Mitochondria-associated degradation (MAD) mediated by the Cdc48 complex and proteasome degrades ubiquitinated mitochondrial outer-membrane proteins. MAD is critical for mitochondrial proteostasis, but it remains poorly characterized. We identified several mitochondrial Cdc48 substrates and developed a genetic screen assay to uncover regulators of the Cdc48-dependent MAD pathway. Surprisingly, we identified Doa1, a substrate-processing factor of Cdc48 that inhibits the degradation of some Cdc48 substrates, as a critical mediator of the turnover of mitochondrial Cdc48 substrates. Deletion ofDOA1causes the accumulation and mislocalization of substrates on mitochondria. Profiling of Cdc48 cofactors shows that Doa1 and Cdc48(-Ufd1-Npl4)form a functional complex mediating MAD. Biochemically, Doa1 interacts with ubiquitinated substrates and facilitates substrate recruitment to the Cdc48(-Ufd1-Npl4)complex. Functionally, Doa1 is critical for cell survival under mitochondrial oxidative stress, but not ER stress, conditions. Collectively, our results demonstrate the essential role of the Doa1-Cdc48(-Ufd1-Npl4)complex in mitochondrial proteostasis and suggest that Doa1 plays dual roles on the Cdc48 complex.
© 2016 Wu et al.