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). 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 downward arrow (X/S). Recombinant rat and yeast MPP catalyze the p --> i step 4 and 40 times faster, respectively, than the i --> m step. In isolated rat mitochondria, p-fxn undergoes a sequence of cleavages, p --> i --> m --> d(1) --> d(2), with d(1) and d(2) representing two C-terminal fragments of m-fxn produced by an unknown protease. 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 nonspecific degradation of i-fxn. Thus, the second of the two processing steps catalyzed by MPP limits the levels of mature frataxin within mitochondria.
Cavadini
P
P
Department of Pediatric & Adolescent Medicine and Biochemistry & Molecular Biology, Mayo Clinic and Foundation, 200 First Street SW, Rochester, Minnesota 55905, USA.
Adamec
J
J
Taroni
F
F
Gakh
O
O
Isaya
G
G
D000595:Amino Acid Sequence; D000818:Animals; D006801:Humans; D033862:Iron-Binding Proteins; D008928:Mitochondria / Q000378:metabolism*; D008969:Molecular Sequence Data; D010447:Peptide Hydrolases / Q000502:physiology*; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism*; D017354:Point Mutation; D051381:Rats; D000096923:Frataxin
D033862:Iron-Binding Proteins; D010447:Peptide Hydrolases; D017853:Phosphotransferases (Alcohol Group Acceptor); D000096923:Frataxin