Abstract
Mitochondria contain hundreds of proteins which are encoded by the nuclear genome and synthesized in the cytosol from where they are imported into the organelle. Sorting signals encoded in the primary and secondary sequence of these proteins mediate the recognition of newly synthesized precursor proteins and their subsequent translocation through the mitochondrial TOM and TIM translocases. Proteins of the mitochondrial matrix employ aminoterminal matrix targeting signals (MTSs), also called presequences, that are necessary and sufficient for their import into mitochondria. In most cases, these MTSs are proteolytically removed from the mature part of precursor proteins subsequent to their translocation into the matrix. Recently, internal MTS-like sequences (iMTS-Ls) were discovered in the mature region of many precursor proteins. Although these sequences are not sufficient for matrix targeting, they strongly increase the import competence of precursors by supporting their interaction with mitochondrial surface receptors. Due to their similarity to N-terminal MTSs, these iMTS-Ls can be identified using mitochondrial targeting prediction tools such as TargetP which was initially trained to recognize MTSs. In this protocol we describe how TargetP can be used to identify iMTS-Ls in protein sequences.