ER-Targeted Beclin 1 Supports Autophagosome Biogenesis in the Absence of ULK1 and ULK2 Kinases

Cells. 2019 May 17;8(5):475. doi: 10.3390/cells8050475.

Abstract

Autophagy transports cytoplasmic material and organelles to lysosomes for degradation and recycling. Beclin 1 forms a complex with several other autophagy proteins and functions in the initiation phase of autophagy, but the exact role of Beclin 1 subcellular localization in autophagy initiation is still unclear. In order to elucidate the role of Beclin 1 localization in autophagosome biogenesis, we generated constructs that target Beclin 1 to the endoplasmic reticulum (ER) or mitochondria. Our results confirmed the proper organelle-specific targeting of the engineered Beclin 1 constructs, and the proper formation of autophagy-regulatory Beclin 1 complexes. The ULK kinases are required for autophagy initiation upstream of Beclin 1, and autophagosome biogenesis is severely impaired in ULK1/ULK2 double knockout cells. We tested whether Beclin 1 targeting facilitated its ability to rescue autophagosome formation in ULK1/ULK2 double knockout cells. ER-targeted Beclin 1 was most effective in the rescue experiments, while mitochondria-targeted and non-targeted Beclin 1 also showed an ability to rescue, but with lower activity. However, none of the constructs was able to increase autophagic flux in the knockout cells. We also showed that wild type Beclin 1 was enriched on the ER during autophagy induction, and that ULK1/ULK2 facilitated the ER-enrichment of Beclin 1 under basal conditions. The results suggest that one of the functions of ULK kinases may be to enhance Beclin 1 recruitment to the ER to drive autophagosome formation.

Keywords: Beclin 1; ULK1; ULK2; autophagy; endoplasmic reticulum; mitochondria.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acids / deficiency
  • Animals
  • Autophagosomes / metabolism*
  • Autophagy
  • Autophagy-Related Protein-1 Homolog / genetics
  • Autophagy-Related Protein-1 Homolog / metabolism*
  • Beclin-1 / metabolism*
  • Endoplasmic Reticulum / metabolism*
  • Gene Knockout Techniques
  • Green Fluorescent Proteins / metabolism
  • HEK293 Cells
  • Humans
  • Intracellular Signaling Peptides and Proteins / genetics
  • Intracellular Signaling Peptides and Proteins / metabolism*
  • Lysosomes / metabolism
  • Mice
  • Mitochondria / metabolism
  • Organelle Biogenesis*
  • Protein Serine-Threonine Kinases / genetics
  • Protein Serine-Threonine Kinases / metabolism*

Substances

  • Amino Acids
  • BECN1 protein, human
  • Beclin-1
  • Intracellular Signaling Peptides and Proteins
  • enhanced green fluorescent protein
  • Green Fluorescent Proteins
  • Autophagy-Related Protein-1 Homolog
  • Protein Serine-Threonine Kinases
  • ULK1 protein, human
  • Ulk2 protein, human