Department of Biochemistry and Medicine, Stanford University School of Medicine, Stanford, United States.
Department of Physiological Chemistry, Genentech, South San Francisco, United States.
Elife. 2021 Oct 26;10:e70504. doi: 10.7554/eLife.70504.
A long-standing mystery in vertebrate Hedgehog signaling is how Patched 1 (PTCH1), the receptor for Hedgehog ligands, inhibits the activity of Smoothened, the protein that transmits the signal across the membrane. We previously proposed (Kinnebrew et al., 2019) that PTCH1 inhibits Smoothened by depleting accessible cholesterol from the ciliary membrane. Using a new imaging-based assay to directly measure the transport activity of PTCH1, we find that PTCH1 depletes accessible cholesterol from the outer leaflet of the plasma membrane. This transport activity is terminated by binding of Hedgehog ligands to PTCH1 or by dissipation of the transmembrane potassium gradient. These results point to the unexpected model that PTCH1 moves cholesterol from the outer to the inner leaflet of the membrane in exchange for potassium ion export in the opposite direction. Our study provides a plausible solution for how PTCH1 inhibits SMO by changing the organization of cholesterol in membranes and establishes a general framework for studying how proteins change cholesterol accessibility to regulate membrane-dependent processes in cells.
脊椎动物 Hedgehog 信号通路中存在一个长期未解之谜，即 Hedgehog 配体的受体 Patched 1（PTCH1）如何抑制跨膜信号转导蛋白 Smoothened 的活性。我们之前提出（Kinnebrew 等人，2019），PTCH1 通过从纤毛膜中耗尽可及胆固醇来抑制 Smoothened。我们使用一种新的基于成像的测定法来直接测量 PTCH1 的转运活性，发现 PTCH1 从质膜的外叶耗尽可及胆固醇。这种转运活性通过 Hedgehog 配体与 PTCH1 的结合或跨膜钾梯度的耗散而终止。这些结果表明了一个出乎意料的模型，即 PTCH1 通过将胆固醇从膜的外叶转移到内叶，以交换钾离子的反向输出，从而移动胆固醇。我们的研究为 PTCH1 如何通过改变膜中胆固醇的组织来抑制 SMO 提供了一个合理的解决方案，并为研究蛋白质如何改变胆固醇的可及性以调节细胞中依赖膜的过程建立了一个通用框架。
