Delayed protein translocation protects mitochondria against toxic CAT-tailed proteins

In the ribosome-associated quality control acting on nuclear-encoded mitochondrial proteins (mitoRQC), Vms1 protects mitochondria from toxic effects of Rqc2-generated C-terminal alanyl and threonyl (CAT) tailed proteins that escaped proteosomal degradation facilitated by E3 ubiquitin ligase Ltn1. Here, we performed a genome-wide screen in yeast to identify novel proteins involved in mitoRQC. We found that Pth2, a peptidyl-tRNA hydrolase in the outer mitochondrial membrane, influences aggregation of mitochondrial-targeted CAT-tailed proteins without majorly affecting the CAT-tailing process itself. Peptidyl-tRNA hydrolase activity is essential during this process, however, the activity of Pth2 can be substituted by another peptidyl-tRNA hydrolase, upon proper localization. Our data suggest that Pth2 acts through modulating protein import into mitochondria, enabling CAT-tailed proteins to get access to the cytosolic chaperones and thus relieving the mitochondrial proteostasis network. Other hits obtained in the screen show that, in general, slowing down protein translocation protects mitochondria against toxic CAT-tailed proteins.