Mitochondria play a major role in the maintenance of cellular homeostasis, e. g. by synthesis of ATP through oxidative phosphorylation. Because of their importance, mitochondrial quality control is extremely important for the cell and damaged mitochondria must be quickly eliminated. One of the most studied processes for removal of damaged mitochondria is PINK1/Parkin-dependent mitophagy, where PINK1 recruits the cytosolic E3 ubiquitin ligase parkin to damaged mitochondria. Parkin then mediates the ubiquitylation of mitochondrial proteins, leading to their degradation. The regulation of parkin during later stages of mitophagy is yet to be determined. This process is linked to Parkinson"s disease (PD), as both, PINK1 and PARKIN genes are linked to recessive, early onset PD. Thus, a thorough insight on the regulation of the main participants can potentially lead to important novel therapeutic targets. Our recent work identified an outside-in progression of this process and ubiquitylation of parkin itself during late stages of mitophagy.

In this study we measure protein turnover during PINK1/Parkin-dependent mitophagy using dynamic SILAC (stable isotope labelling with amino acids in cell culture). HeLa cells expressing WT parkin and/or ligase-dead parkin (C431A) were grown in DMEM medium supplemented with "light" amino acids (Lys0, Arg0). Upon induction of mitophagy by addition of the protonophore carbonyl cyanide m-chlorophenyl hydrazone (CCCP), the medium was exchanged to DMEM containing "heavy" isotopes (Lys8, Arg10). Cells were harvested at six time points ranging from 0 to 24 hours post-induction. Of about 3,500 protein groups identified, protein turnover was determined for about 1,800 protein groups. Upon CCCP treatment, turnover was overall decreased in both cell lines with a few exceptions. Interestingly, several proteins exhibiting an increased turnover differed between WT and C431A parkin cells. One protein that was found to be significantly upregulated during late stages of mitophagy and thus exhibits a faster turnover in the WT cells is ISG15, a small, ubiquitin-like protein. Its upregulation was further confirmed by western blotting as well as by analysis of previous data from our lab. ISG15 is known as a central player in antiviral immunity but its role is not limited to this process. Although conjugation of ISG15 to mitochondrial proteins has been reported, the role of this modification remains to be studied. We are currently investigating potential targets of ISGylation during mitophagy by further examination of data as well as co-immunoprecipitation assays.