Divergent roles of BECN1 in LC3 lipidation and autophagosomal function

Autophagy. 2015;11(5):740-7. doi: 10.1080/15548627.2015.1034404.

Abstract

BECN1/Beclin 1 is regarded as a critical component in the class III phosphatidylinositol 3-kinase (PtdIns3K) complex to trigger autophagy in mammalian cells. Despite its significant role in a number of cellular and physiological processes, the exact function of BECN1 in autophagy remains controversial. Here we created a BECN1 knockout human cell line using the TALEN technique. Surprisingly, the complete loss of BECN1 had little effect on LC3 (MAP1LC3B/LC3B) lipidation, and LC3B puncta resembling autophagosomes by fluorescence microscopy were still evident albeit significantly smaller than those in the wild-type cells. Electron microscopy (EM) analysis revealed that BECN1 deficiency led to malformed autophagosome-like structures containing multiple layers of membranes under amino acid starvation. We further confirmed that the PtdIns3K complex activity and autophagy flux were disrupted in BECN1(-/-) cells. Our results demonstrate the essential role of BECN1 in the functional formation of autophagosomes, but not in LC3B lipidation.

Keywords: ATG, autophagy related; BCL2L1/Bcl-xL, BCL2-like 1; BECN1; BECN1, Beclin 1, autophagy related; BECN1P1/BECN2, Beclin 1, autophagy related, pseudogene 1; BafA1, bafilomycin A1; EM, electron microscopy; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GFP, green fluorescent protein; KO, knockout; LC3; MAP1LC3-I/LC3-I, soluble, proteolytically processed microtubule-associated protein 1 light chain 3; MAP1LC3-II/LC3-II, proteolytically processed and lipid-modified microtubule-associated protein 1 light chain 3; MAP1LC3/LC3, microtubule-associated protein 1 light chain 3; MAP1LC3B/LC3B, microtubule-associated protein 1 light chain 3 β; PIK3C3/VPS34, phosphatidylinositol 3-kinase, catalytic subunit type 3; PIK3R4/VPS15, phosphoinositide-3-kinase, regulatory subunit 4; PtdIns3K; PtdIns3K, phosphatidylinositol 3-kinase; SQSTM1/p62, sequestosome 1; TALEN, transcription activator-like effector nuclease; TUBB, tubulin, β class I; UVRAG, UV radiation resistance associated; ZFYVE1/DFCP1, zinc finger, FYVE domain containing 1.; autophagosome; autophagy.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Apoptosis Regulatory Proteins / deficiency
  • Apoptosis Regulatory Proteins / metabolism*
  • Autophagy*
  • Base Sequence
  • Beclin-1
  • Class III Phosphatidylinositol 3-Kinases / metabolism
  • HeLa Cells
  • Humans
  • Lipids / chemistry*
  • Membrane Proteins / deficiency
  • Membrane Proteins / metabolism*
  • Microtubule-Associated Proteins / metabolism*
  • Molecular Sequence Data
  • Phagosomes / metabolism*
  • Phagosomes / ultrastructure

Substances

  • Apoptosis Regulatory Proteins
  • BECN1 protein, human
  • Beclin-1
  • Lipids
  • MAP1LC3A protein, human
  • Membrane Proteins
  • Microtubule-Associated Proteins
  • Class III Phosphatidylinositol 3-Kinases