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PMID: 24954904 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

WIPI2 links LC3 conjugation with PI3P, autophagosome formation, and pathogen clearance by recruiting Atg12-5-16L1.

Molecular cell ·Vol. 55 ·No. 2 ·2014-07-17 ·Pages 238-52

Dooley HC, Razi M, Polson HE, Girardin SE, Wilson MI, Tooze SA

Abstract

Mammalian cell homeostasis during starvation depends on initiation of autophagy by endoplasmic reticulum-localized phosphatidylinositol 3-phosphate (PtdIns(3)P) synthesis. Formation of double-membrane autophagosomes that engulf cytosolic components requires the LC3-conjugating Atg12-5-16L1 complex. The molecular mechanisms of Atg12-5-16L1 recruitment and significance of PtdIns(3)P synthesis at autophagosome formation sites are unknown. By identifying interacting partners of WIPIs, WD-repeat PtdIns(3)P effector proteins, we found that Atg16L1 directly binds WIPI2b. Mutation experiments and ectopic localization of WIPI2b to plasma membrane show that WIPI2b is a PtdIns(3)P effector upstream of Atg16L1 and is required for LC3 conjugation and starvation-induced autophagy through recruitment of the Atg12-5-16L1 complex. Atg16L1 mutants, which do not bind WIPI2b but bind FIP200, cannot rescue starvation-induced autophagy in Atg16L1-deficient MEFs. WIPI2b is also required for autophagic clearance of pathogenic bacteria. WIPI2b binds the membrane surrounding Salmonella and recruits the Atg12-5-16L1 complex, initiating LC3 conjugation, autophagosomal membrane formation, and engulfment of Salmonella.

MeSH Terms
Amino Acid Sequence Animals Autophagy Autophagy-Related Protein 12 Autophagy-Related Protein 5 Autophagy-Related Proteins Carrier Proteins/chemistry,metabolism,physiology Conserved Sequence HEK293 Cells Host-Pathogen Interactions Humans Intracellular Membranes/metabolism Membrane Proteins/physiology Mice Microtubule-Associated Proteins/metabolism Molecular Sequence Data Phagocytosis Phagosomes/metabolism,microbiology Phosphate-Binding Proteins Phosphatidylinositol Phosphates/metabolism Protein Binding Protein Interaction Domains and Motifs Protein Isoforms/physiology Protein Processing, Post-Translational Protein Transport Salmonella typhimurium/physiology Small Ubiquitin-Related Modifier Proteins/metabolism
Chemicals
ATG12 protein, human ATG16L1 protein, human ATG5 protein, human Autophagy-Related Protein 12 Autophagy-Related Protein 5 Autophagy-Related Proteins Carrier Proteins MAP1LC3A protein, human Membrane Proteins Microtubule-Associated Proteins Phosphate-Binding Proteins Phosphatidylinositol Phosphates Protein Isoforms Small Ubiquitin-Related Modifier Proteins WIPI2 protein, human phosphatidylinositol 3-phosphate
Authors & Affiliations
6 authors, click to expand affiliations / ORCID
Dooley Hannah C
London Research Institute, Cancer Research UK, 44 Lincolns Inn Fields, London WC2A 3LY, UK.
Razi Minoo
London Research Institute, Cancer Research UK, 44 Lincolns Inn Fields, London WC2A 3LY, UK.
Polson Hannah E J
London Research Institute, Cancer Research UK, 44 Lincolns Inn Fields, London WC2A 3LY, UK.
Girardin Stephen E
Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON M5S 1A8, Canada.
Wilson Michael I
The Babraham Institute, Babraham Research Campus, Cambridge CB22 3AT, UK.
Tooze Sharon A
London Research Institute, Cancer Research UK, 44 Lincolns Inn Fields, London WC2A 3LY, UK. Electronic address: sharon.tooze@cancer.org.uk.
References (45)
45 references, click to expand
  1. Loss of the autophagy protein Atg16L1 enhances endotoxin-induced IL-1beta production.
    Nature. 2008 Nov 13;456(7219):264-8 PMID: 18849965
  2. FIP200 regulates targeting of Atg16L1 to the isolation membrane.
    EMBO Rep. 2013 Mar 1;14(3):284-91 PMID: 23392225
  3. Characterization of autophagosome formation site by a hierarchical analysis of mammalian Atg proteins.
    Autophagy. 2010 Aug;6(6):764-76 PMID: 20639694
  4. Structure-based analyses reveal distinct binding sites for Atg2 and phosphoinositides in Atg18.
    J Biol Chem. 2012 Sep 14;287(38):31681-90 PMID: 22851171
  5. The role of Atg proteins in autophagosome formation.
    Annu Rev Cell Dev Biol. 2011;27:107-32 PMID: 21801009
  6. The Atg18-Atg2 complex is recruited to autophagic membranes via phosphatidylinositol 3-phosphate and exerts an essential function.
    J Biol Chem. 2008 Aug 29;283(35):23972-80 PMID: 18586673
  7. Guidelines for the use and interpretation of assays for monitoring autophagy.
    Autophagy. 2012 Apr;8(4):445-544 PMID: 22966490
  8. Autophagy controls Salmonella infection in response to damage to the Salmonella-containing vacuole.
    J Biol Chem. 2006 Apr 21;281(16):11374-83 PMID: 16495224
  9. Global analysis of fission yeast mating genes reveals new autophagy factors.
    PLoS Genet. 2013;9(8):e1003715 PMID: 23950735
  10. Regulating the regulator: post-translational modification of RAS.
    Nat Rev Mol Cell Biol. 2011 Dec 22;13(1):39-51 PMID: 22189424
  11. Dissecting the localization and function of Atg18, Atg21 and Ygr223c.
    Autophagy. 2008 Oct;4(7):896-910 PMID: 18769150
  12. Atg12-Atg5 conjugate enhances E2 activity of Atg3 by rearranging its catalytic site.
    Nat Struct Mol Biol. 2013 Apr;20(4):433-9 PMID: 23503366
  13. The Atg16L complex specifies the site of LC3 lipidation for membrane biogenesis in autophagy.
    Mol Biol Cell. 2008 May;19(5):2092-100 PMID: 18321988
  14. Phosphatidylinositol 3,5-bisphosphate: metabolism and cellular functions.
    Trends Biochem Sci. 2006 Jan;31(1):52-63 PMID: 16364647
  15. A subdomain of the endoplasmic reticulum forms a cradle for autophagosome formation.
    Nat Cell Biol. 2009 Dec;11(12):1433-7 PMID: 19898463
  16. Svp1p defines a family of phosphatidylinositol 3,5-bisphosphate effectors.
    EMBO J. 2004 May 5;23(9):1922-33 PMID: 15103325
  17. Autophagosome formation--the role of ULK1 and Beclin1-PI3KC3 complexes in setting the stage.
    Semin Cancer Biol. 2013 Oct;23(5):301-9 PMID: 23727157
  18. 3D tomography reveals connections between the phagophore and endoplasmic reticulum.
    Autophagy. 2009 Nov;5(8):1180-5 PMID: 19855179
  19. The Atg8 family: multifunctional ubiquitin-like key regulators of autophagy.
    Essays Biochem. 2013;55:51-64 PMID: 24070471
  20. Antibacterial autophagy occurs at PI(3)P-enriched domains of the endoplasmic reticulum and requires Rab1 GTPase.
    Autophagy. 2011 Jan;7(1):17-26 PMID: 20980813
  21. Maturation steps of the Salmonella-containing vacuole.
    Microbes Infect. 2001 Nov-Dec;3(14-15):1299-303 PMID: 11755418
  22. Autophagosome formation from membrane compartments enriched in phosphatidylinositol 3-phosphate and dynamically connected to the endoplasmic reticulum.
    J Cell Biol. 2008 Aug 25;182(4):685-701 PMID: 18725538
  23. Atg16L2, a novel isoform of mammalian Atg16L that is not essential for canonical autophagy despite forming an Atg12–5-16L2 complex.
    Autophagy. 2011 Dec;7(12):1500-13 PMID: 22082872
  24. Golgi-resident small GTPase Rab33B interacts with Atg16L and modulates autophagosome formation.
    Mol Biol Cell. 2008 Jul;19(7):2916-25 PMID: 18448665
  25. Roles of the lipid-binding motifs of Atg18 and Atg21 in the cytoplasm to vacuole targeting pathway and autophagy.
    J Biol Chem. 2010 Apr 9;285(15):11476-88 PMID: 20154084
  26. Cvt18/Gsa12 is required for cytoplasm-to-vacuole transport, pexophagy, and autophagy in Saccharomyces cerevisiae and Pichia pastoris.
    Mol Biol Cell. 2001 Dec;12(12):3821-38 PMID: 11739783
  27. WIPI-1alpha (WIPI49), a member of the novel 7-bladed WIPI protein family, is aberrantly expressed in human cancer and is linked to starvation-induced autophagy.
    Oncogene. 2004 Dec 16;23(58):9314-25 PMID: 15602573
  28. Structural and functional characterization of the two phosphoinositide binding sites of PROPPINs, a β-propeller protein family.
    Proc Natl Acad Sci U S A. 2012 Jul 24;109(30):E2042-9 PMID: 22753491
  29. Dynamic association of the ULK1 complex with omegasomes during autophagy induction.
    J Cell Sci. 2013 Nov 15;126(Pt 22):5224-38 PMID: 24013547
  30. siRNA screening of the kinome identifies ULK1 as a multidomain modulator of autophagy.
    J Biol Chem. 2007 Aug 31;282(35):25464-74 PMID: 17595159
  31. The WD40 repeat PtdIns(3)P-binding protein EPG-6 regulates progression of omegasomes to autophagosomes.
    Dev Cell. 2011 Aug 16;21(2):343-57 PMID: 21802374
  32. Atg18 function in autophagy is regulated by specific sites within its β-propeller.
    J Cell Sci. 2013 Jan 15;126(Pt 2):593-604 PMID: 23230146
  33. Interaction between FIP200 and ATG16L1 distinguishes ULK1 complex-dependent and -independent autophagy.
    Nat Struct Mol Biol. 2013 Feb;20(2):144-9 PMID: 23262492
  34. Mammalian Atg18 (WIPI2) localizes to omegasome-anchored phagophores and positively regulates LC3 lipidation.
    Autophagy. 2010 May;6(4):506-22 PMID: 20505359
  35. Autophagy in human health and disease.
    N Engl J Med. 2013 Feb 14;368(7):651-62 PMID: 23406030
  36. Mammalian Atg2 proteins are essential for autophagosome formation and important for regulation of size and distribution of lipid droplets.
    Mol Biol Cell. 2012 Mar;23(5):896-909 PMID: 22219374
  37. Two-site recognition of phosphatidylinositol 3-phosphate by PROPPINs in autophagy.
    Mol Cell. 2012 Aug 10;47(3):339-48 PMID: 22704557
  38. Amino acid starvation induced by invasive bacterial pathogens triggers an innate host defense program.
    Cell Host Microbe. 2012 Jun 14;11(6):563-75 PMID: 22704617
  39. Mouse Apg16L, a novel WD-repeat protein, targets to the autophagic isolation membrane with the Apg12-Apg5 conjugate.
    J Cell Sci. 2003 May 1;116(Pt 9):1679-88 PMID: 12665549
  40. Recruitment of the autophagic machinery to endosomes during infection is mediated by ubiquitin.
    J Cell Biol. 2013 Oct 14;203(1):115-28 PMID: 24100292
  41. The LC3 recruitment mechanism is separate from Atg9L1-dependent membrane formation in the autophagic response against Salmonella.
    Mol Biol Cell. 2011 Jul 1;22(13):2290-300 PMID: 21525242
  42. Autophagy and the cytoplasm to vacuole targeting pathway both require Aut10p.
    FEBS Lett. 2001 Nov 9;508(1):23-8 PMID: 11707261
  43. Resveratrol-mediated autophagy requires WIPI-1-regulated LC3 lipidation in the absence of induced phagophore formation.
    Autophagy. 2011 Dec;7(12):1448-61 PMID: 22082875
  44. The autophagosome: origins unknown, biogenesis complex.
    Nat Rev Mol Cell Biol. 2013 Dec;14(12):759-74 PMID: 24201109
  45. Temporal analysis of recruitment of mammalian ATG proteins to the autophagosome formation site.
    Autophagy. 2013 Oct;9(10):1491-9 PMID: 23884233
Article Info
Journal
Molecular cell
Abbr.
Mol Cell
ISSN
1097-4164
DOI
10.1016/j.molcel.2014.05.021
Email
sharon.tooze@cancer.org.uk
Published
2014-07-17
Epub
2014-00-19
Pages
238-52
Language
English
Region
United States
NLM ID
9802571
PMCID
PMC4104028
Subset
IM
Grants
Biotechnology and Biological Sciences Research Council · BBS/E/B/0000C235 · United Kingdom
Biotechnology and Biological Sciences Research Council · BBS/E/B/000C0413 · United Kingdom
CIHR · Canada
Cancer Research UK · United Kingdom
External Links
PubMed source DOI: 10.1016/j.molcel.2014.05.021 PMC Full Text View journal details
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