Analysis of mitochondrial translocases TOM and TIM by the patch-clamping technique.

Mitochondrial protein import and sorting relies on sophisticated molecular machineries or translocases, of which channels are integral. Channels are built upon membrane proteins whose functions are driven by conformational changes. This implies that structural and functional information need to be integrated to gain a deep understanding of their dynamic behavior. Patch-clamp approaches are well suited for this purpose. This chapter provides a detailed description and practical guidance for applying the patch-clamp methodology to the electrophysiological characterization of mitochondrial protein import. Implementing the technique to intact mitochondria, mitoplasts, and reconstituted proteoliposomes, combined with genetically modified yeast strains, expands the scope of these studies. Focused on the TOM, TIM23, and TIM22 translocases, an analysis of the patch-clamp contribution to the field is outlined.