Mitochondrial intermediate peptidase

This chapter examines the structural chemistry and the preparation of mitochondrial intermediate peptidase (MIP). MIP denotes cleavage of intermediate-sized mitochondrial proteins to the mature form. MIP activity can be measured by incubation of in vitro translated octapeptide-containing precursors with isolated mitochondria. Under such conditions, the cleavage catalyzed by MIP is at the end of a mitochondrial protein import reaction which also involves outer membrane receptors, outer and inner membrane translocation complexes, molecular chaperones and MPP. If the precursor is incubated directly with mitochondrial matrix or purified enzyme, initial cleavage by MPP is required to observe processing of the intermediate by MIP. This requirement is circumvented when MIP activity is determined using an intermediate protein as the substrate. Intermediates that are translated in vitro from a methionine artificially placed at the octapeptide N-terminus can be processed to the mature form by MIP independent of the presence of MPP. Native RMIP has been purified 2250-fold from rat liver mitochondrial matrix with a final yield of about 2%. Expression of recombinant enzyme has been achieved in S. cerevisiae.