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Most mitochondrial proteins are encoded in the nucleus and synthesized in the cytoplasm as larger precursors containing NH2‐terminal ‘leader’ peptides. To test whether a leader peptide is sufficient to direct mitochondrial import, we fused the cloned nucleotide sequence encoding the leader peptide of the mitochondrial matrix enzyme ornithine transcarbamylase (OTC) with the sequence encoding the cytosolic enzyme dihydrofolate reductase (DHFR). The fused sequence, joined with SV40 regulatory elements, was introduced along with a selectable marker into a mutant CHO cell line devoid of endogenous DHFR. In stable transformants, the predicted 26‐K chimeric precursor protein and two additional proteins, 22 K and 20 K, were detected by immunoprecipitation with anti‐DHFR antiserum. In the presence of rhodamine 6G, an inhibitor of mitochondrial import, only the chimeric precursor was detected. Immunofluorescent staining of stably transformed cells with anti‐DHFR antiserum produced a pattern characteristic of mitochondrial localization of immunoreactive material. When the chimeric precursor was synthesized in a cell‐free system and incubated post‐translationally with isolated rat liver mitochondria, it was imported and converted to a major product of 20 K that associated with mitochondria and was resistant to proteolytic digestion by externally added trypsin. Thus, both in intact cells and in vitro, a leader sequence is sufficient to direct the post‐translational import of a chimeric precursor protein by mitochondria.

Most mitochondrial proteins are encoded in the nucleus and are translated on free cytoplasmic ribosomes as larger precursors containing amino-terminal "leader" sequences, which are removed after the precursors are taken up by mitochondria. We have deduced the complete primary structure of the precursor of a human mitochondrial matrix enzyme, ornithine transcarbamylase (OTC), from the nucleotide sequence of cloned complementary DNA. The amino-terminal leader peptide of OTC is 32 amino acids in length and contains four arginines but no acidic residues. Cleavage of the leader peptide from the "mature" protein occurs between glutamine and asparagine residues. The sequence of mature human OTC resembles that of the subunits of both OTC and aspartate transcarbamylase from Escherichia coli. The biological activity of the cloned OTC complementary DNA was tested by joining it with SV40 (an animal virus) regulatory elements and transfecting cultured HeLa cells, which do not normally express OTC. Both the precursor and mature forms of the OTC subunit were identified; in stable transformants, enzymatic activity was also detected.

A papain-type cysteine endopeptidase with a molecular mass of 35 kDa for the mature enzyme, was purified from germinating castor bean (Ricinus communis L.) endosperm by virtue of its capacity to process the glyoxysomal malate dehydrogenase precursor protein to the mature subunit in vitro (C. Gietl et al., 1997, Plant Physiol 113: 863-871). The cDNA clones from endosperm of germinating seedlings and from developing seeds were isolated and sequence analysis revealed that a very similar or identical peptidase is synthesised in both tissues. Sequencing established a presequence for co-translational targeting into the endoplasmic reticulum, an N-terminal propeptide and a C-terminal KDEL motif for the castor bean cysteine endopeptidase precursor. The 45-kDa pro-enzyme stably present in isolated organelles was enzymatically active. Immunocytochemistry with antibodies raised against the purified cysteine endopeptidase revealed highly specific labelling of ricinosomes, organelles which co-purify with glyoxysomes from germinating Ricinus endosperm. The cysteine endopeptidase from castor bean endosperm, which represents a senescing tissue, is homologous to cysteine endopeptidases from other senescing tissues such as the cotyledons of germinating mung bean (Vigna mungo) and vetch (Vicia sativa), the seed pods of maturing French bean (Phaseolus vulgaris) and the flowers of daylily (Hemerocallis sp.).

Frataxin is a nuclear-encoded mitochondrial protein which is deficient in Friedreich's ataxia, a hereditary neurodegenerative disease. Yeast mutants lacking the yeast frataxin homologue (Yfh1p) show iron accumulation in mitochondria and increased sensitivity to oxidative stress, suggesting that frataxin plays a critical role in mitochondrial iron homeostasis and free radical toxicity. Both Yfh1p and frataxin are synthesized as larger precursor molecules that, upon import into mitochondria, are subject to two proteolytic cleavages, Recent studies have shown that the yeast frataxin homologue (YFH1, gene; Yfh1p, polypeptide) is a nuclear-encoded mitochondrial protein (1-4) and that its deficiency results in mitochondrial iron overload (1, 2, 5), which in turn leads to increased production of free radicals and loss of mitochondrial function (1). Similarly, iron deposits (6), multiple mitochondrial enzyme deficiencies (7), and hypersensitivity to oxidative stress (8) have been reported in studies on Friedreich's ataxia (FRDA), 1 a recessively inherited neurodegenerative disease caused by a deficiency of human frataxin (9, 10). Thus, it is believed that frataxin plays a critical role in mitochondrial iron homeostasis and free radical toxicity and that this function is conserved between yeast and mammals (7, 11). Not surprisingly, Yfh1p and mammalian frataxin share similar pathways of mitochondrial import and processing. The Yfh1p precursor (pYfh1p) is imported by isolated yeast mitochondria and processed to an intermediate (iYfh1p) and a mature size (mYfh1p) form (12). Production of mYfh1p is impaired in mitochondria isolated from yeast with mutations in the mitochondrial Hsp70 homologue Ssq1p, and similar to Yfh1p-deficient yeast (yfh1⌬) (1, 2, 5), ssq1 mutants accumulate large amounts of mitochondrial iron (12), indicating that production of mYfh1p is required for mitochondrial iron homeostasis. The mouse frataxin precursor is also cleaved twice, and missense mutations corresponding to those found in FRDA patients dramatically reduce the efficiency of the second cleavage (13), further demonstrating the importance of proteolytic processing for frataxin function. Mitochondrial processing peptidase (MPP; EC 3.4.24.64) (14) was shown to catalyze conversion of the mouse frataxin precursor to intermediate form, but the peptidase responsible for formation of mature frataxin was not identified (13). Additionally, it has not yet been established whether the intermediate forms of Yfh1p and frataxin represent productive intermediates, in that they are actually processed to the mature form.In this study, we analyze proteolytic processing of Yfh1p and human frataxin and demonstrate that both proteins are processed to the mature form in two sequential steps by MPP. EXPERIMENTAL PROCEDURESYeast Strains, Plasmids, and Media-The strains used in this study are all isogenic derivatives of strain YPH501 (see Table I). Construction of oct1⌬, yfh1⌬, and isogenic 0 strains was described previously (15, 16). For complementation of yfh1⌬ by...