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

Emine Cavdar Koc,William Burkhart,Kevin Blackburn,Arthur Moseley,Hasan Koc,Linda L Spremulli

doi:10.1074/jbc.m003596200

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.

Highlights

Mammalian mitochondrial small subunit ribosomal proteins were separated by two-dimensional polyacrylamide gel electrophoresis
Mammalian mitochondria carry out the synthesis of 13 polypeptides that are essential for oxidative phosphorylation and, for the synthesis of the majority of the ATP used by eukaryotic organisms
Characterization of Bovine Mitochondrial Ribosomal Proteins by Tandem Mass Spectrometry—As a first step toward understanding the protein components of mammalian mitochondrial ribosomes, small subunit proteins were separated by two-dimensional PAGE

Summary

Introduction

Mammalian mitochondrial small subunit ribosomal proteins were separated by two-dimensional polyacrylamide gel electrophoresis. 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 cDNAs and the corresponding amino acid sequences (6 –11). Of these proteins, 12 from the large subunit and 2 from the small subunit are homologs of bacterial ribosomal proteins. The remaining four small subunit proteins fall into new classes of ribosomal proteins

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