Oligomerization of p62 allows for selection of ubiquitinated cargo and isolation membrane during selective autophagy

Abstract

Autophagy is a major pathway for the clearance of harmful material from the cytoplasm. During autophagy cytoplasmic material is delivered into the lysosomal system by organelles called autophagosomes. Autophagosomes form in a de novo manner and, in the course of their formation, isolate cargo material from the rest of the cytoplasm. Cargo specificity is conferred by autophagic cargo receptors that selectively link the cargo to the autophagosomal membrane decorated with ATG8 family proteins such as LC3B. Here we show that the human cargo receptor p62/SQSTM-1 employs oligomerization to stabilize its interaction with LC3B and linear ubiquitin when they are clustered on surfaces. Thus, oligomerization enables p62 to simultaneously select for the isolation membrane and the ubiquitinated cargo. We further show in a fully reconstituted system that the interaction of p62 with ubiquitin and LC3B is sufficient to bend the membrane around the cargo.

Article and author information

Author details

  1. Bettina Wurzer

    Max F. Perutz Laboratories, University of Vienna, Vienna, Austria
    Competing interests
    The authors declare that no competing interests exist.
  2. Gabriele Zaffagnini

    Max F. Perutz Laboratories, University of Vienna, Vienna, Austria
    Competing interests
    The authors declare that no competing interests exist.
  3. Dorotea Fracchiolla

    Max F. Perutz Laboratories, University of Vienna, Vienna, Austria
    Competing interests
    The authors declare that no competing interests exist.
  4. Eleonora Turco

    Max F. Perutz Laboratories, University of Vienna, Vienna, Austria
    Competing interests
    The authors declare that no competing interests exist.
  5. Christine Abert

    Max F. Perutz Laboratories, University of Vienna, Vienna, Austria
    Competing interests
    The authors declare that no competing interests exist.
  6. Julia Romanov

    Max F. Perutz Laboratories, University of Vienna, Vienna, Austria
    Competing interests
    The authors declare that no competing interests exist.
  7. Sascha Martens

    Max F. Perutz Laboratories, University of Vienna, Vienna, Austria
    For correspondence
    sascha.martens@univie.ac.at
    Competing interests
    The authors declare that no competing interests exist.

Reviewing Editor

  1. Randy Schekman, Howard Hughes Medical Institute, University of California, Berkeley, United States

Version history

  1. Received: May 24, 2015
  2. Accepted: September 26, 2015
  3. Accepted Manuscript published: September 28, 2015 (version 1)
  4. Version of Record published: November 30, 2015 (version 2)

Copyright

© 2015, Wurzer 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

  • 6,307
    views
  • 1,792
    downloads
  • 192
    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. Bettina Wurzer
  2. Gabriele Zaffagnini
  3. Dorotea Fracchiolla
  4. Eleonora Turco
  5. Christine Abert
  6. Julia Romanov
  7. Sascha Martens
(2015)
Oligomerization of p62 allows for selection of ubiquitinated cargo and isolation membrane during selective autophagy
eLife 4:e08941.
https://doi.org/10.7554/eLife.08941

Share this article

https://doi.org/10.7554/eLife.08941

Further reading

    1. Biochemistry and Chemical Biology
    2. Structural Biology and Molecular Biophysics
    Marian Brenner, Christoph Zink ... Antje Gohla
    Research Article

    Vitamin B6 deficiency has been linked to cognitive impairment in human brain disorders for decades. Still, the molecular mechanisms linking vitamin B6 to these pathologies remain poorly understood, and whether vitamin B6 supplementation improves cognition is unclear as well. Pyridoxal 5’-phosphate phosphatase (PDXP), an enzyme that controls levels of pyridoxal 5’-phosphate (PLP), the co-enzymatically active form of vitamin B6, may represent an alternative therapeutic entry point into vitamin B6-associated pathologies. However, pharmacological PDXP inhibitors to test this concept are lacking. We now identify a PDXP and age-dependent decline of PLP levels in the murine hippocampus that provides a rationale for the development of PDXP inhibitors. Using a combination of small-molecule screening, protein crystallography, and biolayer interferometry, we discover, visualize, and analyze 7,8-dihydroxyflavone (7,8-DHF) as a direct and potent PDXP inhibitor. 7,8-DHF binds and reversibly inhibits PDXP with low micromolar affinity and sub-micromolar potency. In mouse hippocampal neurons, 7,8-DHF increases PLP in a PDXP-dependent manner. These findings validate PDXP as a druggable target. Of note, 7,8-DHF is a well-studied molecule in brain disorder models, although its mechanism of action is actively debated. Our discovery of 7,8-DHF as a PDXP inhibitor offers novel mechanistic insights into the controversy surrounding 7,8-DHF-mediated effects in the brain.

    1. Biochemistry and Chemical Biology
    2. Stem Cells and Regenerative Medicine
    Parthasarathy Sampathkumar, Heekyung Jung ... Yang Li
    Research Article

    Molecules that facilitate targeted protein degradation (TPD) offer great promise as novel therapeutics. The human hepatic lectin asialoglycoprotein receptor (ASGR) is selectively expressed on hepatocytes. We have previously engineered an anti-ASGR1 antibody-mutant RSPO2 (RSPO2RA) fusion protein (called SWEETS) to drive tissue-specific degradation of ZNRF3/RNF43 E3 ubiquitin ligases, which achieved hepatocyte-specific enhanced Wnt signaling, proliferation, and restored liver function in mouse models, and an antibody–RSPO2RA fusion molecule is currently in human clinical trials. In the current study, we identified two new ASGR1- and ASGR1/2-specific antibodies, 8M24 and 8G8. High-resolution crystal structures of ASGR1:8M24 and ASGR2:8G8 complexes revealed that these antibodies bind to distinct epitopes on opposing sides of ASGR, away from the substrate-binding site. Both antibodies enhanced Wnt activity when assembled as SWEETS molecules with RSPO2RA through specific effects sequestering E3 ligases. In addition, 8M24-RSPO2RA and 8G8-RSPO2RA efficiently downregulate ASGR1 through TPD mechanisms. These results demonstrate the possibility of combining different therapeutic effects and degradation mechanisms in a single molecule.