Two-step processing of human frataxin by mitochondrial processing peptidase - Precursor and intermediate forms are cleaved at different rates

We showed previously that maturation of the human frataxin precursor (p-fxn ) involves two cleavages by the mitochondrial processing peptidase (MPP), T his observation was not confirmed by another group, however, who reported o nly one cleavage. Here, we demonstrate conclusively that MPP cleaves p-fxn in two sequential steps, yielding a 18,826-Da intermediate (i-fxn) and a 17 ,255-Da mature (m-fxn) form, the latter corresponding to endogenous frataxi n in human tissues. The two cleavages occur between residues 41-42 and 55-5 6, and both match the MPP consensus sequence RX down arrow (X/S). Recombina nt rat and yeast MPP catalyze the p --> i step 4 and 40 times faster, respe ctively, than the i --> m step. In isolated rat mitochondria, p-fxn undergo es a sequence of cleavages, p --> i --> m --> d(1) --> d(2), with d(1) and d(2) representing two C-terminal fragments of m-Rm produced by an unknown p rotease, The i --> m step is limiting, and the overall rate of p --> i --> m does not exceed the rate of m --> d(1) --> d(2), such that the levels of m-fxn do not change during incubations as long as 3 h. Inhibition of the i --> m step by a disease-causing frataxin mutation (W173G) leads to nonspeci fic degradation of i-fxn, Thus, the second of the two processing steps cata lyzed by MPP limits the levels of mature frataxin within mitochondria.