Zinc and the iron donor frataxin regulate oligomerization of the scaffold protein to form new Fe-S cluster assembly centers.

Early studies of the bacterial Fe-S cluster assembly system provided structural details for how the scaffold protein and the cysteine desulfurase interact. This work and additional work on the yeast and human systems elucidated a conserved mechanism for sulfur donation but did not provide any conclusive insights into the mechanism for iron delivery from the iron donor, frataxin, to the scaffold. We previously showed that oligomerization is a mechanism by which yeast frataxin (Yfh1) can promote assembly of the core machinery for Fe-S cluster synthesis both in vitro and in cells, in such a manner that the scaffold protein, Isu1, can bind to Yfh1 independent of the presence of the cysteine desulfurase, Nfs1. Here, in the absence of Yfh1, Isu1 was found to exist in two forms, one mostly monomeric with limited tendency to dimerize, and one with a strong propensity to oligomerize. Whereas the monomeric form is stabilized by zinc, the loss of zinc promotes formation of dimer and higher order oligomers. However, upon binding to oligomeric Yfh1, both forms take on a similar symmetrical trimeric configuration that places the Fe-S cluster coordinating residues of Isu1 in close proximity of iron-binding residues of Yfh1. This configuration is suitable for docking of Nfs1 in a manner that provides a structural context for coordinate iron and sulfur donation to the scaffold. Moreover, distinct structural features suggest that in physiological conditions the zinc-regulated abundance of monomeric vs. oligomeric Isu1 yields [Yfh1]·[Isu1] complexes with different Isu1 configurations that afford unique functional properties for Fe-S cluster assembly and delivery.

Mammalian pitrilysin: substrate specificity and mitochondrial targeting.

The substrate specificity of the mitochondrial metallopeptidase proteinase 1 (MP1) was investigated and its mitochondrial targeting signal identified. The substrate specificity of MP1 was examined with physiological peptides as substrates. Although the enzyme exhibits broad substrate specificity, there is a trend for peptides containing 13 or more residues to exhibit K(m) values of 2 muM or less. Three of four peptides containing 11 or fewer residues exhibited K(m) values above 10 muM. Similarly, peptides containing 13 or more residues exhibited k(cat) values below 10 min(-1), while three of four peptides containing 11 or fewer residues exhibited k(cat) values above 30 min(-1). Many of the peptide cleavage sites of MP1 resemble that of the mitochondrial processing protease (MPP); however, MP1 does not process the precursor form of citrate synthase. The enzyme, however, does cleave the released prepeptide from precitrate synthase. A mitochondria localization was shown in MP1 transfected NT2 and HepG2 cells. Deletion of the N-terminal 15 amino acids caused MP1 to be mislocalized to the cytoplasm and nucleus. Furthermore, when fused to green flourescent protein, this 15-amino acid N-terminal sequence directed the fusion protein to the mitochondria.

Functional studies of frataxin.

Mitochondria generate adenosine triphosphate (ATP) but also dangerous reactive oxygen species (ROS). One-electron reduction of dioxygen in the early stages of the electron transport chain yields a superoxide radical that is detoxified by mitochondrial superoxide dismutase to give hydrogen peroxide. The hydroxyl radical is derived from decomposition of hydrogen peroxide via the Fenton reaction, catalyzed by Fe2+ ions. Mitochondria require a constant supply of Fe2+ for heme and iron-sulfur cluster biosyntheses and therefore are particularly susceptible to ROS attack. Two main antioxidant defenses are known in mitochondria: enzymes that catalytically remove ROS, e.g. superoxide dismutase and glutathione peroxidase, and low molecular weight agents that scavenge ROS, including coenzyme Q, glutathione, and vitamins E and C. An effective defensive system, however, should also involve means to control the availability of pro-oxidants such as Fe2+ ions. There is increasing evidence that this function may be carried out by the mitochondrial protein frataxin. Frataxin deficiency is the primary cause of Friedreich's ataxia (FRDA), an autosomal recessive degenerative disease. Frataxin is a highly conserved mitochondrial protein that plays a critical role in iron homeostasis. Respiratory deficits, abnormal cellular iron distribution and increased oxidative damage are associated with frataxin defects in yeast and mouse models of FRDA. The mechanism by which frataxin regulates iron metabolism is unknown. The yeast frataxin homologue (mYfh1p) is activated by Fe(II) in the presence of oxygen and assembles stepwise into a 48-subunit multimer (alpha48) that sequesters >2000 atoms of iron in a ferrihydrite mineral core. Assembly of mYfhlp is driven by two sequential iron oxidation reactions: a fast ferroxidase reaction catalyzed by mYfh1p induces the first assembly step (alpha --> alpha3), followed by a slower autoxidation reaction that promotes the assembly of higher order oligomers yielding alpha48. Depending on the ionic environment, stepwise assembly is associated with the sequestration of < or = 50-75 Fe(II)/subunit. This Fe(II) is initially loosely bound to mYfh1p and can be readily mobilized by chelators or made available to the mitochondrial enzyme ferrochelatase to synthesize heme. However, as iron oxidation and mineralization proceed, Fe(III) becomes progressively inaccessible and a stable iron-protein complex is produced. In conclusion, by coupling iron oxidation with stepwise assembly, frataxin can successively function as an iron chaperon or an iron store. Reduced iron availability and solubility and increased oxidative damage may therefore explain the pathogenesis of FRDA.

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.

Human frataxin maintains mitochondrial iron homeostasis in Saccharomyces cerevisiae.

Frataxin is a nuclear-encoded mitochondrial protein widely conserved among eukaryotes. Human frataxin (fxn) is severely reduced in Friedreich ataxia (FRDA), a frequent autosomal recessive neuro- and cardio-degenerative disease. Whereas the function of fxn is unknown, the yeast frataxin homolog (Yfh1p) has been shown to be involved in mitochondrial iron homeostasis and protection from free radical toxicity. Evidence of iron accumulation and oxidative damage in cardiac tissue from FRDA patients suggests that fxn may have a similar function, but whether yeast and human frataxin actually have interchangeable roles in mitochondrial iron homeostasis is unknown. We show that a wild-type FRDA cDNA can complement Yfh1p-deficient yeast (yfh1 delta) by preventing the mitochondrial iron accumulation and oxidative damage associated with loss of Yfh1p. We analyze the functional effects of two FRDA point mutations, G130V and W173G, associated with a mild and a severe clinical presentation, respectively. The G130V mutation affects protein stability and results in low levels of mature (m) fxn, which are nevertheless sufficient to rescue yfh1 delta yeast. The W173G mutation affects protein processing and stability and results in severe m-fxn deficiency. Expression of the FRDA (W173G) cDNA in yfh1 delta yeast leads to increased levels of mitochondrial iron which are not as elevated as in Yfh1p-deficient cells but are above the threshold for oxidative damage of mitochondrial DNA and iron-sulfur centers, causing a typical yfh1 delta phenotype. These results demonstrate that fxn functions like Yfh1p, providing experimental support to the hypothesis that FRDA is a disorder of mitochondrial iron homeostasis.

Iron-dependent self-assembly of recombinant yeast frataxin: implications for Friedreich ataxia.

Frataxin deficiency is the primary cause of Friedreich ataxia (FRDA), an autosomal recessive cardiodegenerative and neurodegenerative disease. Frataxin is a nuclear-encoded mitochondrial protein that is widely conserved among eukaryotes. Genetic inactivation of the yeast frataxin homologue (Yfh1p) results in mitochondrial iron accumulation and hypersensitivity to oxidative stress. Increased iron deposition and evidence of oxidative damage have also been observed in cardiac tissue and cultured fibroblasts from patients with FRDA. These findings indicate that frataxin is essential for mitochondrial iron homeostasis and protection from iron-induced formation of free radicals. The functional mechanism of frataxin, however, is still unknown. We have expressed the mature form of Yfh1p (mYfh1p) in Escherichia coli and have analyzed its function in vitro. Isolated mYfh1p is a soluble monomer (13,783 Da) that contains no iron and shows no significant tendency to self-associate. Aerobic addition of ferrous iron to mYfh1p results in assembly of regular spherical multimers with a molecular mass of approximately 1. 1 MDa (megadaltons) and a diameter of 13+/-2 nm. Each multimer consists of approximately 60 subunits and can sequester >3,000 atoms of iron. Titration of mYfh1p with increasing iron concentrations supports a stepwise mechanism of multimer assembly. Sequential addition of an iron chelator and a reducing agent results in quantitative iron release with concomitant disassembly of the multimer, indicating that mYfh1p sequesters iron in an available form. In yeast mitochondria, native mYfh1p exists as monomer and a higher-order species with a molecular weight >600,000. After addition of (55)Fe to the medium, immunoprecipitates of this species contain >16 atoms of (55)Fe per molecule of mYfh1p. We propose that iron-dependent self-assembly of recombinant mYfh1p reflects a physiological role for frataxin in mitochondrial iron sequestration and bioavailability.

Functional and genomic analysis of the human mitochondrial intermediate peptidase, a putative protein partner of frataxin.

We showed recently that the yeast mitochondrial intermediate peptidase (YMIP polypeptide; gene symbol, OCT1) promotes mitochondrial iron uptake by catalyzing the maturation of iron-utilizing proteins and exacerbates the mitochondrial iron accumulation that results from loss of yeast frataxin, a mitochondrial protein required for mitochondrial iron efflux. This suggests that the human MIP (HMIP polypeptide; gene symbol MIPEP) may be one of the loci predicted to influence the clinical manifestations of Friedreich's ataxia (FRDA), an autosomal recessive neurodegenerative disease caused by lack of human frataxin. To begin to test this hypothesis, we have characterized HMIP at the functional and genomic levels. We show that HMIP can complement a yeast knock-out mutant lacking YMIP, demonstrating that HMIP and YMIP are functional homologues. The MIPEP gene spans 57 kb and consists of 19 exons that correlate with the functional domains of HMIP. Primer extension analysis has identified a major transcript of the MIPEP gene expressed differentially and predominantly in tissues with high oxygen consumption, while sequence analysis of approximately 2 kb of 5'-flanking DNA has revealed putative Mt1/3/4, NF-kappaB, and AP-1 elements that may regulate MIPEP expression in these tissues. Using a new polymorphic (CA)(n) repeat in intron 4, MIPEP has been genetically mapped within a 7-cM interval between markers D13S283 and D13S217 on 13q12. This work provides the basis for molecular analysis of MIPEP in FRDA and possibly other neurodegenerative diseases.

Processing of artificial peptide-DNA-conjugates by the mitochondrial intermediate peptidase (MIP).

Import of DNA from the cytoplasm into the mitochondrial matrix is an obligatory step for an in organello site-directed mutagenesis or gene therapy approach on mitochondrial DNA diseases. In this context, we have developed an artificial DNA translocation vector that is composed of the mitochondrial signal peptide of the ornithine transcarbamylase (OTC) and a DNA moiety. While this vector is capable of directing attached passenger molecules to the mitochondrial matrix, the recognition of this artificial molecule by the endogenous mitochondrial signal peptide processing machinery as well as the cleavage of the peptide plays a pivotal role in the release of the attached DNA. To study the proteolytic processing of the artificial vector, various signal peptide-DNA-conjugates were treated with purified mitochondrial intermediate peptidase. When the leader peptide is directly linked to the DNA moiety without an intervening spacer, MIP processing is prevented. Cleavage of the peptide can be restored, however, when the first ten amino acid residues of the mature part of OTC are appended at the carboxy-terminal end of the signal peptide. Our results show that artificial peptide-DNA-conjugates are recognized by the mitochondrial proteolytic machinery, and therefore an interference of the peptide with the DNA function can be excluded.

Yeast and human frataxin are processed to mature form in two sequential steps by the mitochondrial processing peptidase.

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, yielding an intermediate and a mature size form. A recent study found that recombinant rat mitochondrial processing peptidase (MPP) cleaves the mouse frataxin precursor to the intermediate but not the mature form (Koutnikova, H., Campuzano, V., and Koenig, M. (1998) Hum. Mol. Gen. 7, 1485-1489), suggesting that a different peptidase might be required for production of mature size frataxin. However, in the present study we show that MPP is solely responsible for maturation of yeast and human frataxin. MPP first cleaves the precursor to intermediate form and subsequently converts the intermediate to mature size protein. In this way, MPP could influence frataxin function and indirectly affect mitochondrial iron homeostasis.

Mitochondrial intermediate peptidase and the yeast frataxin homolog together maintain mitochondrial iron homeostasis in Saccharomyces cerevisiae.

Friedreich's ataxia (FRDA) is a neurodegenerative disease typically caused by a deficiency of frataxin, a mitochondrial protein of unknown function. In Saccharomyces cerevisiae, lack of the yeast frataxin homolog ( YFH1 gene, Yfh1p polypeptide) results in mitochondrial iron accumulation, suggesting that frataxin is required for mitochondrial iron homeostasis and that FRDA results from oxidative damage secondary to mitochondrial iron overload. This hypothesis implies that the effects of frataxin deficiency could be influenced by other proteins involved in mitochondrial iron usage. We show that Yfh1p interacts functionally with yeast mitochondrial intermediate peptidase ( OCT1 gene, YMIP polypeptide), a metalloprotease required for maturation of ferrochelatase and other iron-utilizing proteins. YMIP is activated by ferrous iron in vitro and loss of YMIP activity leads to mitochondrial iron depletion, suggesting that YMIP is part of a feedback loop in which iron stimulates maturation of YMIP substrates and this in turn promotes mitochondrial iron uptake. Accordingly, YMIP is active and promotes mitochondrial iron accumulation in a mutant lacking Yfh1p ( yfh1 [Delta]), while genetic inactivation of YMIP in this mutant ( yfh1 [Delta] oct1 [Delta]) leads to a 2-fold reduction in mitochondrial iron levels. Moreover, overexpression of Yfh1p restores mitochondrial iron homeostasis and YMIP activity in a conditional oct1 ts mutant, but does not affect iron levels in a mutant completely lacking YMIP ( oct1 [Delta]). Thus, we propose that Yfh1p maintains mitochondrial iron homeostasis both directly, by promoting iron export, and indirectly, by regulating iron levels and therefore YMIP activity, which promotes mitochondrial iron uptake. This suggests that human MIP may contribute to the functional effects of frataxin deficiency and the clinical manifestations of FRDA.

3-Hydroxy-3-methylglutaryl coenzyme A lyase: targeting and processing in peroxisomes and mitochondria.

3-Hydroxy-3-methylglutaryl coenzyme A lyase (HL, E.C. 4.1.3.4) has a unique dual localization in both mitochondria and peroxisomes. Mitochondrial HL ( approximately 31.0 kDa) catalyzes the last step of ketogenesis; the function of peroxisomal HL ( approximately 33.5 kDa) is unknown. On density gradient fractionation, normal human lymphoblasts contain both peroxisomal and mitochondrial HL whereas in lymphoblasts from a patient with Zellweger syndrome, in which functional peroxisomes are absent, only the mitochondrial HL isoform was present. To study the kinetics of the dual targeting of HL, we performed pulse-chase experiments in normal and Zellweger cells. Pulse-chase studies revealed a biphasic curve for processing of the HL precursor. The first phase, with a calculated half-life of approximately 3 h in both normal and Zellweger fibroblasts and lymphoblasts and in HepG2 cells, presumably reflects mitochondrial import and processing of the precursor; the second (t1/2, 12-19 h) is present only in normal cells and presumably represents the half-life of peroxisomal HL. The half-life of mature mitochondrial HL was 14 to 19 h in both normal and Zellweger cells. Studies of the HMG-CoA lyase precursor in isolated rat mitochondria showed a rate of processing approximately 2.6-fold lower than that of the ornithine transcarbamylase precursor.

Cloning, expression, and chromosomal assignment of the human mitochondrial intermediate peptidase gene (MIPEP).

The mitochondrial intermediate peptidase of Saccharomyces cerevisiae (YMIP) is a component of the yeast mitochondrial protein import machinery critically involved in the biogenesis of the oxidative phosphorylation (OXPHOS) system. This leader peptidase removes specific octapeptides from the amino terminus of nuclear-encoded OXPHOS subunits and components of the mitochondrial genetic apparatus. To address the biologic role of the human peptidase [MIPEP gene, HMIP polypeptide], we have initiated its molecular and functional characterization. A full-length cDNA was isolated by screening a human liver library using a rat MIP (RMIP) cDNA as a probe. The encoded protein contained a typical mitochondrial leader peptide and showed 92 and 54% homology to RMIP and YMIP, respectively. A survey of human mitochondrial protein precursors revealed that, similar to YMIP, HMIP is primarily involved in the maturation of OXPHOS-related proteins. Northern analysis showed that the MIPEP gene is differentially expressed in human tissues, with the highest levels of expression in the heart, skeletal muscle, and pancreas, three organ systems that are frequently affected in OXPHOS disorders. Using fluorescence in situ hybridization, the MIPEP locus was assigned to 13q12. This information offers the possibility of testing the potential involvement of HMIP in the pathophysiology of nuclear-driven OXPHOS disorders.

Mutations in a putative zinc-binding domain inactivate the mitochondrial intermediate peptidase.

The mitochondrial intermediate peptidase (MIP) cleaves characteristic octapeptides, (F/L/I)XX(T/S/ G)XXXX(decreases), from the N-terminus of many imported mitochondrial proteins. This leader peptidase is activated by divalent cations and inactivated by thiol-blocking agents, properties which are typical of metallo- and cysteine-proteases, respectively. To elucidate the mechanism of action of MIP, we analyzed by site-directed mutagenesis the functional role of a putative zinc-binding domain (F-H-E-X-G-H-(X)2-H-(X)12-G-(X)5-D-(X)2-E-X-P-S-(X)3-E) and two cysteine residues (C131 and C581), which are highly conserved in evolutionarily distant MIP sequences. We show that two histidines and a glutamic acid in the H-E-X-G-H motif and a glutamic acid 25 residues from the second histidine are essential for MIP function in vivo. In contrast, C131 and C581 are important for protein stability but are not required for activity in vivo or in vitro. These findings are consistent with MIP being a metallopeptidase.

Prediction and identification of new natural substrates of the yeast mitochondrial intermediate peptidase.

Most mitochondrial precursor proteins are processed to the mature form in one step by mitochondrial processing peptidase (MPP), while a subset of precursors destined for the matrix or the inner membrane are cleaved sequentially by MPP and mitochondrial intermediate peptidase (MIP). We showed previously that yeast MIP (YMIP) is required for mitochondrial function in Saccharomyces cerevisiae. To further define the role played by two-step processing in mitochondrial biogenesis, we have now characterized the natural substrates of YMIP. A total of 133 known yeast mitochondrial precursors were collected from the literature and analyzed for the presence of the motif RX(decreases)(F/L/I)XX(T/S/G)XXXX(decreases), typical of precursors cleaved by MPP and MIP. We found characteristic MIP cleavage sites in two distinct sets of proteins: respiratory components, including subunits of the electron transport chain and tricarboxylic acid cycle enzymes, and components of the mitochondrial genetic machinery, including ribosomal proteins, translation factors, and proteins required for mitochondrial DNA metabolism. Representative precursors from both sets were cleaved to predominantly mature form by mitochondrial matrix or intact mitochondria from wild-type yeast. In contrast, intermediate-size forms were accumulated upon incubation of the precursors with matrix from mip1 delta yeast or intact mitochondria from mip1ts yeast, indicating that YMIP is necessary for maturation of these proteins. Consistent with the fact that some of these substrates are essential for the maintenance of mitochondrial protein synthesis and mitochondrial DNA replication, mip1 delta yeast undergoes loss of functional mitochondrial genomes.

Cloning of glutaryl-CoA dehydrogenase cDNA, and expression of wild type and mutant enzymes in Escherichia coli.

We have cloned, sequenced, and expressed cDNAs encoding wild type human glutaryl-CoA dehydrogenase subunit, and have expressed a mutant enzyme found in a patient with glutaric acidemia type I. The mutant protein is expressed at the same level as the wild type in Escherichia coli, but has less than 1% of the activity of wild-type dehydrogenase. We also present evidence that the glutaryl-CoA dehydrogenase transcript is alternatively spliced in human fibroblasts and liver; the alternatively spliced mRNA, when expressed in E.coli, encodes a stable but inactive protein. Purified expressed human glutaryl-CoA dehydrogenase has kinetic constants similar to those of the previously purified porcine dehydrogenase. The primary translation product from in vitro transcribed glutaryl-CoA dehydrogenase mRNA is translocated into mitochondria and processed in the same manner as most other nuclear-encoded mitochondrial proteins. Human glutaryl-CoA dehydrogenase shows 53% sequence similarity to porcine medium chain acyl-CoA dehydrogenase, and these similarities were utilized to predict structure-function relationships in glutaryl-CoA dehydrogenase.

Mammalian mitochondrial intermediate peptidase: structure/function analysis of a new homologue from Schizophyllum commune and relationship to thimet oligopeptidases.

Mitochondrial intermediate peptidase (MIP) is a component of the mitochondrial protein import machinery required for maturation of nuclear-encoded precursor proteins targeted to the mitochondrial matrix or inner membrane. We previously characterized this enzyme in rat (RMIP) and Saccharomyces cerevisiae (YMIP) and showed that MIP activity is essential for mitochondrial function in yeast. We have now defined the structure of a new MIP homologue (SMIP) from the basidiomycete fungus Schizophyllum commune. SMIP includes 4 exons of 523, 486, 660, and 629 bp separated by 3 short introns. The predicted SMIP, YMIP, and RMIP sequences share 31-37% identity and 54-57% similarity over 700 amino acids. When SMIP and RMIP were expressed in a yeast mip1 delta mutant, they were both able to rescue the respiratory-deficient phenotype caused by genetic inactivation of YMIP, indicating that the function of this enzyme is conserved in eukaryotes. Moreover, the MIP sequences show 20-24% identity and 40-47% similarity to a family of oligopeptidases from bacteria, yeast, and mammals. MIP and these proteins are characterized by a highly conserved motif, F-H-E-X-G-H-(X)2-H-(X)12-G-(X)5-D-(X)2-E-X-P-S-(X)3-E-X, centered around a zinc-binding site and appear to represent a new family of genes associated with proteolytic processing in the mitochondrial and cytosolic compartments.

MIP1, a new yeast gene homologous to the rat mitochondrial intermediate peptidase gene, is required for oxidative metabolism in Saccharomyces cerevisiae.

Cleavage of amino-terminal octapeptides, F/L/IXXS/T/GXXXX, by mitochondrial intermediate peptidase (MIP) is typical of many mitochondrial precursor proteins imported to the matrix and the inner membrane. We previously described the molecular characterization of rat liver MIP (RMIP) and indicated a putative homolog in the sequence predicted from gene YCL57w of yeast chromosome III. A new yeast gene, MIP1, has now been isolated by screening a Saccharomyces cerevisiae genomic library with an RMIP cDNA probe. MIP1 predicts a protein of 772 amino acids (YMIP), which is 54% similar and 31% identical to RMIP and includes a putative 37-residue mitochondrial leader peptide. RMIP and YMIP contain the sequence LFHEMGHAM HSMLGRT, which includes a zinc-binding motif, HEXXH, while the predicted YCL57w protein contains a comparable sequence with a lower degree of homology. No obvious biochemical phenotype was observed in a chromosomally disrupted ycl57w mutant. In contrast, a mip1 mutant was unable to grow on nonfermentable substrates, while a mip1 ycl57w double disruption did not result in a more severe phenotype. The mip1 mutant exhibited defects of complexes III and IV of the respiratory chain, caused by failure to carry out the second MIP-catalyzed cleavage of the nuclear-encoded precursors for cytochrome oxidase subunit IV (CoxIV) and the iron-sulfur protein (Fe-S) of the bc1 complex to mature proteins. In vivo, intermediate-size CoxIV was accumulated in the mitochondrial matrix, while intermediate-size Fe-S was targeted to the inner membrane. Moreover, mip1 mitochondrial fractions failed to carry out maturation of the human ornithine transcarbamylase intermediate (iOTC), specifically cleaved by RMIP. A CEN plasmid-encoded YMIP protein restored normal MIP activity along with respiratory competence. Thus, YMIP is a functional homolog of RMIP and represents a new component of the yeast mitochondrial import machinery.

Sequence analysis of rat mitochondrial intermediate peptidase: similarity to zinc metallopeptidases and to a putative yeast homologue.

Proteolytic removal of amino-terminal octapeptides from mitochondrial intermediate proteins is a required step for a subgroup of nuclear-encoded mitochondrial precursors and is specifically catalyzed by mitochondrial intermediate peptidase (MIP). We recently reported the purification of MIP from rat liver and showed that the enzyme is a monomer of 75 kDa. We now report the sequence of a full-length rat MIP cDNA. This cDNA codes for a protein of 710 amino acids, including an amino-terminal mitochondrial leader peptide of 33 residues. The region surrounding the mature MIP amino terminus shows a cleavage site typically recognized by the general mitochondrial processing peptidase (MPP). In vitro synthesized MIP precursor is cleaved to mature MIP by purified MPP, and thus MIP is not required for its own proteolytic maturation. Comparison of the deduced MIP sequence with other sequences in the GenBank data base reveals two important similarities. The first is to a sequence encoding a putative MIP homologue in the recently reported sequence of yeast chromosome III. The putative yeast protein is predicted to be 712 amino acids long and includes a putative 23-residue mitochondrial leader peptide also with a MPP processing site. It shows 47% similarity and 24% identity to rat MIP. The second similarity is to members of a subfamily of metallopeptidases that includes rat metalloendopeptidase EC 3.4.24.15 and two bacterial proteases, oligopeptidase A and dipeptidyl carboxypeptidase. A region of greater than 50% similarity over 400 residues between MIP and these proteins is centered around the sequence motif HEXXH, typical of zinc metallopeptidases.