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()(F/L/I)XX(T/S/G)XXXX(), 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 mip1Δ yeast or intact mitochondria from mip1 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, mip1Δ yeast undergoes loss of functional mitochondrial genomes.

[1]  B. Lemire,et al.  Isolation and characterization of a Saccharomyces cerevisiae mutant disrupted for the succinate dehydrogenase flavoprotein subunit. , 1991, The Journal of biological chemistry.

[2]  F. Foury,et al.  A single‐stranded DNA binding protein required for mitochondrial DNA replication in S. cerevisiae is homologous to E. coli SSB. , 1992, The EMBO journal.

[3]  F. Hartl,et al.  Transport into mitochondria and intramitochondrial sorting of the Fe/S protein of ubiquinol-cytochrome c reductase , 1986, Cell.

[4]  R. A. Rollins,et al.  Mammalian mitochondrial intermediate peptidase: structure/function analysis of a new homologue from Schizophyllum commune and relationship to thimet oligopeptidases. , 1995, Genomics.

[5]  P. Edwards,et al.  COQ2 is a candidate for the structural gene encoding para-hydroxybenzoate:polyprenyltransferase. , 1992, The Journal of biological chemistry.

[6]  B. Lemire,et al.  Isolation and nucleotide sequence of the Saccharomyces cerevisiae gene for the succinate dehydrogenase flavoprotein subunit. , 1992, The Journal of biological chemistry.

[7]  A. Tzagoloff,et al.  Characterization of a yeast nuclear gene (MST1) coding for the mitochondrial threonyl-tRNA1 synthetase. , 1985, The Journal of biological chemistry.

[8]  W. Dowhan,et al.  The cytoplasmically‐made subunit IV is necessary for assembly of cytochrome c oxidase in yeast. , 1985, The EMBO journal.

[9]  S. Ackerman,et al.  Mitochondrial translational-initiation and elongation factors in Saccharomyces cerevisiae. , 1991, European journal of biochemistry.

[10]  L. Rosenberg,et al.  Sequence analysis of rat mitochondrial intermediate peptidase: similarity to zinc metallopeptidases and to a putative yeast homologue. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[11]  G von Heijne,et al.  Cleavage-site motifs in mitochondrial targeting peptides. , 1990, Protein engineering.

[12]  A. Vassarotti,et al.  Sequences distal to the mitochondrial targeting sequences are necessary for the maturation of the F1-ATPase beta-subunit precursor in mitochondria. , 1987, The Journal of biological chemistry.

[13]  M. Yaffe,et al.  Import of proteins into yeast mitochondria: the nuclear MAS2 gene encodes a component of the processing protease that is homologous to the MAS1‐encoded subunit. , 1988, The EMBO journal.

[14]  R. Jensen,et al.  MAS1, a gene essential for yeast mitochondrial assembly, encodes a subunit of the mitochondrial processing protease. , 1988, The EMBO journal.

[15]  J. Heitman,et al.  A yeast cyclophilin gene essential for lactate metabolism at high temperature. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[16]  N. Rawlings,et al.  [32] Thimet oligopeptidase and oligopeptidase M or neurolysin , 1995 .

[17]  A. Myers,et al.  Mitochondrial protein synthesis is required for maintenance of intact mitochondrial genomes in Saccharomyces cerevisiae. , 1985, The EMBO journal.

[18]  E. Hurt,et al.  The first twelve amino acids (less than half of the pre‐sequence) of an imported mitochondrial protein can direct mouse cytosolic dihydrofolate reductase into the yeast mitochondrial matrix. , 1985, The EMBO journal.

[19]  R. Schweyen,et al.  The nuclear gene MRS2 is essential for the excision of group II introns from yeast mitochondrial transcripts in vivo. , 1992, The Journal of biological chemistry.

[20]  B. Trumpower,et al.  The mitochondrial targeting presequence of the Rieske iron-sulfur protein is processed in a single step after insertion into the cytochrome bc1 complex in mammals and retained as a subunit in the complex. , 1993, The Journal of biological chemistry.

[21]  T. Fox,et al.  A genetic link between an mRNA-specific translational activator and the translation system in yeast mitochondria. , 1990, Genetics.

[22]  R. Haselbeck,et al.  Isolation, nucleotide sequence, and disruption of the Saccharomyces cerevisiae gene encoding mitochondrial NADP(H)-specific isocitrate dehydrogenase. , 1991, The Journal of biological chemistry.

[23]  S. Nagata,et al.  Molecular cloning and sequence determination of the nuclear gene coding for mitochondrial elongation factor Tu of Saccharomyces cerevisiae. , 1983, Proceedings of the National Academy of Sciences of the United States of America.

[24]  J. Hendrick,et al.  Survey of amino-terminal proteolytic cleavage sites in mitochondrial precursor proteins: leader peptides cleaved by two matrix proteases share a three-amino acid motif. , 1989, Proceedings of the National Academy of Sciences of the United States of America.

[25]  G. Daum,et al.  Import of proteins into mitochondria. Cytochrome b2 and cytochrome c peroxidase are located in the intermembrane space of yeast mitochondria. , 1982, The Journal of biological chemistry.

[26]  K. Baker,et al.  Mitochondrial proteins essential for viability mediate protein import into yeast mitochondria , 1991, Nature.

[27]  L. Rosenberg,et al.  Amino-terminal octapeptides function as recognition signals for the mitochondrial intermediate peptidase. , 1992, The Journal of biological chemistry.

[28]  G. Sancar.  Sequence of the Saccharomyces cerevisiae PHR1 gene and homology of the PHR1 photolyase to E. coli photolyase. , 1985, Nucleic acids research.

[29]  R. D. Gietz,et al.  New yeast-Escherichia coli shuttle vectors constructed with in vitro mutagenized yeast genes lacking six-base pair restriction sites. , 1988, Gene.

[30]  L. Grivell,et al.  The yeast nuclear gene MRF1 encodes a mitochondrial peptide chain release factor and cures several mitochondrial RNA splicing defects. , 1992, Nucleic acids research.

[31]  L. Reed,et al.  Nucleotide sequence for yeast dihydrolipoamide dehydrogenase. , 1988, Proceedings of the National Academy of Sciences of the United States of America.

[32]  T. Fox,et al.  Saccharomyces cerevisiae positive regulatory gene PET111 encodes a mitochondrial protein that is translated from an mRNA with a long 5' leader , 1987, Molecular and cellular biology.

[33]  P. Slonimski,et al.  ABC1, a novel yeast nuclear gene has a dual function in mitochondria: it suppresses a cytochrome b mRNA translation defect and is essential for the electron transfer in the bc 1 complex. , 1991, The EMBO journal.

[34]  E. Sztul,et al.  Import of the malate dehydrogenase precursor by mitochondria. Cleavage within leader peptide by matrix protease leads to formation of intermediate-sized form. , 1988, The Journal of biological chemistry.

[35]  F. Hartl,et al.  Cyclosporin A-binding protein (cyclophilin) of Neurospora crassa. One gene codes for both the cytosolic and mitochondrial forms. , 1988, The Journal of biological chemistry.

[36]  B. Trumpower,et al.  Mutational analysis of assembly and function of the iron-sulfur protein of the cytochromebc1 complex inSaccharomyces cerevisiae , 1993, Journal of bioenergetics and biomembranes.

[37]  D. Jones,et al.  The gene coding for the yeast oligomycin sensitivity-conferring protein. , 1990, The Journal of biological chemistry.

[38]  W. Fenton,et al.  Cleavage of precursors by the mitochondrial processing peptidase requires a compatible mature protein or an intermediate octapeptide , 1991, The Journal of cell biology.

[39]  MSS1, a nuclear-encoded mitochondrial GTPase involved in the expression of COX1 subunit of cytochrome c oxidase. , 1993, Journal of molecular biology.

[40]  R. A. Rollins,et al.  MIP1, a new yeast gene homologous to the rat mitochondrial intermediate peptidase gene, is required for oxidative metabolism in Saccharomyces cerevisiae. , 1994, Molecular and cellular biology.

[41]  L. Thompson,et al.  Gene sequence and primary structure of mitochondrial malate dehydrogenase from Saccharomyces cerevisiae. , 1988, Biochemistry.

[42]  D. Marguet,et al.  Molecular cloning of the yeast mitochondrial aconitase gene (ACO1) and evidence of a synergistic regulation of expression by glucose plus glutamate , 1990, Molecular and cellular biology.

[43]  J. Ohmen,et al.  Divergent overlapping transcripts at the PET122 locus in Saccharomyces cerevisiae , 1990, Molecular and cellular biology.

[44]  S. Ellis,et al.  Structural and functional analyses of a yeast mitochondrial ribosomal protein homologous to ribosomal protein S15 of Escherichia coli. , 1990, Nucleic acids research.

[45]  M. Yaffe.  Analysis of mitochondrial function and assembly. , 1991, Methods in enzymology.

[46]  F. Foury,et al.  PIF1: a DNA helicase in yeast mitochondria. , 1991, The EMBO journal.

[47]  R. Sikorski,et al.  In vitro mutagenesis and plasmid shuffling: from cloned gene to mutant yeast. , 1991, Methods in enzymology.

[48]  R. Labbe-Bois.  The ferrochelatase from Saccharomyces cerevisiae. Sequence, disruption, and expression of its structural gene HEM15. , 1990, The Journal of biological chemistry.

[49]  L. Rosenberg,et al.  Rat liver mitochondrial intermediate peptidase (MIP): purification and initial characterization. , 1992, The EMBO journal.

[50]  J. Hendrick,et al.  Import of rat ornithine transcarbamylase precursor into mitochondria: two-step processing of the leader peptide , 1987, The Journal of cell biology.

[51]  K. Isono,et al.  Cloning and characterization of nuclear genes for two mitochondrial ribosomal proteins in Saccharomyces cerevisiae. , 1990, Nucleic acids research.

[52]  D. Botstein,et al.  A Saccharomyces cerevisiae genomic plasmid bank based on a centromere-containing shuttle vector. , 1987, Gene.