Structural basis for membrane recruitment of ATG16L1 by WIPI2 in Autophagy

  1. Lisa M Strong
  2. Chunmei Chang
  3. Julia F Riley
  4. C Alexander Boecker
  5. Thomas G Flower
  6. Cosmo Z Buffalo
  7. Xuefeng Ren
  8. Andrea KH Stavoe
  9. Erika LF Holzbaur
  10. James H Hurley  Is a corresponding author
  1. University of California, Berkeley, United States
  2. University of Pennsylvania, United States
  3. The University of Texas Health Science Center at Houston McGovern Medical School, United States

Abstract

Autophagy is a cellular process that degrades cytoplasmic cargo by engulfing it in a double membrane vesicle, known as the autophagosome, and delivering it to the lysosome. The ATG12-5-16L1 complex is responsible for conjugating members of the ubiquitin-like ATG8 protein family to phosphatidylethanolamine in the growing autophagosomal membrane, known as the phagophore. ATG12-5-16L1 is recruited to the phagophore by a subset of the phosphatidylinositol 3-phosphate-binding seven bladed â-propeller WIPI proteins. We determined the crystal structure of WIPI2d in complex with the WIPI2 interacting region (W2IR) of ATG16L1 comprising residues 207-230 at 1.85 Å resolution. The structure shows that the ATG16L1 W2IR adopts an alpha helical conformation and binds in an electropositive and hydrophobic groove between WIPI2 â-propeller blades 2 and 3. Mutation of residues at the interface reduces or blocks the recruitment of ATG12-5-16L1 and the conjugation of the ATG8 protein LC3B to synthetic membranes. Interface mutants show a decrease in starvation-induced autophagy. Comparisons across the four human WIPIs suggest that WIPI1 and 2 belong to a W2IR-binding subclass responsible for localizing ATG12-5-16L1 and driving ATG8 lipidation, whilst WIPI3 and 4 belong to a second W34IR-binding subclass responsible for localizing ATG2, and so directing lipid supply to the nascent phagophore. The structure provides a framework for understanding the regulatory node connecting two central events in autophagy initiation, the action of the autophagic PI 3-kinase complex on the one hand, and ATG8 lipidation on the other.

Data availability

Coordinates and structure factors have been deposited in the Protein Data Bank under accession code PDB 7MU2. Protocols have been deposited in protocols.io. Plasmids developed for this study will be deposited at Addgene.org. GUV source data are being deposited in Zenodo.

The following data sets were generated

Article and author information

Author details

  1. Lisa M Strong

    Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States
    Competing interests
    Lisa M Strong, LMS is enrolled as a graduate student at UC Berkeley. The author has no competing interests to declare..
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-4293-8131
  2. Chunmei Chang

    Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States
    Competing interests
    Chunmei Chang, CC is employed at UC Berkeley. The author has no competing interests to declare..
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-5607-7985
  3. Julia F Riley

    University of Pennsylvania, Philadelphia,, United States
    Competing interests
    No competing interests declared.
  4. C Alexander Boecker

    University of Pennsylvania, Philadelphia,, United States
    Competing interests
    C Alexander Boecker, is employed at the University of Pennsylvania. The author has no competing interests to declare..
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-9701-5273
  5. Thomas G Flower

    Molecular Cell Biology, University of California, Berkeley, Berkeley, United States
    Competing interests
    Thomas G Flower, Thomas G. Flower was employed by UC Berkeley at the time he contributed to this study, and is currently employed at Galapagos, Romainville, France. The author has no competing interests to declare..
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-7890-6473
  6. Cosmo Z Buffalo

    Molecular Cell Biology, University of California, Berkeley, Berkeley, United States
    Competing interests
    Cosmo Z Buffalo, CZB is employed by UC Berkeley. The author has no competing interests to declare..
  7. Xuefeng Ren

    Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States
    Competing interests
    Xuefeng Ren, XR is employed by UC Berkeley. The author has no competing interests to declare..
  8. Andrea KH Stavoe

    The University of Texas Health Science Center at Houston McGovern Medical School, Houston, United States
    Competing interests
    Andrea KH Stavoe, is employed by The University of Texas Health Science Center at Houston McGovern Medical School. The author has no competing interests to declare..
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-4073-4565
  9. Erika LF Holzbaur

    Physiology, University of Pennsylvania, Philadelphia, United States
    Competing interests
    Erika LF Holzbaur, ELFH is employed by the University of Pennsylvania. The author has no competing interests to declare..
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-5389-4114
  10. James H Hurley

    Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States
    For correspondence
    jimhurley@berkeley.edu
    Competing interests
    James H Hurley, JHH is employed by UC Berkeley. JHH has a competing interest as a co-founder of Casma Therapeutics..
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-5054-5445

Funding

Aligning Science Across Parkinson's (ASAP-000350)

  • Erika LF Holzbaur
  • James H Hurley

NIGMS (R01 GM111730)

  • James H Hurley

NINDS (R00 NS109286)

  • Andrea KH Stavoe

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

Copyright

© 2021, Strong 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

  • 3,515
    views
  • 559
    downloads
  • 41
    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. Lisa M Strong
  2. Chunmei Chang
  3. Julia F Riley
  4. C Alexander Boecker
  5. Thomas G Flower
  6. Cosmo Z Buffalo
  7. Xuefeng Ren
  8. Andrea KH Stavoe
  9. Erika LF Holzbaur
  10. James H Hurley
(2021)
Structural basis for membrane recruitment of ATG16L1 by WIPI2 in Autophagy
eLife 10:e70372.
https://doi.org/10.7554/eLife.70372

Share this article

https://doi.org/10.7554/eLife.70372

Further reading

    1. Cell Biology
    2. Evolutionary Biology
    Paul Richard J Yulo, Nicolas Desprat ... Heather L Hendrickson
    Research Article

    Maintenance of rod-shape in bacterial cells depends on the actin-like protein MreB. Deletion of mreB from Pseudomonas fluorescens SBW25 results in viable spherical cells of variable volume and reduced fitness. Using a combination of time-resolved microscopy and biochemical assay of peptidoglycan synthesis, we show that reduced fitness is a consequence of perturbed cell size homeostasis that arises primarily from differential growth of daughter cells. A 1000-generation selection experiment resulted in rapid restoration of fitness with derived cells retaining spherical shape. Mutations in the peptidoglycan synthesis protein Pbp1A were identified as the main route for evolutionary rescue with genetic reconstructions demonstrating causality. Compensatory pbp1A mutations that targeted transpeptidase activity enhanced homogeneity of cell wall synthesis on lateral surfaces and restored cell size homeostasis. Mechanistic explanations require enhanced understanding of why deletion of mreB causes heterogeneity in cell wall synthesis. We conclude by presenting two testable hypotheses, one of which posits that heterogeneity stems from non-functional cell wall synthesis machinery, while the second posits that the machinery is functional, albeit stalled. Overall, our data provide support for the second hypothesis and draw attention to the importance of balance between transpeptidase and glycosyltransferase functions of peptidoglycan building enzymes for cell shape determination.

    1. Cell Biology
    2. Developmental Biology
    Pavan K Nayak, Arul Subramanian, Thomas F Schilling
    Research Article

    Mechanical forces play a critical role in tendon development and function, influencing cell behavior through mechanotransduction signaling pathways and subsequent extracellular matrix (ECM) remodeling. Here we investigate the molecular mechanisms by which tenocytes in developing zebrafish embryos respond to muscle contraction forces during the onset of swimming and cranial muscle activity. Using genome-wide bulk RNA sequencing of FAC-sorted tenocytes we identify novel tenocyte markers and genes involved in tendon mechanotransduction. Embryonic tendons show dramatic changes in expression of matrix remodeling associated 5b (mxra5b), matrilin1 (matn1), and the transcription factor kruppel-like factor 2a (klf2a), as muscles start to contract. Using embryos paralyzed either by loss of muscle contractility or neuromuscular stimulation we confirm that muscle contractile forces influence the spatial and temporal expression patterns of all three genes. Quantification of these gene expression changes across tenocytes at multiple tendon entheses and myotendinous junctions reveals that their responses depend on force intensity, duration and tissue stiffness. These force-dependent feedback mechanisms in tendons, particularly in the ECM, have important implications for improved treatments of tendon injuries and atrophy.