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.

Highlights

  • The mechanism of phagophore closure remains unclear due to technical limitations in distinguishing unclosed and closed autophagosomal membranes

  • We demonstrate that proper autophagosomal membrane closure requires the endosomal sorting complexes required for transport (ESCRT)-III component CHMP2A and the AAA-ATPase Vacuolar Protein Sorting-associated 4 (VPS4) activity and that the generation of the outer autophagosomal membrane (OAM) and inner autophagosomal membrane (IAM) by ESCRT-mediated membrane abscission prior to lysosomal recruitment is a critical step in the formation of functional autolysosomes

  • The Membrane-impermeable HaloTag ligand (MIL)+Membrane-permeable HaloTag ligand (MPL)− signals displayed cup- or oval-shaped structures (Fig.1d, e; arrows in h, i) in agreement with phagophore morphology[17], while MIL+MPL+ signals formed nascent autophagosome-like structures in which MIL signals (OAM-associated HT-light chain 3 (LC3)-II) surrounded MPL signals (IAM-associated HT-LC3-II) (Fig. 1f; white arrowheads in h–j) and MIL−MPL+ puncta were consistent with mature autophagosomal structures in which OAM-associated LC3-II has been delipidated (Fig. 1g; blue arrowheads in j)

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Summary

Introduction

The mechanism of phagophore closure remains unclear due to technical limitations in distinguishing unclosed and closed autophagosomal membranes. We report the HaloTag-LC3 autophagosome completion assay that 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. How the phagophore undergoes membrane remodeling to generate the inner and outer membranes of the completed autophagosome remains far from clear[4] and has been hindered by technical challenges associated with distinguishing unclosed and closed autophagosomal membranes[5]. We demonstrate that proper autophagosomal membrane closure requires the ESCRT-III component CHMP2A and the AAA-ATPase Vacuolar Protein Sorting-associated 4 (VPS4) activity and that the generation of the OAM and IAM by ESCRT-mediated membrane abscission prior to lysosomal recruitment is a critical step in the formation of functional autolysosomes

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