Presequence binding to Tim50 studied by photocrosslinking

Although mitochondria possess their own protein synthesis machinery, most of the mitochondrial proteins are encoded by nuclear genes and translated on cytosolic ribosomes. Transport of these proteins into mitochondria requires coordinated action of specific protein translocases of the outer and inner mitochondrial membranes (TOM and TIM23 complexes, respectively). Most of these proteins possess an N-terminal signal, referred to as presequence. Their transport depends on sequential steps of presequence recognition by different components of mitochondrial translocases. However, the details of these interactions are mostly unknown or remain controversial. We used a photocrosslinking approach combined with affinity purification techniques and mass-spectrometry in order to identify interaction partners of the presequence peptides and their binding sites. We show here that Tim50, an essential component of the TIM23 translocase, specifically recognizes presequences and that this interaction is essential for protein import and cell viability. Moreover, we identify the presequence-binding site on Tim50, which is distinct from the Tim23-binding site. Together, these results contribute to the current model of the presequence-recognition pathway and propose a new approach to study interactions of signal peptides with their receptors in vivo.