Patched 1 reduces the accessibility of cholesterol in the outer leaflet of membranes

  1. Maia Kinnebrew
  2. Giovanni Luchetti
  3. Ria Sircar
  4. Sara Frigui
  5. Lucrezia Vittoria Viti
  6. Tomoki Naito
  7. Francis Beckert
  8. Yasunori Saheki
  9. Christian Siebold
  10. Arun Radhakrishnan
  11. Rajat Rohatgi  Is a corresponding author
  1. Stanford University School of Medicine, United States
  2. Oxford University, United Kingdom
  3. Nanyang Technological University, Singapore
  4. University of Texas Southwestern Medical Center, United States

Abstract

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. To directly test the effect of PTCH1 on accessible cholesterol, we measured the transport activity of PTCH1 using an imaging-based assay to follow the kinetics of cholesterol extraction from the plasma membrane of live cells by methyl-β-cyclodextrin. PTCH1 depletes accessible cholesterol in the outer leaflet of the membrane in a manner regulated by its ligand Sonic Hedgehog and the transmembrane potassium gradient. We propose that PTCH1 moves cholesterol from the outer to the inner leaflet of the membrane in exchange for potassium ion export. Our results show that proteins can change accessible cholesterol levels in membranes to regulate signaling reaction.

Data availability

No dataset was generated or used during this study (such as deep sequencing data, mass spectrometry data, structural coordinates or maps, genetic data or clinical trial data) that required deposition in a repository such as GenBank, the PDB, mass spec data repositories, or clinical data repositories. We have provided original, uncropped scans of immunoblots shown in Figures 2B, 4B, and Figure 3-figure supplement 1 in the Source Data Files. All other data generated are included in this study, with replicates and statistics described in the figure legends and methods.

Article and author information

Author details

  1. Maia Kinnebrew

    Department of Biochemistry, Stanford University School of Medicine, Stanford, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-7344-8231
  2. Giovanni Luchetti

    Department of Biochemistry, Stanford University School of Medicine, Stanford, United States
    Competing interests
    No competing interests declared.
  3. Ria Sircar

    Department of Biochemistry, Stanford University School of Medicine, Stanford, United States
    Competing interests
    No competing interests declared.
  4. Sara Frigui

    Department of Biochemistry, Stanford University School of Medicine, Stanford, United States
    Competing interests
    No competing interests declared.
  5. Lucrezia Vittoria Viti

    Oxford University, Oxford, United Kingdom
    Competing interests
    No competing interests declared.
  6. Tomoki Naito

    Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore, Singapore
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-8393-3601
  7. Francis Beckert

    Department of Biochemistry, Stanford University School of Medicine, Stanford, United States
    Competing interests
    No competing interests declared.
  8. Yasunori Saheki

    Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore, Singapore
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-1229-6668
  9. Christian Siebold

    Oxford University, Oxford, United Kingdom
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-6635-3621
  10. Arun Radhakrishnan

    Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, United States
    Competing interests
    Arun Radhakrishnan, Arun Radhakrishnan is a reviewing editor for eLife..
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-7266-7336
  11. Rajat Rohatgi

    Department of Biochemistry, Stanford University School of Medicine, Stanford, United States
    For correspondence
    rrohatgi@stanford.edu
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-7609-8858

Funding

Cancer Research UK (C20724)

  • Christian Siebold

Ministry of Education, Singapore (MOE-T2EP30120-0002)

  • Yasunori Saheki

National Science Foundation (Predoctoral Fellowship)

  • Maia Kinnebrew

Ford Foundation (Predoctoral Fellowship)

  • Giovanni Luchetti

Cancer Research UK (A26752)

  • Christian Siebold

European Research Council (647278)

  • Christian Siebold

National Institutes of Health (GM118082)

  • Rajat Rohatgi

National Institutes of Health (GM106078)

  • Rajat Rohatgi

National Institutes of Health (HL20948)

  • Arun Radhakrishnan

Welch Foundation (I-1793)

  • Arun Radhakrishnan

Leducq Foundation (19CVD04)

  • Arun Radhakrishnan

Ministry of Education, Singapore (MOE2017-T2-2-001)

  • Yasunori Saheki

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

  1. Duojia Pan, UT Southwestern Medical Center and HHMI, United States

Publication history

  1. Received: May 27, 2021
  2. Accepted: October 25, 2021
  3. Accepted Manuscript published: October 26, 2021 (version 1)
  4. Version of Record published: December 8, 2021 (version 2)

Copyright

© 2021, Kinnebrew et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

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  1. Maia Kinnebrew
  2. Giovanni Luchetti
  3. Ria Sircar
  4. Sara Frigui
  5. Lucrezia Vittoria Viti
  6. Tomoki Naito
  7. Francis Beckert
  8. Yasunori Saheki
  9. Christian Siebold
  10. Arun Radhakrishnan
  11. Rajat Rohatgi
(2021)
Patched 1 reduces the accessibility of cholesterol in the outer leaflet of membranes
eLife 10:e70504.
https://doi.org/10.7554/eLife.70504

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