NMR identification of the Tom20 binding segment in mitochondrial presequences

Many mitochondrial proteins are synthesized in the cytosol as precursors wi th N-terminal presequences, and are imported into mitochondria with the aid of translocator protein complexes containing presequence-binding proteins. Tom20, a receptor protein which functions in an early step of the mitochon drial protein import, recognizes presequences with disvergent amino acid se quences. Here, we report the identification of the segments involved in bin ding to Tom20 in mitochondrial presequences. We monitored the chemical shif t perturbation of the NMR signals of five different N-15-labeled presequenc e peptides by the addition of the cytosolic receptor domain of rat or yeast Tom20. The perturbed segments occupy different positions, either near the N terminus or at the C terminus, in the presequences. Spin label experiment s revealed that this is not due to different orientations of the presequenc e peptides bound to Tom20. The results presented here will offer a starting point to perform detailed analyses of Tom20-binding elements by systematic amino acid replacements. (C) 2001 Academic Press.