We showed previously that maturation of the human
frataxin precursor (p-fxn) involves two cleavages by the
mitochondrial processing peptidase (MPP). This observation
was not confirmed by another group, however,
who reported only 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 frataxin in human tissues. The two cleavages
occur between residues 41–42 and 55–56, and both
match the MPP consensus sequence RX ↓ (X/S). Recombinant
rat and yeast MPP catalyze the pài step 4 and 40
times faster, respectively, than the i à m step. In isolated
rat mitochondria, p-fxn undergoes a sequence of
cleavages, p à i à m à d1 à d2, with d1 and d2 representing
two C-terminal fragments of m-fxn produced by
an unknown protease. The iàm step is limiting, and the
overall rate of p à i à m does not exceed the rate of mà
d1 à d2, such that the levels of m-fxn do not change
during incubations as long as 3 h. Inhibition of the iàm
step by a disease-causing frataxin mutation (W173G)
leads to nonspecific degradation of i-fxn. Thus, the second
of the two processing steps catalyzed by MPP limits
the levels of mature frataxin within mitochondria
