An autophagy assay reveals the ESCRT-III component CHMP2A as a regulator of phagophore closure.
basic_science · Level V
Where this comes from
- Record sourced from PubMed, PMID 30030437.
- Also identified by DOI 10.1038/s41467-018-05254-w and PMC identifier 6054611.
- Licence recorded as CC BY.
- The licence permits redistribution, so the abstract is shown in full and the full text is available from the publisher.
Abstract
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.
Medical subject headings
- Autophagy
- Endosomal Sorting Complexes Required for Transport
- Endosomes
- Gene Expression Regulation
- Lysosomes