ESCRT-mediated phagophore sealing during mitophagy

ABSTRACT

Inactivation of the endosomal sorting complex required for transport (ESCRT) machinery has been reported to cause autophagic defects, but the exact functions of ESCRT proteins in macroautophagy/autophagy remain incompletely understood. Using live-cell fluorescence microscopy we found that the filament-forming ESCRT-III subunit CHMP4B was recruited transiently to nascent autophagosomes during starvation-induced autophagy and mitophagy, with residence times of about 1 and 2 min, respectively. Correlative light microscopy and electron tomography revealed CHMP4B recruitment at a late step in mitophagosome formation. The autophagosomal dwell time of CHMP4B was strongly increased by depletion of the regulatory ESCRT-III subunit CHMP2A. Using a novel optogenetic closure assay we observed that depletion of CHMP2A inhibited phagophore sealing during mitophagy. Consistent with this, depletion of CHMP2A and other ESCRT-III subunits inhibited both PRKN/PARKIN-dependent and -independent mitophagy. We conclude that the ESCRT machinery mediates phagophore closure, and that this is essential for mitophagic flux.

Abbreviations: BSA: bovine serum albumin; CHMP: chromatin-modifying protein; CLEM: correlative light and electron microscopy; EGFP: enhanced green fluorescent protein; ESCRT: endosomal sorting complex required for transport; HEPES: 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid; HRP: horseradish peroxidase; ILV: intralumenal vesicle; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; LOV2: light oxygen voltage 2; MLS: mitochondrial localization sequence; MT-CO2: mitochondrially encoded cytochrome c oxidase II; O+A: oligomycin and antimycin A; PBS: phosphate-buffered saline; PIPES: piperazine-N,N-bis(2-ethanesulfonic acid); PRKN/PARKIN: parkin RBR E3 ubiquitin protein ligase; RAB: RAS-related in brain; SD: standard deviation; SEM: standard error of the mean; TOMM20: TOMM20: translocase of outer mitochondrial membrane 20; VCL: vinculin; VPS4: vacuolar protein sorting protein 4; Zdk1: Zdark 1; TUBG: Tubulin gamma chain

KEYWORDS:

• ESCRT
• macroautophagy
• membrane sealing
• mitophagy
• phagophore

Acknowledgments

We thank Eva Rønning, Anne Engen, Ulrikke Dahl Brinch and Ingrid Kjos for expert technical assistance, and Tor Erik Rusten for providing Drosophila specimens. The Core Facilities for Advanced Light Microscopy and Advanced Electron Microscopy at Oslo University Hospital are acknowledged for access to relevant microscopes. Y.Z. and H.Sp. were supported by postdoctoral and doctoral fellowships, respectively, from the Research Council of Norway (grant number 249794). H.St. and A.S. were supported by project grants from the Norwegian Cancer Society (grant numbers 182698 and 171318, respectively). H.St. was also supported by a grant from the South-Eastern Norway Regional Health Authority (grant number 2016087). This work was partly supported by the Research Council of Norway through its Centres of Excellence funding scheme, project number 262652.

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

This work was supported by the Helse Sør-Øst RHF [2016087];Kreftforeningen [171318];Kreftforeningen [182698];Norges Forskningsråd [249794, 262652].