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 mip1Δ 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.     

Cloning, Expression, and Chromosomal Assignment of the Human Mitochondrial Intermediate Peptidase Gene (MIPEP)

The mitochondrial intermediate peptidase ofSaccharomyces 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 fluorescencein situhybridization, 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.     

Mrs3p, Mrs4p, and frataxin provide iron for Fe-S cluster synthesis in mitochondria

Yeast Mrs3p and Mrs4p are evolutionarily conserved mitochondrial carrier proteins that transport iron into mitochondria under some conditions. Yeast frataxin (Yfh1p), the homolog of the human protein implicated in Friedreich ataxia, is involved in iron homeostasis. However, its precise functions are controversial. Anaerobically grown triple mutant cells (Deltamrs3/4/Deltayfh1) displayed a severe growth defect corrected by in vivo iron supplementation. Because anaerobically grown cells do not synthesize heme, and they do not experience oxidative stress, this growth defect was most likely due to Fe-S cluster deficiency. Fe-S cluster formation was assessed in anaerobically grown cells shifted to air for a brief period. In isolated mitochondria, Fe-S clusters were detected on newly imported yeast ferredoxin precursor and on endogenous aconitase by means of [35S]cysteine labeling and native gel separation. New cluster formation was dependent on iron addition to mitochondria, and the iron concentration dependence was shifted dramatically upward in the Deltamrs3/4 mutant, indicating a role of Mrs3/4p in iron transport. The frataxin mutant strain lacked protein import capacity because of low mitochondrial membrane potential, although this was partially restored by growth in the presence of high iron. Under these conditions, a kinetic defect in new Fe-S cluster formation was still noted. Import of frataxin into frataxin-minus isolated mitochondria promptly corrected the Fe-S cluster assembly defect without further iron addition. These findings show that Mrs3/4p transports iron into mitochondria, whereas frataxin makes iron already within mitochondria available for Fe-S cluster synthesis.     

Conserved functions of Arabidopsis mitochondrial late-acting maturation factors in the trafficking of iron‑sulfur clusters

Numerous proteins require iron?sulfur (Fe-S) clusters as cofactors for their function. Their biogenesis is a multi-step process occurring in the cytosol and mitochondria of all eukaryotes and additionally in plastids of photosynthetic eukaryotes. A basic model of Fe-S protein maturation in mitochondria has been obtained based on studies achieved in mammals and yeast, yet some molecular details, especially of the late steps, still require investigation. In particular, the late-acting biogenesis factors in plant mitochondria are poorly understood. In this study, we expressed the factors belonging to NFU, BOLA, SUFA/ISCA and IBA57 families in the respective yeast mutant strains. Expression of the Arabidopsis mitochondrial orthologs was usually sufficient to rescue the growth defects observed on specific media and/or to restore the abundance or activity of the defective Fe-S or lipoic acid-dependent enzymes. These data demonstrate that the plant mitochondrial counterparts, including duplicated isoforms, likely retained their ancestral functions. In contrast, the SUFA1 and IBA57.2 plastidial isoforms cannot rescue the lysine and glutamate auxotrophies of the respective isa1-isa2Δ and iba57Δ strains or of the isa1-isa2-iba57Δ triple mutant when expressed in combination. This suggests a specialization of the yeast mitochondrial and plant plastidial factors in these late steps of Fe-S protein biogenesis, possibly reflecting substrate-specific interactions in these different compartments.     

The yeast vacuolar proton-translocating ATPase contains a subunit homologous to the Manduca sexta and bovine e subunits that is essential for function

The yeast cwh36Delta mutant was identified in a screen for yeast mutants exhibiting a Vma(-) phenotype suggestive of loss of vacuolar proton-translocating ATPase (V-ATPase) activity. The mutation disrupts two genes, CWH36 and a recently identified open reading frame on the opposite strand, YCL005W-A. We demonstrate that disruption of YCL005W-A is entirely responsible for the Vma(-) growth phenotype of the cwh36Delta mutant. YCL005W-A encodes a homolog of proteins associated with the Manduca sexta and bovine chromaffin granule V-ATPase. The functional significance of these proteins for V-ATPase activity had not been tested, but we show that the protein encoded by YCL005W-A, which we call Vma9p, is essential for V-ATPase activity in yeast. Vma9p is localized to the vacuole but fails to reach the vacuole in a mutant lacking one of the integral membrane subunits of the V-ATPase. Vma9p is associated with the yeast V-ATPase complex in vacuolar membranes, as demonstrated by co-immunoprecipitation with known V-ATPase subunits and glycerol gradient fractionation of solubilized vacuolar membranes. Based on this evidence, we propose that Vma9p is a genuine subunit of the yeast V-ATPase and that e subunits may be a functionally essential part of all eukaryotic V-ATPases.     

A nuclear mutant of Saccharomyces cerevisiae deficient in mitochondrial DNA replication and polymerase activity

We have isolated a thermosensitive mutant which is transformed into a population of cells devoid of mitochondrial DNA (rho 0 cells) at 35 degrees C and is deficient in mitochondrial (mt) DNA polymerase activity. A single recessive nuclear mutation (mip1) is responsible for rho 0 phenotype and mtDNA polymerase deficiency in vitro. At 25 degrees C (or 30 degrees C) a dominant suppressor mutation (SUP) masks the deficiency in vivo. The meiotic segregants (mip1 sup) which do not harbor the suppressor have a rho 0 phenotype both at 25 and 35 degrees C. They have no mtDNA polymerase activity, in contrast with MIP rho 0 mutants of mitochondrial inheritance which do exhibit mtDNA polymerase activity. In the thermosensitive mutant (mip1 SUP), the replication of mtDNA observed in vivo at 30 degrees C is completely abolished at 35 degrees C. In the meiotic segregants (mip1 sup), no mtDNA replication takes place at 30 and 35 degrees C. The synthesis of nuclear DNA is not affected. DNA polymerases may have replicative and/or repair activity. There is no evidence that mip mutants are deficient in mtDNA repair. In contrast the MIP gene product is strictly required for the replication of mtDNA and for the expression of the mtDNA polymerase activity. This enzyme might be the replicase of mtDNA.     

Isolation and characterization of the putative nuclear modifier gene MTO1 involved in the pathogenesis of deafness-associated mitochondrial 12 S rRNA A1555G mutation

The human mitochondrial 12 S rRNA A1555G mutation has been found to be associated with aminoglycoside-induced and non-syndromic deafness. However, putative nuclear modifier gene(s) have been proposed to regulate the phenotypic expression of this mutation. In yeast, the mutant alleles of MTO1, encoding a mitochondrial protein, manifest respiratory-deficient phenotype only when coupled with the mitochondrial 15 S rRNA P(R)454 mutation corresponding to human A1555G mutation. This suggests that the MTO1-like modifier gene may influence the phenotypic expression of human A1555G mutation. Here we report the identification of full-length cDNA and elucidation of genomic organization of the human MTO1 homolog. Human Mto1 is an evolutionarily conserved protein that implicates a role in the mitochondrial tRNA modification. Functional conservation of this protein is supported by the observation that isolated human MTO1 cDNA can complement the respiratory deficient phenotype of yeast mto1 cells carrying P(R)454 mutation. MTO1 is ubiquitously expressed in various tissues, but with a markedly elevated expression in tissues of high metabolic rates including cochlea. These observations suggest that human MTO1 is a structural and functional homolog of yeast MTO1. Thus, it may play an important role in the pathogenesis of deafness-associated A1555G mutation in 12 S rRNA gene or mutations in tRNA genes.     

The Mammalian Proteins MMS19, MIP18, and ANT2 Are Involved in Cytoplasmic Iron-Sulfur Cluster Protein Assembly

Iron-sulfur (Fe-S) clusters are essential cofactors of proteins with a wide range of biological functions. A dedicated cytosolic Fe-S cluster assembly (CIA) system is required to assemble Fe-S clusters into cytosolic and nuclear proteins. Here, we show that the mammalian nucleotide excision repair protein homolog MMS19 can simultaneously bind probable cytosolic iron-sulfur protein assembly protein CIAO1 and Fe-S proteins, confirming that MMS19 is a central protein of the CIA machinery that brings Fe-S cluster donor proteins and the receiving apoproteins into proximity. In addition, we show that mitotic spindle-associated MMXD complex subunit MIP18 also interacts with both CIAO1 and Fe-S proteins. Specifically, it binds the Fe-S cluster coordinating regions in Fe-S proteins. Furthermore, we show that ADP/ATP translocase 2 (ANT2) interacts with Fe-S apoproteins and MMS19 in the CIA complex but not with the individual proteins. Together, these results elucidate the composition and interactions within the late CIA complex.     

Mutation in the Fe-S scaffold protein Isu bypasses frataxin deletion

Frataxin is a conserved mitochondrial protein deficient in patients with Friedreich's ataxia. Frataxin has been implicated in control of iron homoeostasis and Fe-S cluster assembly. In yeast or human mitochondria, frataxin interacts with components of the Fe-S cluster synthesis machinery, including the cysteine desulfurase Nfs1, accessory protein Isd11 and scaffold protein Isu. In the present paper, we report that a single amino acid substitution (methionine to isoleucine) at position 107 in the mature form of Isu1 restored many deficient functions in Δyfh1 or frataxin-depleted yeast cells. Iron homoeostasis was improved such that soluble/usable mitochondrial iron was increased and accumulation of insoluble/non-usable iron within mitochondria was largely prevented. Cytochromes were returned to normal and haem synthesis was restored. In mitochondria carrying the mutant Isu1 and no frataxin, Fe-S cluster enzyme activities were improved. The efficiency of new Fe-S cluster synthesis in isolated mitochondria was markedly increased compared with frataxin-negative cells, although the response to added iron was minimal. The M107I substitution in the highly conserved Isu scaffold protein is typically found in bacterial orthologues, suggesting that a unique feature of the bacterial Fe-S cluster machinery may be involved. The mechanism by which the mutant Isu bypasses the absence of frataxin remains to be determined, but could be related to direct effects on Fe-S cluster assembly and/or indirect effects on mitochondrial iron availability.     

The iron-sulphur protein Ind1 is required for effective complex I assembly

NADH:ubiquinone oxidoreductase (complex I) of the mitochondrial inner membrane is a multi-subunit protein complex containing eight iron-sulphur (Fe-S) clusters. Little is known about the assembly of complex I and its Fe-S clusters. Here, we report the identification of a mitochondrial protein with a nucleotide-binding domain, named Ind1, that is required specifically for the effective assembly of complex I. Deletion of the IND1 open reading frame in the yeast Yarrowia lipolytica carrying an internal alternative NADH dehydrogenase resulted in slower growth and strongly decreased complex I activity, whereas the activities of other mitochondrial Fe-S enzymes, including aconitase and succinate dehydrogenase, were not affected. Two-dimensional gel electrophoresis, in vitro activity tests and electron paramagnetic resonance signals of Fe-S clusters showed that only a minor fraction (approximately 20%) of complex I was assembled in the ind1 deletion mutant. Using in vivo and in vitro approaches, we found that Ind1 can bind a [4Fe-4S] cluster that was readily transferred to an acceptor Fe-S protein. Our data suggest that Ind1 facilitates the assembly of Fe-S cofactors and subunits of complex I.     

Characterization of MSM1, the structural gene for yeast mitochondrial methionyl-tRNA synthetase

Respiratory-deficient mutants of Saccharomyces cerevisiae assigned to pet complementation group G72 are impaired in mitochondrial protein synthesis. The loss of this activity has been correlated with the inability of the mutants to acylate the two methionyl-tRNAs of yeast mitochondria. A nuclear gene (MSM1) capable of complementing the respiratory deficiency has been cloned by transformation of the G72 mutant C122/U3 with a yeast genomic library. In situ disruption of the MSM1 gene in a wild-type haploid strain of yeast induces a respiratory-deficient phenotype but does not affect the ability of the mutant to grow on fermentable substrates indicating that the product of MSM1 functions only in mitochondrial protein synthesis. Mitochondrial extracts prepared from the mutant with the disrupted copy of MSM1 were found to be defective in acylation of the two mitochondrial methionyl-tRNAs thereby confirming the identity of MSM1 as the structural gene for the mitochondrial methionyl-tRNA synthetase. The sequence of the protein encoded by MSM1 is similar to the Escherichia coli and yeast cytoplasmic methionyl-tRNA synthetases. Based on the primary-sequence similarities of the three proteins, the mitochondrial enzyme appears to be more related to the bacterial than to the yeast cytoplasmic methionyl-tRNA synthetase.     

MINOS1 is a conserved component of mitofilin complexes and required for mitochondrial function and cristae organization

The inner membrane of mitochondria is especially protein rich and displays a unique morphology characterized by large invaginations, the mitochondrial cristae, and the inner boundary membrane, which is in proximity to the outer membrane. Mitochondrial inner membrane proteins appear to be not evenly distributed in the inner membrane, but instead organize into functionally distinct subcompartments. It is unknown how the organization of the inner membrane is achieved. We identified MINOS1/MIO10 (C1orf151/YCL057C-A), a conserved mitochondrial inner membrane protein. mio10-mutant yeast cells are affected in growth on nonfermentable carbon sources and exhibit altered mitochondrial morphology. At the ultrastructural level, mutant mitochondria display loss of inner membrane organization. Proteomic analyses reveal MINOS1/Mio10 as a novel constituent of Mitofilin/Fcj1 complexes in human and yeast mitochondria. Thus our analyses reveal new insight into the composition of the mitochondrial inner membrane organizing machinery.     

Loss of Cardiolipin Leads to Perturbation of Mitochondrial and Cellular Iron Homeostasis

Cardiolipin (CL) is the signature phospholipid of mitochondrial membranes, where it is synthesized locally and plays a critical role in mitochondrial bioenergetic functions. The importance of CL in human health is underscored by the observation that perturbation of CL biosynthesis causes the severe genetic disorder Barth syndrome. To fully understand the cellular response to the loss of CL, we carried out genome-wide expression profiling of the yeast CL mutant crd1Δ. Our results show that the loss of CL in this mutant leads to increased expression of iron uptake genes accompanied by elevated levels of mitochondrial iron and increased sensitivity to iron and hydrogen peroxide. Previous studies have shown that increased mitochondrial iron levels result from perturbations in iron-sulfur (Fe-S) cluster biogenesis. Consistent with an Fe-S defect, deletion of ISU1, one of two ISU genes that encode the mitochondrial Fe-S scaffolding protein essential for the synthesis of Fe-S clusters, led to synthetic growth defects with the crd1Δ mutant. We further show that crd1Δ cells have reduced activities of mitochondrial Fe-S enzymes (aconitase, succinate dehydrogenase, and ubiquinol-cytochrome c oxidoreductase), as well as cytosolic Fe-S enzymes (sulfite reductase and isopropylmalate isomerase). Increased expression of ATM1 or YAP1 did not rescue the Fe-S defects in crd1Δ. These findings show for the first time that CL is required for Fe-S biogenesis to maintain mitochondrial and cellular iron homeostasis.     

Mitochondrial DNA defects in Saccharomyces cerevisiae caused by functional interactions between DNA polymerase gamma mutations associated with disease in human

The yeast mitochondrial DNA (mtDNA) replicase Mip1 has been used as a model to generate five mutations equivalent to POLG mutations associated with a broad spectrum of diseases in human. All mip1 mutations, alone or in combination in cis or in trans, increase mtDNA instability as measured by petite frequency and Ery(R) mutant accumulation. This phenotype is associated with decreased Mip1 levels in mitochondrial extracts and/or decreased polymerase activity. We have demonstrated that (1) in the mip1(G651S) (hG848S) mutant the high mtDNA instability and increased frequency of point Ery(R) mutations is associated with low Mip1 levels and polymerase activity; (2) in the mip1(A692T-E900G) (hA889T-hE1143G) mutant, A692T is the major contributor to mtDNA instability by decreasing polymerase activity, and E900G acts synergistically by decreasing Mip1 levels; (3) in the mip1(H734Y)/mip1(G807R) (hH932Y/hG1051R) mutant, H734Y is the most deleterious mutation and acts synergistically with G807R as a result of its dominant character; (4) the mip1(E900G) (h1143G) mutation is not neutral but results in a temperature-sensitive phenotype associated with decreased Mip1 levels, a property explaining its synergistic effect with mutations impairing the polymerase activity. Thus, the human E1143G mutation is not a true polymorphism.     

A yeast nuclear gene, MRS1, involved in mitochondrial RNA splicing: nucleotide sequence and mutational analysis of two overlapping open reading frames on opposite strands.

We have cloned a 1.6-kb fragment of yeast nuclear DNA, which complements pet- mutant MK3 (mrs1). This mutant was shown to be defective in mitochondrial RNA splicing: the excision of intron 3 from the mitochondrial COB pre-RNA is blocked. The DNA sequence of the nuclear DNA fragment revealed two open reading frames (ORF1 with 1092 bp; ORF2 with 735 bp) on opposite strands, which overlap by 656 bp. As shown by in vitro mutagenesis, ORF1, but not ORF2, is responsible for complementation of the splice defect. Hence, ORF1 represents the nuclear MRS1 gene. Disruption of the gene (both ORFs) in the chromosomal DNA of the respiratory competent yeast strain DBY747 (long form COB gene) leads to a stable pet- phenotype and to the accumulation of the same mitochondrial RNA precursors as in strain MK3. The amino acid sequence of the putative ORF1 product does not exhibit any homology with other known proteins, except for a small region of homology with the gene product of another nuclear yeast gene involved in mitochondrial RNA splicing, CBP2. The function of the MRS1 (ORF1) gene in mitochondrial RNA splicing and the significance of the overlapping ORFs in this gene are discussed.     

RNA silencing of mitochondrial m-Nfs1 reduces Fe-S enzyme activity both in mitochondria and cytosol of mammalian cells

In prokaryotes and yeast, the general mechanism of biogenesis of iron-sulfur (Fe-S) clusters involves activities of several proteins among which IscS and Nfs1p provide, through cysteine desulfuration, elemental sulfide for Fe-S core formation. Although these proteins have been well characterized, the role of their mammalian homolog in Fe-S cluster biogenesis has never been evaluated. We report here the first functional study that implicates the putative cysteine desulfurase m-Nfs1 in the biogenesis of both mitochondrial and cytosolic mammalian Fe-S proteins. Depletion of m-Nfs1 in cultured fibroblasts through small interfering RNA-based gene silencing significantly inhibited the activities of mitochondrial NADH-ubiquinone oxidoreductase (complex I) and succinate-ubiquinone oxidoreductase (complex II) of the respiratory chain, as well as aconitase of the Krebs cycle, with no alteration in their protein levels. Activity of cytosolic xanthine oxidase, which holds a [2Fe-2S] cluster, was also specifically reduced, and iron-regulatory protein-1 was converted from its [4Fe-4S] aconitase form to its apo- or RNA-binding form. Reduction of Fe-S enzyme activities occurred earlier and more markedly in the cytosol than in mitochondria, suggesting that there is a mechanism that primarily dedicates m-Nfs1 to the biogenesis of mitochondrial Fe-S clusters in order to maintain cell survival. Finally, depletion of m-Nfs1, which conferred on apo-IRP-1 a high affinity for ferritin mRNA, was associated with the down-regulation of the iron storage protein ferritin.     

On the effect of cholesterol on the fate of CYP11A1 imported into yeast mitochondria in vivo

It has earlier been shown that CYP11A1 (cytochrome P450scc precursor), synthesized in yeast cells, is imported into yeast mitochondria. However, in large part the foreign protein undergoes degradation or aggregates. In this work, we tried to prevent aggregation of CYP11A1 and stimulate its insertion into the mitochondrial inner membrane by substituting cholesterol (a substrate for cytochrome P450scc) for ergosterol in yeast cells. To this end, an ergosterol-deficient Saccharomyces cerevisiae mutant, growing in the presence of cholesterol and expressing a modified bovine CYP11A1 gene, was used. Under defined conditions, the mitochondrial respiratory system developed in this yeast and CYP11A1 with the CoxIV targeting presequence was imported into the mitochondria, being then proteolytically processed. However, substitution of cholesterol for ergosterol did not result in lowered aggregation of the imported CYP11A1 and its increased content in the SMP fraction. Hence, the presence of cholesterol is not instrumental in proper intramitochondrial compartmentalization and folding of CYP11A1.     

Adrenodoxin reductase homolog (Arh1p) of yeast mitochondria required for iron homeostasis

Arh1p is an essential mitochondrial protein of yeast with reductase activity. Here we show that this protein is involved in iron metabolism. A yeast strain was constructed in which the open reading frame was placed under the control of a galactose-regulated promoter. Protein expression was induced by galactose and repressed to undetectable levels in the absence of galactose, although cells grew quite well in the absence of inducer. Under noninducing conditions, cellular iron uptake was dysregulated, exhibiting a failure to repress in response to medium iron. Iron trafficking within the cell was also disturbed. Exposure of Arh1p-depleted cells to increasing iron concentrations during growth led to drastic increases in mitochondrial iron, indicating a loss of homeostatic control. Activity of aconitase, a prototype Fe-S protein, was deficient at all concentrations of mitochondrial iron, although the protein level was unaltered. Heme protein deficiencies were exacerbated in the iron-loaded mitochondria, suggesting a toxic side effect of accumulated iron. Finally, a time course correlated the cellular depletion of Arh1p with the coordinated appearance of various mutant phenotypes including dysregulated cellular iron uptake, deficiency of Fe-S protein activities in mitochondria and cytoplasm, and deficiency of hemoproteins. Thus, Arh1p is required for control of cellular and mitochondrial iron levels and for the activities of Fe-S cluster proteins.