1. Biochemistry and Chemical Biology
  2. Cell Biology
Download icon

Quantitative proteomics reveals the selectivity of ubiquitin-binding autophagy receptors in the turnover of damaged lysosomes by lysophagy

  1. Vinay V Eapen
  2. Sharan Swarup
  3. Melissa J Hoyer
  4. Joao A Paulo
  5. Wade Harper  Is a corresponding author
  1. Harvard Medical School, United States
Research Article
  • Cited 1
  • Views 1,776
  • Annotations
Cite this article as: eLife 2021;10:e72328 doi: 10.7554/eLife.72328

Abstract

Removal of damaged organelles via the process of selective autophagy constitutes a major form of cellular quality control. Damaged organelles are recognized by a dedicated surveillance machinery, leading to the assembly of an autophagosome around the damaged organelle, prior to fusion with the degradative lysosomal compartment. Lysosomes themselves are also prone to damage and are degraded through the process of lysophagy. While early steps involve recognition of ruptured lysosomal membranes by glycan-binding Galectins and ubiquitylation of transmembrane lysosomal proteins, many steps in the process, and their inter-relationships, remain poorly understood, including the role and identity of cargo receptors required for completion of lysophagy. Here, we employ quantitative organelle capture and proximity biotinylation proteomics of autophagy adaptors, cargo receptors, and Galectins in response to acute lysosomal damage, thereby revealing the landscape of lysosome-associated proteome remodeling during lysophagy. Among proteins dynamically recruited to damaged lysosomes were ubiquitin-binding autophagic cargo receptors. Using newly developed lysophagic flux reporters including Lyso-Keima, we demonstrate that TAX1BP1, together with its associated kinase TBK1, are both necessary and sufficient to promote lysophagic flux in both HeLa cells and induced neurons (iNeurons). While the related receptor OPTN can drive damage-dependent lysophagy when overexpressed, cells lacking either OPTN or CALCOCO2 still maintain significant lysophagic flux in HeLa cells. Mechanistically, TAX1BP1-driven lysophagy requires its N-terminal SKICH domain, which binds both TBK1 and the autophagy regulatory factor RB1CC1, and requires upstream ubiquitylation events for efficient recruitment and lysophagic flux. These results identify TAX1BP1 as a central component in the lysophagy pathway and provide a proteomic resource for future studies of the lysophagy process.

Data availability

All proteomic .RAW files have been deposited in the PRIDE component of Proteome xchange with the identifier PXDO27476, and will be released upon publication

The following data sets were generated

Article and author information

Author details

  1. Vinay V Eapen

    Harvard Medical School, Boston, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-8023-387X
  2. Sharan Swarup

    Harvard Medical School, Boston, United States
    Competing interests
    No competing interests declared.
  3. Melissa J Hoyer

    Harvard Medical School, Boston, United States
    Competing interests
    No competing interests declared.
  4. Joao A Paulo

    Harvard Medical School, Boston, United States
    Competing interests
    No competing interests declared.
  5. Wade Harper

    Harvard Medical School, Boston, United States
    For correspondence
    wade_harper@hms.harvard.edu
    Competing interests
    Wade Harper, J.W.H. is a consultant and founder of Caraway Therapeutics and a founding scientific advisory board member of Interline Therapeutics.Reviewing editor, eLife.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-6944-7236

Funding

aligning science across parkinsons (ASAP-000282)

  • Wade Harper

National Institutes of Health (NS083524)

  • Wade Harper

National Institutes of Health (NS110395)

  • Wade Harper

National Institutes of Health (DK098285)

  • Joao A Paulo

Jane Coffin Childs Memorial Fund for Medical Research

  • Melissa J Hoyer

Jane Coffin Childs Memorial Fund for Medical Research

  • Vinay V Eapen

Canadian Institutes of Health Research

  • Sharan Swarup

Ned Goodnow Fund

  • Wade Harper

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

Reviewing Editor

  1. Ivan Dikic, Institute of Biochemistry II, Germany

Publication history

  1. Preprint posted: July 19, 2021 (view preprint)
  2. Received: July 20, 2021
  3. Accepted: September 25, 2021
  4. Accepted Manuscript published: September 29, 2021 (version 1)
  5. Version of Record published: October 18, 2021 (version 2)

Copyright

© 2021, Eapen 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

  • 1,776
    Page views
  • 341
    Downloads
  • 1
    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)

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

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

Further reading

    1. Biochemistry and Chemical Biology
    Astrid Kollewe et al.
    Research Article Updated

    The transient receptor potential melastatin-subfamily member 7 (TRPM7) is a ubiquitously expressed membrane protein consisting of ion channel and protein kinase domains. TRPM7 plays a fundamental role in the cellular uptake of divalent cations such as Zn2+, Mg2+, and Ca2+, and thus shapes cellular excitability, plasticity, and metabolic activity. The molecular appearance and operation of TRPM7 channels in native tissues have remained unresolved. Here, we investigated the subunit composition of endogenous TRPM7 channels in rodent brain by multi-epitope affinity purification and high-resolution quantitative mass spectrometry (MS) analysis. We found that native TRPM7 channels are high-molecular-weight multi-protein complexes that contain the putative metal transporter proteins CNNM1-4 and a small G-protein ADP-ribosylation factor-like protein 15 (ARL15). Heterologous reconstitution experiments confirmed the formation of TRPM7/CNNM/ARL15 ternary complexes and indicated that complex formation effectively and specifically impacts TRPM7 activity. These results open up new avenues towards a mechanistic understanding of the cellular regulation and function of TRPM7 channels.

    1. Biochemistry and Chemical Biology
    2. Chromosomes and Gene Expression
    Fang Huang et al.
    Research Article

    The positive transcription elongation factor b (P-TEFb) is a critical co-activator for transcription of most cellular and viral genes, including those of HIV. While P-TEFb is regulated by 7SK snRNA in proliferating cells, P-TEFb is absent due to diminished levels of CycT1 in quiescent and terminally differentiated cells, which has remained unexplored. In these cells, we found that CycT1 not bound to CDK9 is rapidly degraded. Moreover, productive CycT1:CDK9 interactions are increased by PKC mediated phosphorylation of CycT1 in human cells. Conversely, dephosphorylation of CycT1 by PP1 reverses this process. Thus, PKC inhibitors or removal of PKC by chronic activation results in P-TEFb disassembly and CycT1 degradation. This finding not only recapitulates P-TEFb depletion in resting CD4+ T cells but also in anergic T cells. Importantly, our studies reveal mechanisms of P-TEFb inactivation underlying T cell quiescence, anergy, and exhaustion as well as proviral latency and terminally differentiated cells.