Resveratrol-mediated autophagy requires WIPI-1-regulated LC3 lipidation in the absence of induced phagophore formation

Autophagy. 2011 Dec;7(12):1448-61. doi: 10.4161/auto.7.12.17802.

Abstract

Canonical autophagy is positively regulated by the Beclin 1/phosphatidylinositol 3-kinase class III (PtdIns3KC3) complex that generates an essential phospholipid, phosphatidylinositol 3-phosphate (PtdIns(3)P), for the formation of autophagosomes. Previously, we identified the human WIPI protein family and found that WIPI-1 specifically binds PtdIns(3)P, accumulates at the phagophore and becomes a membrane protein of generated autophagosomes. Combining siRNA-mediated protein downregulation with automated high through-put analysis of PtdIns(3)P-dependent autophagosomal membrane localization of WIPI-1, we found that WIPI-1 functions upstream of both Atg7 and Atg5, and stimulates an increase of LC3-II upon nutrient starvation. Resveratrol-mediated autophagy was shown to enter autophagic degradation in a noncanonical manner, independent of Beclin 1 but dependent on Atg7 and Atg5. By using electron microscopy, LC3 lipidation and GFP-LC3 puncta-formation assays we confirmed these results and found that this effect is partially wortmannin-insensitive. In line with this, resveratrol did not promote phagophore localization of WIPI-1, WIPI-2 or the Atg16L complex above basal level. In fact, the presence of resveratrol in nutrient-free conditions inhibited phagophore localization of WIPI-1. Nevertheless, we found that resveratrol-mediated autophagy functionally depends on canonical-driven LC3-II production, as shown by siRNA-mediated downregulation of WIPI-1 or WIPI-2. From this it is tempting to speculate that resveratrol promotes noncanonical autophagic degradation downstream of the PtdIns(3)P-WIPI-Atg7-Atg5 pathway, by engaging a distinct subset of LC3-II that might be generated at membrane origins apart from canonical phagophore structures.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Androstadienes / pharmacology
  • Animals
  • Autophagy / drug effects*
  • Autophagy-Related Protein 12
  • Autophagy-Related Protein 5
  • Autophagy-Related Protein 7
  • Autophagy-Related Proteins
  • Carrier Proteins / drug effects
  • Carrier Proteins / metabolism*
  • Cell Line
  • Green Fluorescent Proteins / metabolism
  • Humans
  • Lipids / chemistry*
  • Membrane Proteins / metabolism*
  • Mice
  • Microtubule-Associated Proteins / metabolism*
  • Phagosomes / drug effects
  • Phagosomes / metabolism*
  • Phagosomes / ultrastructure
  • Phosphatidylinositol Phosphates / metabolism
  • Protein Transport / drug effects
  • Recombinant Fusion Proteins / metabolism
  • Resveratrol
  • Small Ubiquitin-Related Modifier Proteins / metabolism
  • Stilbenes / pharmacology*
  • Ubiquitin-Activating Enzymes / metabolism
  • Wortmannin

Substances

  • ATG12 protein, human
  • ATG5 protein, human
  • Androstadienes
  • Atg5 protein, mouse
  • Atg7 protein, mouse
  • Autophagy-Related Protein 12
  • Autophagy-Related Protein 5
  • Autophagy-Related Proteins
  • Carrier Proteins
  • Lipids
  • Membrane Proteins
  • Microtubule-Associated Proteins
  • Phosphatidylinositol Phosphates
  • Recombinant Fusion Proteins
  • Small Ubiquitin-Related Modifier Proteins
  • Stilbenes
  • WIPI-1 protein, human
  • ZFYVE1 protein, human
  • light chain 3, human
  • phosphatidylinositol 3-phosphate
  • Green Fluorescent Proteins
  • Atg7 protein, human
  • Autophagy-Related Protein 7
  • Ubiquitin-Activating Enzymes
  • Resveratrol
  • Wortmannin