Mammalian Mitochondrial Ribosomal Proteins (4): AMINO ACID SEQUENCING, CHARACTERIZATION, AND IDENTIFICATION OF CORRESPONDING GENE SEQUENCES

Thomas W O'Brien,Jiguo Liu,James E Sylvester,Edward B Mougey,Nathan Fischel-Ghodsian,Bernd Thiede,Brigitte Wittmann-Liebold,Hanns-Rüdiger Graack

doi:10.1074/jbc.m909762199

Abstract

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.

Highlights

Human oxidative phosphorylation disorders have beenThe peptide sequences reported in this paper have been submitted to the MIPS Data Bank with accession numbers S78761 (MRP-L18bov, fragments), S78772 (MRP-L43bov, fragments), S78762 (MRP-S12bov, fragment), S78763 (MRP-S18bov, fragment), and S78766 (MRP-S28bov, fragment)
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

Summary

Human oxidative phosphorylation disorders have been

The peptide sequences reported in this paper have been submitted to the MIPS Data Bank with accession numbers S78761 (MRP-L18bov, fragments), S78772 (MRP-L43bov, fragments), S78762 (MRP-S12bov, fragment), S78763 (MRP-S18bov, fragment), and S78766 (MRP-S28bov, fragment). 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. 12). Mammalian Mitochondrial Ribosomal Proteins 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 (15, 16). 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.

EXPERIMENTAL PROCEDURES

RESULTS AND DISCUSSION

New New New

Yeast mitochondria

We hope that the discovery of the complete set of mammalian