Cell-Free Reconstitution of Autophagic Membrane Formation

Methods Mol Biol. 2019:1880:135-148. doi: 10.1007/978-1-4939-8873-0_7.

Abstract

Autophagy is a catabolic pathway for bulk turnover of cytoplasmic components through the lysosome. Completion of autophagy requires a sophisticated membrane remodeling process. The early steps involve autophagic membrane precursor generation from the intracellular membranes. The intricate protein-membrane interactions underlying autophagic membrane precursor generation have been a focus of attention but yet poorly defined. Here, we summarize the procedure of a cell-free system we have established to dissect the molecular mechanism of early autophagic membrane generation.

Keywords: Autophagosome; Autophagy; Cell-free reconstitution; LC3 lipidation.

Publication types

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

MeSH terms

  • Animals
  • Autophagosomes / metabolism*
  • Autophagy*
  • Autophagy-Related Protein 5 / genetics
  • Autophagy-Related Protein 5 / metabolism*
  • Cell Fractionation / methods
  • Cell Membrane / metabolism
  • Cell-Free System / metabolism*
  • Cells, Cultured
  • Cytosol / metabolism
  • HEK293 Cells
  • Humans
  • Lipid Metabolism*
  • Mice
  • Mice, Knockout
  • Microtubule-Associated Proteins / metabolism*

Substances

  • Autophagy-Related Protein 5
  • MAP1LC3A protein, human
  • Map1lc3b protein, mouse
  • Microtubule-Associated Proteins