Distinct roles for two N-terminal cleaved domains in mitochondrial import of the yeast frataxin homolog, Yfh1p

The yeast frataxin homolog (Yfh1p) participates in mitochondrial iron homeo stasis, The phenotypic defects of the Delta yfh1 mutant include drastic acc umulation of iron in mitochondria and slow growth, The Yfh1p precursor prot ein contains two N-terminal domains that are sequentially cleaved by the ma trix processing peptidase on import into mitochondria, generating the matur e protein. We have precisely mapped these two cleavage sites, Mutations blo cking the first or the second cleavage of Yfh1p do not interfere with its i n vitro import or with its ability to complement phenotypes of the Delta yf h1 mutant strain. Distinct roles have been ascertained for the two cleaved domains of Yfh1p, The first cleaved domain (domain I) is sufficient for in vitro mitochondrial import of a non-mitochondrial passenger protein. Howeve r, neither domain I nor other matrix-targeting signals alone can support ef ficient in vitro import of mature Yfh1p, The second cleaved domain (domain II) is required as a spacer between a targeting signal and mature Yfh1p, Li kewise, when Yfh1p constructs lacking domain I or II are expressed in vivo, they fail to attain appreciable steady-state amounts in mitochondria and c annot complement phenotypes of the Delta yfh1 mutant.