Topogenic sequence recognition at TIM complexes revealed by a stendomycin-bound structure

In the mitochondrial inner membrane (IM), topogenesis of imported proteins is mediated by TIM23 and TIM22 complexes. TIM23 translocates soluble polypeptides across the IM into the matrix, whereas TIM22 inserts polytopic membrane proteins into the IM. Although functionally distinct, both rely on homologous subunits, Tim17 in TIM23 and Tim22 in TIM22. The underlying mechanisms, however, remain elusive. Here we use structural and functional approaches with yeast Tim17, Tim22 and the TIM23 inhibitor stendomycin. Cryogenic-electron microscopy shows that stendomycin binds to the protein translocation cavity of Tim17, mimicking α-helical topogenic sequences. While Tim22 does not bind stendomycin, a single mutation in its equivalent cavity suffices to enable binding. The cavities of Tim17 and Tim22 are largely interchangeable without disrupting their functions. Lastly, stendomycin triggers a collapse of the membrane potential, likely via its Tim17- or Tim22-dependent translocation across the IM. These findings reveal a mechanistic overlap between protein translocases and insertases.