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

Version 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.

Metrics

  • 2,483
    Page views
  • 559
    Downloads
  • 26
    Citations

Article citation count generated by polling the highest count across the following sources: Crossref, PubMed Central, Scopus.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  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

Share this article

https://doi.org/10.7554/eLife.70504

Further reading

    1. Cell Biology
    Fabian Link, Alyssa Borges ... Markus Engstler
    Research Article

    Endocytosis is a common process observed in most eukaryotic cells, although its complexity varies among different organisms. In Trypanosoma brucei, the endocytic machinery is under special selective pressure because rapid membrane recycling is essential for immune evasion. This unicellular parasite effectively removes host antibodies from its cell surface through hydrodynamic drag and fast endocytic internalization. The entire process of membrane recycling occurs exclusively through the flagellar pocket, an extracellular organelle situated at the posterior pole of the spindle-shaped cell. The high-speed dynamics of membrane flux in trypanosomes do not seem compatible with the conventional concept of distinct compartments for early endosomes (EE), late endosomes (LE), and recycling endosomes (RE). To investigate the underlying structural basis for the remarkably fast membrane traffic in trypanosomes, we employed advanced techniques in light and electron microscopy to examine the three-dimensional architecture of the endosomal system. Our findings reveal that the endosomal system in trypanosomes exhibits a remarkably intricate structure. Instead of being compartmentalized, it constitutes a continuous membrane system, with specific functions of the endosome segregated into membrane subdomains enriched with classical markers for EE, LE, and RE. These membrane subdomains can partly overlap or are interspersed with areas that are negative for endosomal markers. This continuous endosome allows fast membrane flux by facilitated diffusion that is not slowed by multiple fission and fusion events.

    1. Cell Biology
    2. Neuroscience
    Haibin Yu, Dandan Liu ... Kai Yuan
    Research Article

    O-GlcNAcylation is a dynamic post-translational modification that diversifies the proteome. Its dysregulation is associated with neurological disorders that impair cognitive function, and yet identification of phenotype-relevant candidate substrates in a brain-region specific manner remains unfeasible. By combining an O-GlcNAc binding activity derived from Clostridium perfringens OGA (CpOGA) with TurboID proximity labeling in Drosophila, we developed an O-GlcNAcylation profiling tool that translates O-GlcNAc modification into biotin conjugation for tissue-specific candidate substrates enrichment. We mapped the O-GlcNAc interactome in major brain regions of Drosophila and found that components of the translational machinery, particularly ribosomal subunits, were abundantly O-GlcNAcylated in the mushroom body of Drosophila brain. Hypo-O-GlcNAcylation induced by ectopic expression of active CpOGA in the mushroom body decreased local translational activity, leading to olfactory learning deficits that could be rescued by dMyc overexpression-induced increase of protein synthesis. Our study provides a useful tool for future dissection of tissue-specific functions of O-GlcNAcylation in Drosophila, and suggests a possibility that O-GlcNAcylation impacts cognitive function via regulating regional translational activity in the brain.