Members of the autophagy class III phosphatidylinositol 3-kinase complex I interact with GABARAP and GABARAPL1 via LIR motifs

Autophagy. 2019 Aug;15(8):1333-1355. doi: 10.1080/15548627.2019.1581009. Epub 2019 Mar 4.

Abstract

Autophagosome formation depends on a carefully orchestrated interplay between membrane-associated protein complexes. Initiation of macroautophagy/autophagy is mediated by the ULK1 (unc-51 like autophagy activating kinase 1) protein kinase complex and the autophagy-specific class III phosphatidylinositol 3-kinase complex I (PtdIns3K-C1). The latter contains PIK3C3/VPS34, PIK3R4/VPS15, BECN1/Beclin 1 and ATG14 and phosphorylates phosphatidylinositol to generate phosphatidylinositol 3-phosphate (PtdIns3P). Here, we show that PIK3C3, BECN1 and ATG14 contain functional LIR motifs and interact with the Atg8-family proteins with a preference for GABARAP and GABARAPL1. High resolution crystal structures of the functional LIR motifs of these core components of PtdIns3K-C1were obtained. Variation in hydrophobic pocket 2 (HP2) may explain the specificity for the GABARAP family. Mutation of the LIR motif in ATG14 did not prevent formation of the PtdIns3K-C1 complex, but blocked colocalization with MAP1LC3B/LC3B and impaired mitophagy. The ULK-mediated phosphorylation of S29 in ATG14 was strongly dependent on a functional LIR motif in ATG14. GABARAP-preferring LIR motifs in PIK3C3, BECN1 and ATG14 may, via coincidence detection, contribute to scaffolding of PtdIns3K-C1 on membranes for efficient autophagosome formation. Abbreviations: ATG: autophagy-related; BafA1: bafilomycin A1; GABARAP: GABA type A receptor-associated protein; GABARAPL1: GABA type A receptor associated protein like 1; GFP: enhanced green fluorescent protein; KO: knockout; LDS: LIR docking site; LIR: LC3-interacting region; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; PIK3C3: phosphatidylinositol 3-kinase catalytic subunit type 3; PIK3R4: phosphoinositide-3-kinase regulatory subunit 4; PtdIns3K: phosphatidylinositol 3-kinase; PtdIns3P: phosphatidylinositol-3-phosphate; SQSTM1/p62: sequestosome 1; VPS: Vacuolar protein sorting; ULK: unc-51 like autophagy activating kinase.

Keywords: ATG14; Autophagy; BECN1; GABARAP; LIR; PIK3C3.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adaptor Proteins, Signal Transducing / metabolism*
  • Adaptor Proteins, Vesicular Transport / metabolism
  • Amino Acid Motifs
  • Amino Acid Sequence
  • Apoptosis Regulatory Proteins / metabolism*
  • Autophagy*
  • Autophagy-Related Protein 8 Family / metabolism
  • Autophagy-Related Proteins / metabolism
  • Beclin-1 / chemistry
  • Beclin-1 / metabolism
  • Class III Phosphatidylinositol 3-Kinases / chemistry*
  • Class III Phosphatidylinositol 3-Kinases / metabolism*
  • HCT116 Cells
  • Humans
  • Hydrophobic and Hydrophilic Interactions
  • Microtubule-Associated Proteins / metabolism*
  • Mitophagy
  • Models, Molecular
  • Peptides / chemistry
  • Protein Binding

Substances

  • ATG14 protein, human
  • Adaptor Proteins, Signal Transducing
  • Adaptor Proteins, Vesicular Transport
  • Apoptosis Regulatory Proteins
  • Autophagy-Related Protein 8 Family
  • Autophagy-Related Proteins
  • Beclin-1
  • GABARAP protein, human
  • GABARAPL1 protein, human
  • MAP1LC3A protein, human
  • Microtubule-Associated Proteins
  • Peptides
  • Class III Phosphatidylinositol 3-Kinases