1. Cell Biology

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. Department of Molecular and Cell Biology, University of California, Berkeley, United States
  2. California Institute for Quantitative Biosciences, University of California, Berkeley, United States
  3. Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, United States
  4. Department of Physiology, University of Pennsylvania Perelman School of Medicine, United States
  5. Department of Neurobiology and Anatomy, The University of Texas Health Science Center at Houston McGovern Medical School, United States
https://doi.org/10.7554/eLife.70372
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Cite this article
  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
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  3. Download .RIS
9 figures, 1 table and 3 additional files

Figures

Figure 1
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WIPI2d:ATG16L1 W2IR structure.

Structure of WIPI2d bound to ATG16L1 W2IR. (A) Annotated WIPI2d and ATG16L1 domain schematics. WIPI2d construct for crystallography is shown and W2IR from ATG16L1. (B, C) The ribbon diagram of the …

Figure 2
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Interactions at the interface.

Analysis of the Interface. (A) Overall electrostatic surface and (B) closer view of electrostatic surface with ATG16 W2IR shown as a cartoon and key residues labeled. (C) Overall hydrophobic surface …

Figure 3
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WIPI2d Interfacial mutants decrease ATG16L1 binding.

Key interacting residues shown as sticks in cartoon representation of WIPI2d:ATG16L1 interface shown from (A) the WIPI2d face or (B) down the ATG16L1 helix. (C) Inputs for the (D) Pull-down assays …

Figure 3—source data 1

Uncropped SDS-PAGE gels for Figure 3.

Uncropped gel used in Figure 3C,D with lanes labeled similarly.

https://cdn.elifesciences.org/articles/70372/elife-70372-fig3-data1-v3.zip
Figure 4
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WIPI2d mutants disrupt E3 recruitment and LC3 lipidation on GUVs.

(A) The schematic drawing illustrates the reaction setting. Colors indicate fluorescent protein-fused components. Components in gray are not labeled but are present in the reaction. (B) …

Figure 5
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Altering the electrostatic interface of WIPI2 impairs starvation-induced autophagy in MEFs.

(A) Representative maximum projections of LC3 staining and either Halo or Halo-WIPI2 signal in WT or WIPI2 knockout (KO) cells (indicated on left) following 2 hr of starvation and 100 nM BafA …

Figure 6
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Reconstitution of membrane recruitment of WIPI2d mutants.

(A) Representative confocal images of GUVs showing membrane binding of mCherry-WIPI2d. PI3KC3-C1, mCherry-WIPI2d WT or mutant, E3-GFP were incubated with GUVs (64.8% DOPC: 20% DOPE: 5% DOPS: 10% …

Figure 7
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Comparing WIPI2d and WIPI3 structures and binding modes.

Comparison of WIPI2d and WIPI3. Alignment of WIPI2d and WIPI3 (A) structure and (B) sequence based on structures with W2IR residues denoted with white squares, W34IR with black, and from both with …

Figure 8
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Comparison of WIPI2 and Atg21 binding to Atg16.

(A) Structural alignment of WIPI2 (PDB: 7MU2) and Atg21 (PDB: 6RGO; indigo) structures bound to Atg16. (B) Sequence alignment of Atg16 ß-propeller binding residues based on structure. Residues for …

Figure 9
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WIPI1-4 comparison.

Comparison of electrostatic surface potential of (A) WIPI1-4. (B) Hydrophobic surface of WIPI1 with predicted ATG16L1 W2IR shown as cartoon. (C, D) Alignment of WIPI2d crystal structure and WIPI1 …

Tables

Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Cell line (Homo sapiens)HeLa human epithelial cell lineATCCCCL-2 Authenticated by STR profiling; tested negative for mycoplasma
Cell line (Homo sapiens)WIPI2-KO cells: HeLa cell line gene-edited to knockout WIPI2 expressionFischer et al., 2020 
Recombinant DNA reagentpHTC HaloTagPromegaG7711 
Recombinant DNA reagentHalo-WIPI2-WT (Homo sapiens) plasmid for transfectionStavoe et al., 2019Addgene 175025Available from Addgene
Recombinant DNA reagentHalo-WIPI2-H85E (Homo sapiens) plasmid for transfectionModified from Halo-WIPI2-WT in Stavoe et al., 2019Addgene 175027Available from Addgene
Recombinant DNA reagentHalo-WIPI2-K88E (Homo sapiens) plasmid for transfectionModified from Halo-WIPI2-WT in Stavoe et al., 2019Addgene 175028Available from Addgene
Recombinant DNA reagentHalo-WIPI2-I92E(Homo sapiens) plasmid for transfectionModified from Halo-WIPI2-WT in Stavoe et al., 2019Addgene 175029Available from Addgene
Recombinant DNA reagentHalo-WIPI2-R108E (Homo sapiens) plasmid for transfectionStavoe et al., 2019Addgene 176004Available from Addgene
Recombinant DNA reagentHalo-WIPI2-H85/K88/I92E (Homo sapiens) plasmid for transfectionModified from Halo-WIPI2-WT in Stavoe et al., 2019Addgene 175033Available from Addgene
AntibodyAnti-LC3B, (Rabbit polyclonal) primary antibodyAbcamCat.#ab48394IF (1:1000)
AntibodyAnti-Rabbit AlexaFluor488, (Goat polyclonal) secondary antibodyThermoFisherCat.#A11034IF (1:1000)
Chemical compound, drugTMRDirect Halo LigandPromegaCat.#G299137.5 nM final concentration
Software, algorithmFIJIPMID:22743772 
Software, algorithmIlastikPMID:31570887 
Software, algorithmAdobe Illustrator 2021Adobe 
Software, algorithmPrism 9GraphPad 
Other35 mm #1.5 glass bottom imaging dishesMatTekCat.# P35G-1.5–20 C 
OtherEBSSThermoFisherCat.# 24010043 
Cell line (Homo sapiens)HEK GnTiATCCCRL-3022 
Recombinant DNA reagentpCAG-WIPI2d-cs-TEVFracchiolla et al., 2020Addgene 171419 
Recombinant DNA reagentpCAG-WIPI2d10-364Δ263–295-cs-TEVThis paperAddgene 171830Materials and methods section: Plasmids
Recombinant DNA reagentpCAG-WIPI2dH85E-cs-TEVThis paperAddgene 171831Materials and methods section: Plasmids
Recombinant DNA reagentpCAG-WIPI2dK88E-cs-TEVThis paperAddgene 171832Materials and methods section: Plasmids
Recombinant DNA reagentpCAG-WIPI2dI92E-cs-TEVThis paperAddgene 171833Materials and methods section: Plasmids
Recombinant DNA reagentpCAG-WIPI2dC93E-cs-TEVThis paperAddgene 171834Materials and methods section: Plasmids
Recombinant DNA reagentpCAG-WIPI2dR108E-cs-TEVThis paperAddgene 171835Materials and methods section: Plasmids
Recombinant DNA reagentpCAG-WIPI2dR125E-cs-TEVThis paperAddgene 171836Materials and methods section: Plasmids
Recombinant DNA reagentpCAG-WIPI2dK128E-cs-TEVThis paperAddgene 171837Materials and methods section: Plasmids
Recombinant DNA reagentpCAG-mcherry-WIPI2d-cs-TEVFracchiolla et al., 2020Addgene 178912 
Recombinant DNA reagentpCAG-mcherry-WIPI2dH85E-cs-TEVThis paperAddgene 171838Materials and methods section: Plasmids
Recombinant DNA reagentpCAG-mcherry-WIPI2dK88E-cs-TEVThis paperAddgene 171839Materials and methods section: Plasmids
Recombinant DNA reagentpCAG-mcherry-WIPI2dI92E-cs-TEVThis paperAddgene 171840Materials and methods section: Plasmids
Recombinant DNA reagentpCAG-mcherry-WIPI2dC93E-cs-TEVThis paperAddgene 171841Materials and methods section: Plasmids
Recombinant DNA reagentpCAG-mcherry-WIPI2dR108E-cs-TEVThis paperAddgene 171842Materials and methods section: Plasmids
Recombinant DNA reagentpCAG-mcherry-WIPI2dR125E-cs-TEVThis paperAddgene 171843Materials and methods section: Plasmids
Recombinant DNA reagentpCAG-mcherry-WIPI2dK128E-cs-TEVThis paperAddgene 171844Materials and methods section: Plasmids
Recombinant DNA reagentpLEXm-GST-TEV-ATG14 Addgene 99329 
Recombinant DNA reagentpCAG-TSF-TEV-BECN1 Addgene 99328 
Recombinant DNA reagentpCAG-TSF-TEV-VPS34 Addgene 99327 
Recombinant DNA reagentpCAG-VPS15Stjepanovic et al., 2017
Addgene 99326 
Recombinant DNA reagentpGBdest-ATG12-10xHis-TEV-ATG5-10xHis-TEVcs-ATG16L1-GFP-TEVcs-StrepII, ATG7, ATG10Fracchiolla et al., 2020Addgene 169077 
Recombinant DNA reagentpFast BacHT(B)–6xHis-TEV-ATG7Fracchiolla et al., 2020 
Recombinant DNA reagentpET Duet-1-6xHis-TEV-ATG3Fracchiolla et al., 2020Addgene 169079 
Recombinant DNA reagentpET Duet-1-6xHis-TEV-mCherry-LC3B-Gly(∆5 C)Zaffagnini et al., 2018
Addgene 169168 
Other96–2 well,INTELLI-PLATE (original) trayMolecular Dimensions, Maumee, OH 
OtherGreiner pre-greased 24 well Combo Plate (SBS format) with lidMolecular Dimensions, Maumee, OH 
Software, algorithmNikon Elements microscope imaging software 4.60Nikon Corporation, Tokyo, Japanhttps://www.nikoninstruments.com/Products/Software/NIS-Elements-Advanced-Research/NIS-Elements-Viewer
OtherGlutathione Sepharose 4B GST-tagged protein purification resinGE healthcare, Chicago, ILCat# 17075605 
OtherStrep-Tactin Superflow high capacity 50 % suspensionIBA Lifesciences,Göttingen, GermanyCat# 2-1208-010 
Software, algorithmphenix.refinePMID:20124702,
22505256, 31588918		RRID:SCR_016736 
Software, algorithmXDSPMID:20124692RRID:SCR_015652 
Software, algorithmPOINTLESSPMID:21460446RRID:SCR_014218 
Software, algorithmPHASERPMID:19461840RRID:SCR_014219 
Software, algorithmPyMolPyMol (pymol.org)RRID:SCR_000305 
Software, algorithmAPBSPMID:11517324RRID:SCR_008387 
Software, algorithmSWISS-MODELPMID:29788355RRID:SCR_018123 
Software, algorithmCootPMID:20383002RRID:SCR_014222 

Additional files

Supplementary file 1

Data collection and refinement statistics.

https://cdn.elifesciences.org/articles/70372/elife-70372-supp1-v3.docx
Supplementary file 2

Oligos used for cloning.

https://cdn.elifesciences.org/articles/70372/elife-70372-supp2-v3.docx
Transparent reporting form
https://cdn.elifesciences.org/articles/70372/elife-70372-transrepform1-v3.docx

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