TY - JOUR
T1 - An autophagy assay reveals the ESCRT-III component CHMP2A as a regulator of phagophore closure
AU - Takahashi, Yoshinori
AU - He, Haiyan
AU - Tang, Zhenyuan
AU - Hattori, Tatsuya
AU - Liu, Ying
AU - Young, Megan M.
AU - Serfass, Jacob M.
AU - Chen, Longgui
AU - Gebru, Melat
AU - Chen, Chong
AU - Wills, Carson A.
AU - Atkinson, Jennifer M.
AU - Chen, Han
AU - Abraham, Thomas
AU - Wang, Hong Gang
PY - 2018/12/1
Y1 - 2018/12/1
N2 - 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.
AB - 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.
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U2 - 10.1038/s41467-018-05254-w
DO - 10.1038/s41467-018-05254-w
M3 - Article
C2 - 30030437
AN - SCOPUS:85050602750
VL - 9
JO - Nature Communications
JF - Nature Communications
SN - 2041-1723
IS - 1
M1 - 2855
ER -