The C-terminal region of ATG101 bridges ULK1 and PtdIns3K complex in autophagy initiation

Autophagy. 2018;14(12):2104-2116. doi: 10.1080/15548627.2018.1504716. Epub 2018 Aug 16.

Abstract

The initiation of macroautophagy/autophagy is tightly regulated by the upstream ULK1 kinase complex, which affects many downstream factors including the PtdIns3K complex. The phosphorylation of the right position at the right time on downstream molecules is governed by proper complex formation. One component of the ULK1 complex, ATG101, known as an accessory protein, is a stabilizer of ATG13 in cells. The WF finger region of ATG101 plays an important role in the recruitment of WIPI1 (WD repeat domain, phosphoinositide interacting protein 1) and ZFYVE1 (zinc finger FYVE-type containing 1). Here, we report that the C-terminal region identified in the structure of the human ATG101-ATG13HORMA complex is responsible for the binding of the PtdIns3K complex. This region adopts a β-strand conformation in free ATG101, but either an α-helix or random coil in our ATG101-ATG13HORMA complex, which protrudes from the core and interacts with other molecules. The C-terminal deletion of ATG101 shows a significant defect in the interaction with PtdIns3K components and subsequently impairs autophagosome formation. This result clearly presents an additional role of ATG101 for bridging the ULK1 and PtdIns3K complexes in the mammalian autophagy process. Abbreviations: ATG: autophagy related; BECN1: beclin 1; GFP: green fluorescent protein; HORMA: Hop1p/Rev7p/MAD2; HsATG13HORMA: HORMA domain of ATG13 from Homo sapiens; KO: knockout; MAD2: mitotic arrest deficient 2 like 1; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; PIK3C3/VPS34: phosphatidylinositol 3-kinase catalytic subunit type 3; PIK3R4/VPS15: phosphoinositide-3-kinase regulatory subunit 4; PtdIns3K: phosphatidylinositol 3-kinase; RB1CC1/FIP200: RB1 inducible coiled-coil 1; SAXS: small-angle X-ray scattering; ScAtg13HORMA: HORMA domain of Atg13 from Sccharomyces cerevisiae; SEC-SAXS: size-exclusion chromatography with small-angle X-ray scattering; SpAtg13HORMA: HORMA domain of Atg13 from Schizosaccharomyces pombe; SQSTM1/p62: sequestosome 1; ULK1: unc51-like autophagy activating kinase 1; UVRAG: UV radiation resistance associated; WIPI1: WD repeat domain: phosphoinositide interacting 1; ZFYVE1/DFCP1: zinc finger FYVE-type containing 1.

Keywords: ATG101; ATG13; C-terminal region; PtdIns3K complex; ULK1 complex; structure.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Autophagy / physiology*
  • Autophagy-Related Protein-1 Homolog / chemistry
  • Autophagy-Related Protein-1 Homolog / metabolism*
  • Autophagy-Related Proteins / chemistry*
  • Autophagy-Related Proteins / metabolism*
  • Class III Phosphatidylinositol 3-Kinases / chemistry
  • Class III Phosphatidylinositol 3-Kinases / metabolism*
  • Crystallography, X-Ray
  • HEK293 Cells
  • Humans
  • Intracellular Signaling Peptides and Proteins / chemistry
  • Intracellular Signaling Peptides and Proteins / metabolism*
  • Male
  • Models, Molecular
  • Multiprotein Complexes / chemistry
  • Multiprotein Complexes / metabolism
  • Protein Binding
  • Protein Interaction Domains and Motifs / physiology
  • Scattering, Small Angle
  • Tumor Cells, Cultured
  • Vacuolar Sorting Protein VPS15 / metabolism*
  • Vesicular Transport Proteins / chemistry*
  • Vesicular Transport Proteins / metabolism*
  • X-Ray Diffraction

Substances

  • ATG101 protein, human
  • Autophagy-Related Proteins
  • Intracellular Signaling Peptides and Proteins
  • Multiprotein Complexes
  • Vesicular Transport Proteins
  • Class III Phosphatidylinositol 3-Kinases
  • Autophagy-Related Protein-1 Homolog
  • PIK3R4 protein, human
  • ULK1 protein, human
  • Vacuolar Sorting Protein VPS15

Grants and funding

This work was supported by National Research Foundation of Korea (NRF) grants from the Korean government (NRF-2016R1E1A1A01942623, BRL grant: No. 2015041919, and International Cooperation Program: No. 2015K2A2A6002008 to H.K.S.; NRF-2017R1A2B4011732 to H.C.).