Endosomes facilitate mitochondrial clearance by enhancing Parkin recruitment to mitochondria

Abstract

13Mitochondrial quality control and homeostasis is ensured by removal of the damaged 14 mitochondria involving lysosomal and proteasomal degradation mechanisms. Though 15 much remains to be elucidated, recent findings suggest important roles for endosomal 16 machinery in the elimination of damaged mitochondria. In this study, we report a novel 17 function for a FYVE-like domain containing endosomal ubiquitin ligase, CARP2 in 18 mitophagy. Our results show that endosomal CARP2 associates with damaged 19 mitochondria, and this association precedes that of Parkin, whose recruitment is known 20 to be essential for mitochondrial clearance. Association of Parkin to damaged 21 mitochondria was substantially reduced in CARP2 KO cells upon CCCP treatment. We 22 also demonstrate that Mitofusin-2 degradation, which is a prerequisite for Parkin 23 recruitment during damage, was inhibited in CARP2-deficient cells. Hence, we conclude 24 that endosomal-associated CARP2 facilitates mitophagy by regulating Parkin recruitment 25 and activation by targeting Mitofusin-2. Our study proposes a model on how 26 heterogenous endocytic machinery can interact with damaged mitochondria and unravel 27 a new mechanism for mitochondrial clearance. 28 29 30 31 32 33 Stringent quality control mechanisms ensure organelle and cellular homeostasis. 34 Signaling pathways that regulate mitochondrial quality and function are believed to play 35 critical roles in diverse physiological processes including cell survival and their 36 dysregulation results in the development of numerous pathological conditions like 37 neurological disorders, cancer and myopathies (Arun, Liu and Donmez, 2015; Ahuja, 38 2018; Porporato et al., 2018). Autophagy is a conserved intracellular catabolic process in 39 which cytoplasmic contents, including damaged mitochondria, are sequestered in double-40 membrane autophagosomes and delivered to the lysosomal compartment for 41 degradation (Dikic, 2017). Selective elimination of dysfunctional and damaged 42 mitochondria involving autophagic machinery and lysosomes is known as mitophagy 43 (Montava-Garriga and Ganley, 2020). 44 Though cells can employ various other cellular tools like ESCRT machinery (Hammerling 45 et al., 2017), MDVs (Mitochondrial Derived Vesicles) (Sugiura et al., 2014; McLelland et 46 al., 2016) etc., to remove mitochondrial contents, mitophagy mediated by PINK1/Parkin 47 is relatively well studied (Narendra et al., 2008(Narendra et al., , 2010 Durcan and Fon, 2015). In 48 depolarized mitochondria, PINK1 accumulated on the outer mitochondrial membrane 49 phosphorylates Parkin which in turn pleiotropically ubiquitinates Mfn2 resulting in its 50 degradation. Mfn2 degradation mediated by Parkin is believed to be a critical step in 51 Parkin stabilization and mitophagy, as Mfn2 regulates physical attachment of 52 mitochondria to ER. Many details of the sequence of events involved in Parkin 53 recruitment, stabilization and identity of proteins are not available. 54 Recent reports, however, suggested a role...

CARP2 regulates the Golgi dynamics upon EGF stimulation

“…Amino acid cysteine at positions 5 and 6 are the residues predicted to be involved in palmitoylation. Our lab along with other groups showed that mutation of C5,6 to A disrupts the CARP2 localization to intracellular vesicles 25 . We have used this variant of CARP2 to assess the importance of CARP2 endosomal localization for Golgi dispersal.…”

“…Epidermal Growth factor (E9644, Sigma). Polyclonal CARP2 antibody raised in Rabbit (generated in our laboratory) 25 .…”