Tom40 Functions as a Channel for Protein Retrotranslocation in the Mitochondria-Associated Degradation (MAD) Pathway

The mitochondria-associated degradation pathway (MAD) mediates removal and elimination of unfolded mitochondrial proteins by the ubiquitin-proteasome system (UPS). Here, we reconstituted retrotranslocation of MAD substrates from the mitochondrial matrix across mitochondrial inner and outer membranes in cell-free systems. This retrotranslocation is ATP- dependent but membrane potential-independent. We also identified a role for the TOM complex, the protein import channel in the mitochondrial outer membrane (MOM), in this process. Inhibition of protein translocation across the Tom40p channel inhibits retrotranslocation of MAD substrates in vitro. Deletion of the TOM5 subunit of the TOM complex inhibits retrotranslocation in vitro, and results in increased sensitivity to oxidative stress and decreased association of a MAD substrate with Cdc48p, a subunit of the segregase complex that removes proteins from mitochondria and targets them for proteasomal degradation in MAD, in vivo. Our studies indicate that unfolded proteins in the mitochondrial matrix are retrotranslocated across both mitochondrial membranes and the TOM complex is the retrotranslocation channel in the MOM in MAD.