A selectivity filter in Tim17 mediates intramitochondrial protein sorting

Mitochondria import hundreds of proteins into the matrix and the inner membrane (IM). The translocase of the IM of mitochondria (TIM23 complex) sorts these proteins using topogenic signals in their structure and directs them into the matrix or inserts them into the IM. How these signals are deciphered is unknown. Here we show that Tim17, the protein-conducting subunit of the TIM23 complex, contains a highly conserved selectivity filter for incoming proteins. Using a fluorescence-based sorting reporter, we identified the mitochondrial oxidoreductase Dmo2 (akin to DMAC1 in humans) as factor for intramitochondrial protein sorting. Molecular dynamics simulations show that a Dmo2-dependent disulfide bond into Tim17 constitutes a negatively charged selectivity filter at the vestibule of the TIM23 complex that arrests membrane proteins and facilitates the lateral insertion. Our work provides a molecular rationale as to how the TIM23 translocase differentiates between soluble and membrane proteins.