Approaches to the study of Atg8-mediated membrane dynamics in vitro

Methods Cell Biol. 2012;108:93-116. doi: 10.1016/B978-0-12-386487-1.00005-5.

Abstract

Macro-autophagy is the intracellular stress-response pathway by which the cell packages portions of the cytosol for delivery into the lysosome. This "packaging" is carried out by the de novo formation of a new organelle called the autophagosome that grows and encapsulates cytosolic material for eventual lysosomal degradation. How autophagosomes form, including especially how the membrane expands and eventually closes upon itself is an area of intense study. One factor implicated in both membrane expansion and membrane fusion is the ubiquitin-like protein, Atg8. During autophagy, Atg8 becomes covalently bound to phosphatidylethanolamine (PE) on the pre-autophagosomal membrane and remains bound through the maturation process of the autophagosome. In this chapter, we discuss two approaches to the in vitro reconstitution of this lipidation reaction. We then describe methods to study Atg8-PE mediated membrane tethering and fusion, two functions implicated in Atg8's role in autophagosome maturation.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Autophagy
  • Autophagy-Related Protein 7
  • Autophagy-Related Protein 8 Family
  • Autophagy-Related Proteins
  • Biocatalysis
  • Cell Membrane / chemistry*
  • Cross-Linking Reagents / chemistry
  • Cryoelectron Microscopy
  • Humans
  • Kinetics
  • Light
  • Liposomes / chemistry
  • Maleimides / chemistry
  • Membrane Fusion
  • Membrane Lipids / chemistry
  • Microtubule-Associated Proteins / biosynthesis
  • Microtubule-Associated Proteins / chemistry*
  • Microtubule-Associated Proteins / isolation & purification
  • Models, Biological
  • Nephelometry and Turbidimetry
  • Particle Size
  • Phagosomes / chemistry
  • Phosphatidylethanolamines / chemistry
  • Recombinant Proteins / biosynthesis
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / isolation & purification
  • Saccharomyces cerevisiae Proteins / biosynthesis
  • Saccharomyces cerevisiae Proteins / chemistry*
  • Saccharomyces cerevisiae Proteins / isolation & purification
  • Scattering, Radiation
  • Ubiquitin-Conjugating Enzymes / chemistry

Substances

  • ATG7 protein, S cerevisiae
  • ATG8 protein, S cerevisiae
  • Autophagy-Related Protein 8 Family
  • Autophagy-Related Proteins
  • Cross-Linking Reagents
  • Liposomes
  • Maleimides
  • Membrane Lipids
  • Microtubule-Associated Proteins
  • Phosphatidylethanolamines
  • Recombinant Proteins
  • Saccharomyces cerevisiae Proteins
  • maleimide
  • phosphatidylethanolamine
  • Ubiquitin-Conjugating Enzymes
  • Autophagy-Related Protein 7
  • ATG3 protein, S cerevisiae