Abstract
Mitophagy, the selective degradation of mitochondria by autophagy, affects defective mitochondria following damage or stress. At the onset of mitophagy, parkin ubiquitylates proteins on the mitochondrial outer membrane. While the role of parkin at the onset of mitophagy is well understood, less is known about its activity during later stages in the process. Here, we used HeLa cells expressing catalytically active or inactive parkin to perform temporal analysis of the proteome, ubiquitylome, and phosphoproteome during 18 h after induction of mitophagy by mitochondrial uncoupler carbonyl cyanide m-chlorophenyl hydrazine. Abundance profiles of proteins downregulated in parkin-dependent manner revealed a stepwise and “outside–in” directed degradation of mitochondrial subcompartments. While ubiquitylation of mitochondrial outer membrane proteins was enriched among early parkin-dependent targets, numerous mitochondrial inner membrane, matrix, and cytosolic proteins were also found ubiquitylated at later stages of mitophagy. Phosphoproteome analysis revealed a possible crosstalk between phosphorylation and ubiquitylation during mitophagy on key parkin targets, such as voltage-dependent anion channel 2.
Highlights
We report dynamics of the mitochondrial proteome during parkin-dependent mitophagy. Our data point to outside–in degradation of mitochondria. Inner membrane and matrix proteins are ubiquitylated after 12 and 18 h of mitophagy. Interplay of phosphorylation and ubiquitylation leads to degradation of VDAC2
While ubiquitylation of mitochondrial outer membrane proteins was enriched among early parkin-dependent targets, numerous mitochondrial inner membrane, matrix, and cytosolic proteins were found ubiquitylated at later stages of mitophagy
During the course of mitophagy, some mitochondrial outer membrane (MOM) proteins are known to be eliminated at early stages, whereas some mitochondrial proteins remain ubiquitylated after intermediate depolarization times on damaged mitochondria [12,13,14]
Summary
In Brief We used a quantitative proteomics approach to study dynamics of the mitochondrial proteome, ubiquitylome, and phosphoproteome during early (2–6 h) and late stages (12–18 h) of mitophagy. After extensive parkin-dependent ubiquitylation events, ubiquitin-binding autophagy receptor proteins are recruited, which trigger the engulfment of damaged mitochondria by the autophagosome This eventually promotes mitochondrial degradation after the autophagosome fused. Even though it is well accepted that damaged mitochondria are engulfed as a single whole entity [19, 20], an alternative hypothesis suggests a sequential subcompartment degradation In this context, Yoshii et al [21] demonstrated that autophagosomes engulf complete mitochondria after 6 h of depolarization, but MOM-ruptured and mitochondrial inner membrane (MIM)-ruptured mitochondria are engulfed after extensive depolarization times (12 h carbonyl cyanide m-chlorophenyl hydrazine [CCCP]). We identify novel parkin and downstream-acting substrates under mitophagy conditions; suggest that parkin-dependent mitochondrial degradation is tightly regulated in space and time; and indicate a potential crosstalk between ubiquitylation and phosphorylation able to influence specific protein degradation during PINK1/parkindependent mitophagy. We suggest an outside– in degradation of mitochondria, initiated by parkin activity during mitophagy
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