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Mammalian Mitochondrial Ribosomal Proteins (4)

AMINO ACID SEQUENCING, CHARACTERIZATION, AND IDENTIFICATION OF CORRESPONDING GENE SEQUENCES*
Open AccessPublished:June 16, 2000DOI:https://doi.org/10.1074/jbc.M909762199
      Mitochondrial ribosomal proteins (MRPs) are required for the translation of all 13 mitochondrial encoded genes in humans. It has been speculated that mutations and polymorphisms in the human MRPs may be a primary cause of some oxidative phosphorylation disorders or modulate the severity and tissue specificity of pathogenic mitochondrial DNA mutations. Although the sequences of most of the yeast MRPs are known, only very few mammalian and nearly no human MRPs have been completely characterized. MRPs differ greatly in sequence, and sometimes biochemical properties, between different species, not allowing easy recognition by sequence homology. Therefore, the Mammalian Mitochondrial Ribosomal Consortium is using a direct approach of purifying individual mammalian (bovine) MRPs, determining their N-terminal and/or internal peptide sequences using different protein sequencing techniques, and using the resulting sequence information for screening expressed sequence tags and genomic data bases to determine human, mouse, and rat homologues of the bovine proteins. Two proteins of the large and three proteins of the small ribosomal subunit have been analyzed in this manner. Three of them represent “new,” i.e. formerly unknown mammalian mitochondrial ribosomal protein classes. Only one of these three different MRPs shows significant sequence similarities to known ribosomal proteins. In one case, the corresponding human genomic DNA sequences were found in the data bases, and the exon/intron structure was determined.
      MRP
      mitochondrial ribosomal protein(s)
      PAGE
      polyacrylamide gel electrophoresis
      bp
      base pair(s)
      MISP
      mitochondrial import signal peptide
      ORF
      open reading frame
      EST
      expressed sequence tag
      Human oxidative phosphorylation disorders have been shown over the last decade to be the cause of a great variety of inherited and acquired diseases, including such different clinical entities as systemic neuromuscular disorders, diabetes, aplastic anemia, deafness, and degenerative disorders by either mitochondrial or nuclear gene mutations (
      • Wallace D.C.
      ). It has been speculated that mutations or polymorphisms in proteins involved in mitochondrial RNA processing and translation can be involved in some of these diseases, either as the primary cause or as factors modulating the severity and/or tissue specificity of them (
      • Fischel-Ghodsian N.
      ).
      The proper expression of the mitochondrial encoded protein genes depends on the nuclear-encoded components of the mitochondrial translational system (
      • Costanzo M.C.
      • Fox T.D.
      ). In yeast, knockout mutants of nuclear encoded MRPs1 lose their mitochondrial DNA, changing the mitochondrial genetic status to ρo (
      • Myers A.M.
      • Pape L.K.
      • Tzagoloff A.
      ), an effect also observed in mammalian nuclear mutations affecting mitochondria (
      • Grossman L.I.
      • Shoubridge E.A.
      ). Mammalian mitochondrial ribosomes differ significantly from other known bacterial and eukaryotic cytoplasmic ribosomes when analyzed by biochemical methods (
      • Matthews D.E.
      • Hessler R.A.
      • Denslow N.D.
      • Edwards J.S.
      • O'Brien T.W.
      ,
      • Graack H.-R.
      • Grohmann L.
      • Choli T.
      ,
      • O'Brien T.W.
      • Denslow N.D.
      • Anders J.C.
      • Courtney B.C.
      ,
      • Cahill A.
      • Baio D.L.
      • Cunningham C.C.
      ). Numbers and sizes of rRNA molecules are reduced as compared with bacterial ribosomes, and numbers of ribosomal proteins are elevated (
      • Kitakawa M.
      • Isono K.
      ). However, the differences turned out to be even greater than expected when molecular biological and protein biochemical investigations began to reveal the amino acid and gene sequences of MRPs. These efforts are supported by the recent genome projects. Most of the yeast MRPs were identified by the comparison of peptide sequences obtained from purified mature yeast MRPs by N-terminal sequencing, with the protein sequences deduced from the yeast genome project data (reviewed in Ref.
      • Graack H.-R.
      • Wittmann-Liebold B.
      ). However, the assumption that yeast MRPs and mammalian MRPs are similar to each other, as is the case for yeast and rat cytoplasmic ribosomal proteins, and that it may be possible to identify mammalian MRPs by using yeast MRP probes as screening devices is not valid. When the current project of mammalian MRP gene identification was launched in 1997, only very few mammalian MRPs were known on the molecular biological level, rather by chance than by a systematic approach. The mammalian mitochondrial homologue of the bacterial L3 ribosomal protein was identified as an overexpressed protein in Mahlavu hepatocarcinoma cells (
      • Ou J.-H.
      • Yen T.S.B.
      • Wang Y.-F.
      • Kam W.K.
      • Rutter W.J.
      ,
      • Graack H.-R.
      • Grohmann L.
      • Kitakawa M.
      • Schäfer K.-L.
      • Kruft V.
      ). The mammalian homologues RPMS12 of the EcoS12 protein, a protein strongly conserved through evolution, were cloned by sequence similarities to the EcoS12 ribosomal protein and the S12 homologue of Drosophila melanogaster mitochondria (
      • Shah Z.H.
      • O'Dell K.M.C.
      • Miller S.C.M.
      • An X.
      • Jacobs H.T.
      ,
      • Johnson D.F.
      • Hamon M.
      • Fischel-Ghodsian N.
      ). L23MRP was identified by its sequence similarity to the EcoL23 ribosomal protein (
      • Tsang P.
      • Gilles F.
      • Yuan L.
      • Kuo Y.H.
      • Lupu F.
      • Samara G.
      • Moosikasuwan J.
      • Goye A.
      • Zelenetz A.D.
      • Tycko B.
      ). The mammalian mitochondrial homologue of the strongly conserved EcoL7/L12gene was cloned as a delayed-early expressed gene (
      • Marty L.
      • Fort P.
      ). An HSMRPS14 cDNA similar to the EcoS14 ribosomal protein was cloned (GenBankTM accession number Z99297) but not further characterized. However, none of the identified mammalian MRPs was “new” in terms of lacking sequence similarities to known ribosomal proteins.
      To characterize mammalian MRPs systematically, the Mammalian Mitochondrial Ribosomal Consortium was formed, and the initial primary experimental approach was to be based on the N-terminal sequencing of purified mature bovine MRPs. By using the obtained peptide sequence information, EST and genomic DNA data bases are screened, and cDNA sequences are assembled in silico. This approach takes advantage of the existing bovine model for MRPs (
      • Matthews D.E.
      • Hessler R.A.
      • Denslow N.D.
      • Edwards J.S.
      • O'Brien T.W.
      ,
      • O'Brien T.W.
      • Denslow N.D.
      ) and the rapidly growing sequence data bases of human and other organisms. The obtained sequences were characterized by comparison to known ribosomal protein sequences, and corresponding genomic DNA sequences were identified. Nineteen different groups of homologous mammalian MRPs have been determined so far (
      • Goldschmidt-Reisin S.
      • Kitakawa M.
      • Herfurth E.
      • Grohmann L.
      • Wittmann-Liebold B.
      • Graack H.-R.
      ,
      • Graack H.-R.
      • Bryant M.L.
      • O'Brien T.W.
      ,
      • O'Brien T.W.
      • Fiesler S.E.
      • Denslow N.D.
      • Thiede B.
      • Wittmann-Liebold B.
      • Sylvester J.E.
      • Mougey E.B.
      • Graack H.-R.
      ).
      J. Anders, H.-R. Graack, J. Liu, and T. W. O'Brien, manuscript in preparation.
      2J. Anders, H.-R. Graack, J. Liu, and T. W. O'Brien, manuscript in preparation.
      Only 10 of them show significant sequence similarities to yeast MRPs and/or bacterial ribosomal proteins. This paper describes the identification of five mammalian MRPs.

      REFERENCES

        • Schieber G.L.
        • O'Brien T.W.
        J. Biol. Chem. 1982; 257: 8781-8787
        • Wallace D.C.
        Science. 1999; 283: 1482-1488
        • Fischel-Ghodsian N.
        Mol. Genet. Metab. 1998; 65: 97-104
        • Costanzo M.C.
        • Fox T.D.
        Annu. Rev. Genet. 1990; 24: 91-113
        • Myers A.M.
        • Pape L.K.
        • Tzagoloff A.
        EMBO J. 1985; 4: 2087-2092
        • Grossman L.I.
        • Shoubridge E.A.
        BioEssays. 1996; 18: 983-991
        • Matthews D.E.
        • Hessler R.A.
        • Denslow N.D.
        • Edwards J.S.
        • O'Brien T.W.
        J. Biol. Chem. 1982; 257: 8788-8794
        • Graack H.-R.
        • Grohmann L.
        • Choli T.
        FEBS Lett. 1988; 242: 4-8
        • O'Brien T.W.
        • Denslow N.D.
        • Anders J.C.
        • Courtney B.C.
        Biochim. Biophys. Acta. 1990; 1050: 174-178
        • Cahill A.
        • Baio D.L.
        • Cunningham C.C.
        Anal. Biochem. 1995; 232: 47-55
        • Kitakawa M.
        • Isono K.
        Biochimie (Paris). 1991; 73: 813-825
        • Graack H.-R.
        • Wittmann-Liebold B.
        Biochem. J. 1998; 329: 433-448
        • Ou J.-H.
        • Yen T.S.B.
        • Wang Y.-F.
        • Kam W.K.
        • Rutter W.J.
        Nucleic Acids Res. 1987; 15: 8919-8934
        • Graack H.-R.
        • Grohmann L.
        • Kitakawa M.
        • Schäfer K.-L.
        • Kruft V.
        Eur. J. Biochem. 1992; 206: 373-380
        • Shah Z.H.
        • O'Dell K.M.C.
        • Miller S.C.M.
        • An X.
        • Jacobs H.T.
        Gene (Amst.). 1997; 204: 55-62
        • Johnson D.F.
        • Hamon M.
        • Fischel-Ghodsian N.
        Genomics. 1998; 52: 363-368
        • Tsang P.
        • Gilles F.
        • Yuan L.
        • Kuo Y.H.
        • Lupu F.
        • Samara G.
        • Moosikasuwan J.
        • Goye A.
        • Zelenetz A.D.
        • Tycko B.
        Hum. Mol. Genet. 1995; 4: 1499-1507
        • Marty L.
        • Fort P.
        J. Biol. Chem. 1996; 271: 11468-11476
        • O'Brien T.W.
        • Denslow N.D.
        Methods Enzymol. 1996; 264: 237-248
        • Goldschmidt-Reisin S.
        • Kitakawa M.
        • Herfurth E.
        • Grohmann L.
        • Wittmann-Liebold B.
        • Graack H.-R.
        J. Biol. Chem. 1998; 273: 34828-34836
        • Graack H.-R.
        • Bryant M.L.
        • O'Brien T.W.
        Biochemistry. 1999; 38: 16569-16577
        • O'Brien T.W.
        • Fiesler S.E.
        • Denslow N.D.
        • Thiede B.
        • Wittmann-Liebold B.
        • Sylvester J.E.
        • Mougey E.B.
        • Graack H.-R.
        J. Biol. Chem. 1999; 274: 36043-36051
        • Otto A.
        • Thiede B.
        • Müller E.-C.
        • Scheler C.
        • Wittmann-Liebold B.
        • Jungblut P.
        Electrophoresis. 1996; 17: 1643-1650
        • Altschul S.F.
        • Madden T.L.
        • Schaffer A.A.
        • Zhang J.
        • Zhang Z.
        • Miller W.
        • Lipman D.J.
        Nucleic Acids Res. 1997; 25: 3389-3402
        • GCG Wisconsin Package
        GCG Wisconsin Software Package, Version 9.1. Genetics Computer Group, Inc., Madison, WI1997
        • Nielsen H.
        • Engelbrecht J.
        • Brunak S.
        • von Heijne G.
        Protein Eng. 1997; 10: 1-6
        • Martinez R.
        • Venturelli D.
        • Perrotti D.
        • Veronese M.L.
        • Kastury K.
        • Druck T.
        • Huebner K.
        • Calabretta B.
        Cancer Res. 1997; 57: 1180-1187
        • Nagase T.
        • Miyajima N.
        • Tanaka A.
        • Sazuka T.
        • Seki N.
        • Sato S.
        • Tabata S.
        • Ishikawa K.I.
        • Kawarabayasi Y.
        • Kotani H.
        • Nomura N.
        DNA Res. 1995; 2: 37-43
        • Nagase T.
        • Seki N.
        • Ishikawa K.
        • Ohira M.
        • Kawarabayasi Y.
        • Ohara O.
        • Tanaka A.
        • Kotani H.
        • Miyajima N.
        • Nomura N.
        DNA Res. 1996; 3: 321-329
        • Branda S.S.
        • Isaya G.
        J. Biol. Chem. 1995; 270: 27366-27373
        • Koc E.C.
        • Blackburn K.
        • Burkhat W.
        • Spremulli L.L.
        Biochem. Biophys. Res. Commun. 1999; 266: 141-146
        • Wool I.G.
        • Chan Y.-L.
        • Glück A.
        Biochem. Cell Biol. 1995; 73: 933-947
        • Pietromonaco S.F.
        • Hessler R.A.
        • O'Brien T.W.
        J. Mol. Evol. 1986; 24: 110-117
        • Matthews D.E.
        • Hessler R.A.
        • O'Brien T.W.
        FEBS Lett. 1978; 86: 76-80
        • Herold M.
        • Nierhaus K.H.
        J. Biol. Chem. 1987; 262: 8826-8833