Ubp3 mediates dynamic deubiquitination of mitochondria upon induction of mitophagy
Ensuring quality and maintenance of mitochondria within eukaryotic cells is important for cellular fitness. One critical pathway that needs to be tightly controlled to achieve this is mitophagy, which is negatively regulated by the deubiquitinase Ubp3 in Saccharomyces cerevisiae. Here we show that beyond this established role of Ubp3, it is critical to mediate substantial temporal changes of the mitochondrial ubiquitin landscape during mitophagy. Isolated mitochondria displayed extensive ubiquitination under steady-state conditions, whereas rapamycin-mediated induction of mitophagy/autophagy led to a progressive elimination of mitochondrial ubiquitination. A systematic screening analysis revealed that Ubp3 and Ubp4 are crucial regulators of rapamycin-induced mitochondrial deubiquitination. Deletion of Bre5, a cofactor of Ubp3, or of the UBI4 gene, encoding Ubi4 required for mitophagy, did not impair deubiquitination of mitochondria during mitophagy, indicating that Ubp3 has additional functions. We further showed that Ubi4 acts epistatic to Ubp3 for regulating mitophagy, supporting that mitophagy does not simply depend on the extent of mitochondrial ubiquitination, but rather on specific ubiquitinated substrates. Consistent with the role of Ubp3 in vivo, purified Ubp3 or its catalytic domain alone were sufficient to efficiently deubiquitinate isolated mitochondria in vitro in a Bre5-independent manner. Together, these findings highlight Ubp3 as a prominent rheostat of mitochondrial ubiquitin remodelling, revealing mitochondrial ubiquitin storage and its dynamic release as another layer of stress regulation by ubiquitin-dependent quality control pathways.