A Proteomics Approach to the Identification of Mammalian Mitochondrial Small Subunit Ribosomal Proteins

Abstract

Mammalian mitochondrial small subunit ribosomal proteins were separated by two-dimensional polyacrylamide gel electrophoresis. The proteins in six individual spots were subjected to in-gel tryptic digestion. Peptides were separated by capillary liquid chromatography, and the sequences of selected peptides were obtained by electrospray tandem mass spectrometry. The peptide sequences obtained were used to screen human expressed sequence tag data bases, and complete consensus cDNAs were assembled. Mammalian mitochondrial small subunit ribosomal proteins from six different classes of ribosomal proteins were identified. Only two of these proteins have significant sequence similarities to ribosomal proteins from prokaryotes. These proteins correspond to Escherichia coli S10 and S14. Homologs of two human mitochondrial proteins not found in prokaryotes were observed in the genomes of Drosophila melanogaster and Caenorhabditis elegans. A homolog of one of these proteins was observed in D. melanogaster but not in C. elegans, while a homolog of the other was present in C. elegans but not in D. melanogaster. A homolog of one of the ribosomal proteins not found in prokaryotes was tentatively identified in the yeast genome. This latter protein is the first reported example of a ribosomal protein that is shared by mitochondrial ribosomes from lower and higher eukaryotes that does not have a homolog in prokaryotes.Mammalian mitochondria carry out the synthesis of 13 polypeptides that are essential for oxidative phosphorylation and, hence, for the synthesis of the majority of the ATP used by eukaryotic organisms. The protein synthesizing system of mammalian mitochondria has a number of interesting features that are not observed in the corresponding systems in prokaryotes or the cell cytoplasm (1). The ribosomes present in mammalian mitochondria are 55-60 S particles and are composed of small (28 S) and a large (39 S) subunits (2). They are characterized by a low percentage of rRNA and a compensating increase in the number of ribosomal proteins (3).Considerable progress has been made on the identification of the mitochondrial ribosomal proteins in yeast. About 50 different mitochondrial ribosomal proteins (MRPs) 1 have been identified in this organism (4). Additional protein components in yeast mitochondrial ribosomes remain to be determined. Surprisingly, less than half of the mitochondrial ribosomal proteins in yeast show significant sequence identities to the ribosomal proteins of other systems (4). Analysis of the protein composition of mammalian mitochondrial ribosomes indicates that they have more proteins than observed in bacterial ribosomes (5). Limited information is available on the identities of these proteins and on their relationships to bacterial ribosomal proteins. Recently, 18 proteins of the large subunit and 5 proteins of the small subunit of the mammalian mitochondrial ribosome have been characterized primarily by peptide sequencing coupled to the extensive use of the EST data bases to deduce the full-length c...

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