An autophagy assay reveals the ESCRT-III component CHMP2A as a regulator of phagophore closure

Nat Commun. 2018 Jul 20;9(1):2855. doi: 10.1038/s41467-018-05254-w.


The mechanism of phagophore closure remains unclear due to technical limitations in distinguishing unclosed and closed autophagosomal membranes. Here, we report the HaloTag-LC3 autophagosome completion assay that specifically detects phagophores, nascent autophagosomes, and mature autophagic structures. Using this assay, we identify the endosomal sorting complexes required for transport (ESCRT)-III component CHMP2A as a critical regulator of phagophore closure. During autophagy, CHMP2A translocates to the phagophore and regulates the separation of the inner and outer autophagosomal membranes to form double-membrane autophagosomes. Consistently, inhibition of the AAA-ATPase VPS4 activity impairs autophagosome completion. The ESCRT-mediated membrane abscission appears to be a critical step in forming functional autolysosomes by preventing mislocalization of lysosome-associated membrane glycoprotein 1 to the inner autophagosomal membrane. Collectively, our work reveals a function for the ESCRT machinery in the final step of autophagosome formation and provides a useful tool for quantitative analysis of autophagosome biogenesis and maturation.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • ATPases Associated with Diverse Cellular Activities / metabolism
  • Autophagy*
  • Carrier Proteins
  • Cell Membrane / metabolism
  • Endosomal Sorting Complexes Required for Transport / metabolism*
  • Endosomes / metabolism*
  • Gene Expression Regulation*
  • Green Fluorescent Proteins / metabolism
  • HeLa Cells
  • Humans
  • Lysosomes / metabolism*
  • RNA, Small Interfering / metabolism
  • Vacuolar Proton-Translocating ATPases / metabolism


  • CHMP2A protein, human
  • Carrier Proteins
  • Endosomal Sorting Complexes Required for Transport
  • RNA, Small Interfering
  • Green Fluorescent Proteins
  • Vacuolar Proton-Translocating ATPases
  • ATPases Associated with Diverse Cellular Activities
  • VPS4A protein, human