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Maturation of frataxin within mammalian and yeast mitochondria: one-step processing by matrix processing peptidase
1Department of Physiology, University of Pennsylvania School of Medicine, D403 Richards Building, 3700 Hamilton Walk, Philadelphia, PA 19104-6085, USA.
Abstract:
Friedreich's ataxia is a neurodegenerative disease caused by mutations in the nuclear gene encoding frataxin (FRDA). FRDA is synthesized with an N-terminal signal sequence, which is removed after import into mitochondria. We have shown that FRDA was imported efficiently into isolated mammalian or yeast mitochondria. In both cases, the processing cleavage that removed the N-terminal signal sequence occurred in a single step on import, generating mature products of identical mobility. The processing cleavage could be reconstituted by incubating the FRDA preprotein with rat or yeast matrix processing peptidase (MPP) expressed in Escherichia coli. We used these assays to evaluate the import and processing of an altered form of FRDA containing the disease-causing I154F mutation. No effects on import or maturation of this mutated FRDA were observed. Likewise, no effects were observed on import and maturation of the yeast frataxin homolog (Yfh1p) carrying a homologous I130F mutation. These results argue against the possibility that the I154F mutation interferes with FRDA function via effects on maturation. Other mutations can be screened for effects on FRDA biogenesis as described here, by evaluating import into isolated mitochondria and by testing maturation with purified MPP.
Insights
Friedreich's ataxia (FRDA) is a neurodegenerative disease. This study shows that a common mutation does not affect FRDA protein import or maturation in mitochondria, suggesting other mechanisms are at play.
Area of Science:
- Molecular Biology
- Neurogenetics
- Mitochondrial Biology
Background:
- Friedreich's ataxia (FRDA) is a neurodegenerative disorder caused by mutations in the nuclear gene for frataxin (FRDA).
- Frataxin is synthesized as a precursor protein with an N-terminal signal sequence for mitochondrial import.
- Understanding FRDA biogenesis is crucial for elucidating disease mechanisms.
Purpose of the Study:
- To investigate the impact of the I154F mutation on frataxin (FRDA) import and maturation.
- To establish assays for screening FRDA biogenesis and maturation.
- To explore the role of mitochondrial matrix processing peptidase (MPP) in FRDA processing.
Main Methods:
- Import of FRDA and yeast frataxin homolog (Yfh1p) into isolated mammalian and yeast mitochondria.
- In vitro processing assays using purified rat or yeast MPP.
- Analysis of protein import and maturation using SDS-PAGE.
Main Results:
- FRDA was efficiently imported and processed into mature protein in isolated mitochondria.
- Mitochondrial matrix processing peptidase (MPP) reconstituted the single-step cleavage of the FRDA signal sequence.
- The disease-associated I154F mutation in FRDA, and a homologous mutation in Yfh1p, did not affect mitochondrial import or maturation.
Conclusions:
- The I154F mutation does not appear to impair FRDA function by affecting its mitochondrial import or maturation.
- Established assays allow for screening of other FRDA mutations for effects on biogenesis and maturation.
- Further research is needed to understand the pathogenic mechanisms of FRDA mutations affecting FRDA function.