Protein import and processing reconstituted with isolated rat liver mitochondria and recombinant mitochondrial...

Most mitochondrial proteins are synthesized in the cytoplasm as larger precursors carrying N-terminal matrix-targeting presequences, and are subsequently transported to the mitochondria. The presequence mediates the interaction between the precursor polypeptide and components of the mitochondrial protein import machinery, a complex apparatus that is responsible for translocation of the precursor across the two mitochondrial membranes. Once the precursor has reached the mitochondrial matrix, the presequence is removed by the general mitochondrial processing peptidase (MPP). Some precursors undergo additional processing steps carried out by specialized processing peptidases. For most mitochondrial proteins, however, cleavage by MPP is the step that precedes folding and assembly into the native form. We describe methods to isolate import-competent mitochondria from rat liver and to perform import reactions with precursor proteins synthesized in vitro by coupled transcription-translation. We also describe methods to perform in vitro processing reactions of mitochondrial precursors by recombinant MPP and to identify the cleavage sites used by this enzyme.