Abstract:
Mitochondria fulfill several key functions in cellular energy metabolism and signaling, such as the generation of ATP by oxidative phosphorylation and life-death decisions. Dysfunctional mitochondria pose a threat to cellular homeostasis and have to be quickly removed by the cell. PINK1/Parkin-dependent mitophagy is an important pathway for the mitochondrial quality control (MQC) mechanism to protect cells against pathogenic accumulation of dysfunctional mitochondria. During the initiation of this multistep process post-translational modifications (PTMs), such as phosphorylation by the PINK1 kinase and ubiquitylation by the E3-ubiquitin ligase parkin, function together on ubiquitin, the ubiquitin like (UBL) domain of parkin and outer mitochondrial membrane (OMM) proteins. Quantitative proteomics has already been used to investigate the crosstalk between PTMs during early stages of this process. However, late stages of mitophagy that ultimately lead to the degradation of mitochondria remain understudied.
To obtain a deeper insight into parkin-mediated mitochondrial degradation, I initially established a subcellular fractionation workflow to analyze mitochondrial proteins as well as their ubiquitylation and phosphorylation dynamics by quantitative proteomics after induction of mitophagy in HeLa cells. To this end, I combined cytoplasmic, membrane-bound and soluble nuclear protein fractions, which enabled analysis of exclusive mitophagy-relevant fractions and met the requirements of high quantity input material for subsequent phosphoproteome and ubiquitylome analyses. I then applied different chemical labeling strategies, such as dimethyl labeling and tandem mass tags in combination with high-resolution orbitrap mass spectrometry to study the regulation of protein degradation, ubiquitylation and phosphorylation during early (2-6 h post induction) and late stages (12-18 h post induction) of mitophagy in parkin wild-type and ligase-dead HeLa cells, which were generated and treated with the protonophore Carbonyl cyanide m-chlorophenyl hydrazone (CCCP) in the group of Philipp Kahle. Integration and extensive bioinformatic analysis of all three proteomic datasets revealed that mitochondrial degradation proceeds stepwise and protein ubiquitylation precedes protein degradation. Unexpectedly, protein phosphorylation revealed only a minor effect during this process. In the presence of functional parkin, mitochondrial proteins are ubiquitylated followed by degradation in an outside-in fashion on the mitochondrion. This begins on the OMM and proceeds inwards to the inner
Summary Mitochondrial biology
IV
mitochondrial membrane (IMM) and finally the mitochondrial matrix. Several OMM proteins displayed a similar pattern of modification by ubiquitylation, with an overall minor cross-talk between phosphorylation and ubiquitin-dependent protein degradation.
Further analysis of the mechanisms underlying the stepwise degradation of mitochondrial subcompartments revealed its strong dependency on lysosomal, as well as on proteasomal activity. By application of quantitative proteomics techniques, I could validate the biochemical experiments from the Kahle group that pointed to a general dependency of mitochondrial protein degradation on proteasomal activity. In a temporal study involving inhibition of the proteasome at various timepoints upon induction of mitophagy and subsequent proteome measurements, I could confirm that especially OMM proteins like TOM70 fail to be degraded.
In summary, this thesis provides new insights on the mechanisms involved in parkin-dependent mitophagy and will serve as a resource for future work on this essential cellular process.
Proteomics