Lipid profiles of autophagic structures isolated from wild type and Atg2 mutant Drosophila

Biochim Biophys Acta Mol Cell Biol Lipids. 2021 Mar;1866(3):158868. doi: 10.1016/j.bbalip.2020.158868. Epub 2020 Dec 15.

Abstract

Autophagy is mediated by membrane-bound organelles and it is an intrinsic catabolic and recycling process of the cell, which is very important for the health of organisms. The biogenesis of autophagic membranes is still incompletely understood. In vitro studies suggest that Atg2 protein transports lipids presumably from the ER to the expanding autophagic structures. Autophagy research has focused heavily on proteins and very little is known about the lipid composition of autophagic membranes. Here we describe a method for immunopurification of autophagic structures from Drosophila melanogaster (an excellent model to study autophagy in a complete organism) for subsequent lipidomic analysis. Western blots of several organelle markers indicate the high purity of the isolated autophagic vesicles, visualized by various microscopy techniques. Mass spectrometry results show that phosphatidylethanolamine (PE) is the dominant lipid class in wild type (control) membranes. We demonstrate that in Atg2 mutants (Atg2-), phosphatidylinositol (PI), negatively charged phosphatidylserine (PS), and phosphatidic acid (PA) with longer fatty acyl chains accumulate on stalled, negatively charged phagophores. Tandem mass spectrometry analysis of lipid species composing the lipid classes reveal the enrichment of unsaturated PE and phosphatidylcholine (PC) in controls versus PI, PS and PA species in Atg2-. Significant differences in the lipid profiles of control and Atg2- flies suggest that the lipid composition of autophagic membranes dynamically changes during their maturation. These lipidomic results also point to the in vivo lipid transport function of the Atg2 protein, pointing to its specific role in the transport of short fatty acyl chain PE species.

Keywords: Atg2; Autophagic membranes; Autophagy; Drosophila melanogaster; Lipid composition; Lipidomics.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Animals
  • Autophagosomes / chemistry
  • Autophagosomes / genetics
  • Autophagosomes / metabolism*
  • Autophagy
  • Autophagy-Related Proteins / genetics
  • Autophagy-Related Proteins / metabolism*
  • Drosophila Proteins / genetics
  • Drosophila Proteins / metabolism*
  • Drosophila melanogaster / genetics
  • Drosophila melanogaster / metabolism*
  • Female
  • Intracellular Membranes / chemistry
  • Intracellular Membranes / metabolism
  • Lipid Metabolism
  • Lipids / analysis*
  • Male
  • Mutation

Substances

  • Atg2 protein, Drosophila
  • Autophagy-Related Proteins
  • Drosophila Proteins
  • Lipids