Mitochondria-specific photoactivation to monitor local sphingosine metabolism and function

  1. Suihan Feng
  2. Takeshi Harayama
  3. Sylvie Montessuit
  4. Fabrice PA David
  5. Nicolas Winssinger
  6. Jean-Claude Martinou
  7. Howard Riezman  Is a corresponding author
  1. University of Geneva, Switzerland
  2. Ecole Polytechnique Fédérale de Lausanne, Switzerland

Abstract

Photoactivation ('uncaging') is a powerful approach for releasing bioactive small-molecules in living cells. Current uncaging methods are limited by the random distribution of caged molecules within cells. We have developed a mitochondria-specific photoactivation method, which permitted us to release free sphingosine inside mitochondria and thereafter monitor local sphingosine metabolism by lipidomics. Our results indicate that sphingosine was quickly phosphorylated into sphingosine 1-phosphate (S1P) driven by sphingosine kinases. In time-course studies, the mitochondria-specific uncaged sphingosine demonstrated distinct metabolic patterns compared to globally-released sphingosine, and did not induce calcium spikes. Our data provide direct evidence that sphingolipid metabolism and signaling are highly dependent on the subcellular location and opens up new possibilities to study the effects of lipid localization on signaling and metabolic fate.

Article and author information

Author details

  1. Suihan Feng

    Department of Biochemistry, University of Geneva, Geneva, Switzerland
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-9205-0050
  2. Takeshi Harayama

    Department of Biochemistry, University of Geneva, Geneva, Switzerland
    Competing interests
    The authors declare that no competing interests exist.
  3. Sylvie Montessuit

    Department of Cell Biology, University of Geneva, Geneva, Switzerland
    Competing interests
    The authors declare that no competing interests exist.
  4. Fabrice PA David

    Gene Expression Core Facility, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland
    Competing interests
    The authors declare that no competing interests exist.
  5. Nicolas Winssinger

    National Centre of Competence in Research (NCCR) in Chemical Biology, University of Geneva, Geneva, Switzerland
    Competing interests
    The authors declare that no competing interests exist.
  6. Jean-Claude Martinou

    Department of Cell Biology, University of Geneva, Geneva, Switzerland
    Competing interests
    The authors declare that no competing interests exist.
  7. Howard Riezman

    Department of Biochemistry, University of Geneva, Geneva, Switzerland
    For correspondence
    howard.riezman@unige.ch
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-4680-9422

Funding

Swiss National Science Foundation (CRSII3-154405)

  • Howard Riezman

National Centre for Competence in Research in Chemical Biology (51NF40-160589)

  • Nicolas Winssinger
  • Howard Riezman

Japan Society for the Promotion of Science

  • Takeshi Harayama

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

Reviewing Editor

  1. Suzanne R Pfeffer, Stanford University School of Medicine, United States

Ethics

Animal experimentation: All experimental procedures were performed according to guidelines provided by the Animal Welfare Act and Animal welfare ordinance, the Rectors' Conference of the Swiss Universities (CRUS) policy and the Swiss Academy of Medical Sciences / Swiss Academy of Sciences' Ethical Principles and Guidelines for Experiments on Animals, and were approved by the Geneva Cantonal Veterinary Authority (authorization number: 28038/GE86/16).

Version history

  1. Received: December 24, 2017
  2. Accepted: January 24, 2018
  3. Accepted Manuscript published: January 29, 2018 (version 1)
  4. Version of Record published: February 20, 2018 (version 2)

Copyright

© 2018, Feng 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

  • 3,652
    views
  • 699
    downloads
  • 58
    citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.

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. Suihan Feng
  2. Takeshi Harayama
  3. Sylvie Montessuit
  4. Fabrice PA David
  5. Nicolas Winssinger
  6. Jean-Claude Martinou
  7. Howard Riezman
(2018)
Mitochondria-specific photoactivation to monitor local sphingosine metabolism and function
eLife 7:e34555.
https://doi.org/10.7554/eLife.34555

Share this article

https://doi.org/10.7554/eLife.34555

Further reading

    1. Biochemistry and Chemical Biology
    2. Plant Biology
    Henning Mühlenbeck, Yuko Tsutsui ... Cyril Zipfel
    Research Article

    Transmembrane signaling by plant receptor kinases (RKs) has long been thought to involve reciprocal trans-phosphorylation of their intracellular kinase domains. The fact that many of these are pseudokinase domains, however, suggests that additional mechanisms must govern RK signaling activation. Non-catalytic signaling mechanisms of protein kinase domains have been described in metazoans, but information is scarce for plants. Recently, a non-catalytic function was reported for the leucine-rich repeat (LRR)-RK subfamily XIIa member EFR (elongation factor Tu receptor) and phosphorylation-dependent conformational changes were proposed to regulate signaling of RKs with non-RD kinase domains. Here, using EFR as a model, we describe a non-catalytic activation mechanism for LRR-RKs with non-RD kinase domains. EFR is an active kinase, but a kinase-dead variant retains the ability to enhance catalytic activity of its co-receptor kinase BAK1/SERK3 (brassinosteroid insensitive 1-associated kinase 1/somatic embryogenesis receptor kinase 3). Applying hydrogen-deuterium exchange mass spectrometry (HDX-MS) analysis and designing homology-based intragenic suppressor mutations, we provide evidence that the EFR kinase domain must adopt its active conformation in order to activate BAK1 allosterically, likely by supporting αC-helix positioning in BAK1. Our results suggest a conformational toggle model for signaling, in which BAK1 first phosphorylates EFR in the activation loop to stabilize its active conformation, allowing EFR in turn to allosterically activate BAK1.

    1. Biochemistry and Chemical Biology
    2. Cell Biology
    Ya-Juan Wang, Xiao-Jing Di ... Ting-Wei Mu
    Research Article Updated

    Protein homeostasis (proteostasis) deficiency is an important contributing factor to neurological and metabolic diseases. However, how the proteostasis network orchestrates the folding and assembly of multi-subunit membrane proteins is poorly understood. Previous proteomics studies identified Hsp47 (Gene: SERPINH1), a heat shock protein in the endoplasmic reticulum lumen, as the most enriched interacting chaperone for gamma-aminobutyric acid type A (GABAA) receptors. Here, we show that Hsp47 enhances the functional surface expression of GABAA receptors in rat neurons and human HEK293T cells. Furthermore, molecular mechanism study demonstrates that Hsp47 acts after BiP (Gene: HSPA5) and preferentially binds the folded conformation of GABAA receptors without inducing the unfolded protein response in HEK293T cells. Therefore, Hsp47 promotes the subunit-subunit interaction, the receptor assembly process, and the anterograde trafficking of GABAA receptors. Overexpressing Hsp47 is sufficient to correct the surface expression and function of epilepsy-associated GABAA receptor variants in HEK293T cells. Hsp47 also promotes the surface trafficking of other Cys-loop receptors, including nicotinic acetylcholine receptors and serotonin type 3 receptors in HEK293T cells. Therefore, in addition to its known function as a collagen chaperone, this work establishes that Hsp47 plays a critical and general role in the maturation of multi-subunit Cys-loop neuroreceptors.