p62/SQSTM1 is required for Parkin-induced mitochondrial clustering but not mitophagy; VDAC1 is dispensable for both

Abstract

Mitochondria sustain damage with aging, and the resulting mitochondrial dysfunction has been implicated in a number of diseases including Parkinson disease. We recently demonstrated that the E3 ubiquitin ligase Parkin, which is linked to recessive forms of parkinsonism, causes a dramatic increase in mitophagy and a change in mitochondrial distribution, following its translocation from the cytosol to mitochondria. Investigating how Parkin induces these changes may offer insight into the mechanisms that lead to the sequestration and elimination of damaged mitochondria. We report that following Parkin’s translocation from the cytosol to mitochondria, Parkin (but not a pathogenic mutant) promotes the K63-linked polyubiquitination of mitochondrial substrate(s) and recruits the ubiquitin- and LC3-binding protein, p62/SQSTM1, to mitochondria. After its recruitment, p62/SQSTM1 mediates the aggregation of dysfunctional mitochondria through polymerization via its PB1 domain, in a manner analogous to its aggregation of polyubiquitinated proteins. Surprisingly and in contrast to what has been recently reported for ubiquitin-induced pexophagy and xenophagy, p62 appears to be dispensable for mitophagy. Similarly, mitochondrial-anchored ubiquitin is sufficient to recruit p62 and promote mitochondrial clustering, but does not promote mitophagy. Although VDAC1 (but not VDAC2) is ubiquitinated following mitochondrial depolarization, we find VDAC1 cannot fully account for the mitochondrial K63-linked ubiquitin immunoreactivity observed following depolarization, as it is also observed in VDAC1/3-/- mouse embryonic fibroblasts. Additionally, we find VDAC1 and VDAC3 are dispensable for the recruitment of p62, mitochondrial clustering and mitophagy. These results demonstrate that mitochondria are aggregated by p62, following its recruitment by Parkin in a VDAC1-independent manner. They also suggest that proteins other than p62 are likely required for mitophagy downstream of Parkin substrates other than VDAC1.

p62/SQSTM1 at the interface of aging, autophagy, and disease

“…Interestingly, this would also suggest that when proteasome activity is impaired, p62/SQSTM1 could deliver proteasome substrates to the lysosome through the autophagy machinery, even though no evidence for such a process has been found. p62/SQSTM1 promotes selective autophagy of proteins, protein aggregates, organelles, and bacteria (Narendra et al 2010a;Geisler et al 2010a;Pankiv et al 2007;Zheng et al 2009). Autophagy substrates are usually tagged with noncanonical poly-ubiquitin chains (K63 or K27 linkages) that are brought into sequestosomes by associating with the UBA domain of p62/SQSTM1.…”

“…Mitochondria are tagged for degradation by two E3 ligases: ring finger protein 185 (Rnf185) and parkin (Narendra et al 2010a;Geisler et al 2010a;Tang et al 2011). Both enzymes catalyze the formation of noncanonical ubiquitin chains (K63 and K27 linkages) on mitochondrial outer membrane proteins.…”

“…This is recognized as a signal to recruit the E3 ligase parkin from the cytosol to the mitochondria (Narendra et al 2010b). Here, parkin tags several mitochondrial outer membrane proteins with K63-and K27-linked poly-ubiquitin chains (Narendra et al 2010a;Geisler et al 2010a;Okatsu et al 2010). Once the mitochondrial proteins are tagged with ubiquitin, p62/SQSTM1 associates with them and drives the whole organelle into sequestosomes that are then cleared by autophagy (Narendra et al 2010a;Geisler et al 2010a).…”

“…Here, parkin tags several mitochondrial outer membrane proteins with K63-and K27-linked poly-ubiquitin chains (Narendra et al 2010a;Geisler et al 2010a;Okatsu et al 2010). Once the mitochondrial proteins are tagged with ubiquitin, p62/SQSTM1 associates with them and drives the whole organelle into sequestosomes that are then cleared by autophagy (Narendra et al 2010a;Geisler et al 2010a). Interestingly, p62/SQSTM1 may be dispensable for parkin mediated mitophagy, but its presence is necessary to cluster the damaged mitochondria away from the remainder of the mitochondrial network (Narendra et al 2010a) and protect it from further damage.…”

“…Activating autophagy has beneficial effects in many models of age-related pathologies, especially metabolic syndrome, cardiovascular dysfunction (Mizushima et al 2008;Choi et al 2013), and neurodegenerative diseases such as Alzheimer's (Spilman et al 2010), Huntington's (Ravikumar et al 2004), and Parkinson's disease (Narendra et al 2010a;Geisler et al 2010a;Burman et al 2012). In particular, substrate-specific autophagy plays an important role in removing protein aggregates and damaged organelles that associate with these pathologies; substrate-specific autophagy is mediated by tagging targets with ubiquitin, and substrates are delivered to the autophagosomes through adaptor proteins, prominently the multifunctional protein scaffold p62/ SQSTM1.…”