Artificial targeting of autophagy components to mitochondria reveals both conventional and unconventional mitophagy pathways.

ABSTRACT

Macroautophagy/autophagy enables lysosomal degradation of a diverse array of intracellular material. This process is essential for normal cellular function and its dysregulation is implicated in many diseases. Given this, there is much interest in understanding autophagic mechanisms of action in order to determine how it can be best targeted therapeutically. In mitophagy, the selective degradation of mitochondria via autophagy, mitochondria first need to be primed with signals that allow the recruitment of the core autophagy machinery to drive the local formation of an autophagosome around the target mitochondrion. To determine how the recruitment of different core autophagy components can drive mitophagy, we took advantage of the mito-QC mitophagy assay (an outer mitochondrial membrane-localized tandem mCherry-GFP tag). By tagging autophagy proteins with an anti-mCherry (or anti-GFP) nanobody, we could recruit them to mitochondria and simultaneously monitor levels of mitophagy. We found that targeting ULK1, ATG16L1 and the different Atg8-family proteins was sufficient to induce mitophagy. Mitochondrial recruitment of ULK1 and the Atg8-family proteins induced a conventional mitophagy pathway, requiring RB1CC1/FIP200, PIK3C3/VPS34 activity and ATG5. Surprisingly, the mitophagy pathway upon recruitment of ATG16L1 proceeded independently of ATG5, although it still required RB1CC1 and PIK3C3/VPS34 activity. In this latter pathway, mitochondria were alternatively delivered to lysosomes via uptake into early endosomes.Abbreviation aGFP anti-GFP nanobody; amCh anti-mCherry nanobody; ATG autophagy related; ATG16L1 autophagy related 16 like 1; AUTAC/AUTOTAC autophagy-targeting chimera; BafA1 bafilomycin A1; CALCOCO2/NDP52 calcium binding and coiled-coil domain 2; CCCP carbonyl cyanide m-chlorophenylhydrazone; COX4/COX IV cytochrome c oxidase subunit 4; DFP deferiprone; DMSO dimethyl sulfoxide; GABARAP GABA type A receptor-associated protein; GABARAPL1 GABA type A receptor associated protein like 1; HSPD1/HSP60 heat shock protein family D (Hsp60) member 1; HRP horseradish peroxidase; HTRA2/OMI HtrA serine peptidase 2; IB immunoblotting; IF immunofluorescence; KO knockout; LAMP1 lysosomal associated membrane protein 1; LIR LC3-interacting region; MAP1LC3/LC3 microtubule associated protein 1 light chain 3; MEF mouse embryonic fibroblast; NBR1 NBR1 autophagy cargo receptor; OMM outer mitochondrial membrane; OPA1 OPA1 mitochondrial dynamin like GTPase; OPTN optineurin; (D)PBS (Dulbecco's) phosphate-buffered saline; PD Parkinson disease; PFA paraformaldehyde; POI protein of interest; PtdIns3K class III phosphatidylinositol 3-kinase; PtdIns3P phosphatidylinositol-3-phosphate; RAB RAB, member RAS oncogene family; RB1CC1/FIP200 RB1 inducible coiled-coil 1; SQSTM1 sequestosome 1; TAX1BP1 Tax1 binding protein 1; ULK unc-51 like autophagy activating kinase 1; VPS vacuolar protein sorting; WIPI WD repeat domain, phosphoinositide interacting.