7K65). The ECDs, closely apposed in PTCH1 to form a conduit for sterol transport, in DISP1-A are splayed apart. Conserved ferredoxin-like α + β open-faced sandwich folds are highlighted (lime for ECD1 and pink for ECD2). Magnified views in spectral sequence from N to C termini of the α + β open-faced sandwich folds in DISP1-A ECD1 (bottom left) and ECD2 (bottom right). Distal structures inserted into the peripheral loops of the ferredoxin-like folds are structurally unrelated to each other or to distal PTCH1 ECD structures. Red symbols indicate the five N-linked glycosylation sites, three in ECD1 (N362, N390 and N475) and two in ECD2 (N834 and N915), which can be inferred from additional densities that extend from N-X-S/T sequences in the extracellular loops. Panels d, e, f, see Supplementary Fig. 1 for gel source data.