Identification of four proteins from the small subunit of the mammalian mitochondrial ribosome using a proteomics approach

Abstract

Proteins in the small subunit of the mammalian mitochondrial ribosome were separated by two-dimensional polyacrylamide gel electrophoresis. Four individual proteins were subjected to in-gel Endoprotease Lys-C digestion. The sequences of selected proteolytic peptides were obtained by electrospray tandem mass spectrometry. Peptide sequences obtained from in-gel digestion of individual spots were used to screen human, mouse, and rat expressed sequence tag databases, and complete consensus cDNAs for these species were deduced in silico. The corresponding protein sequences were characterized by comparison to known ribosomal proteins in protein databases. Four different classes of mammalian mitochondrial small subunit ribosomal proteins were identified. Only two of these proteins have significant sequence similarities to ribosomal proteins from prokaryotes. These proteins are homologs to Escherichia coli S9 and S5 proteins. The presence of these newly identified mitochondrial ribosomal proteins are also investigated in the Drosophila melanogaster, Caenorhabditis elegans, and in the genomes of several fungi.

Fig. 1.

Separation of bovine 28 S subunit proteins by two-dimensional gel electrophoresis using a NEPHGE-gel system developed previously (Cahill et al. 1995) for the separation of rat mitochondrial ribosomal proteins. The gel spots chosen for the in-gel tryptic digests are shown in ovals.

Fig. 2.

Product ion spectrum of the tryptic peptide at m/z 831.8 from MRP-S27. Because there were no exact matches in the protein or EST databases, sequence was called de novo and is shown using the single letter amino acid symbols. Ions are labeled according to the nomenclature of Roepstorff and Fohlman (Roepstorff and Folhman 1984), with the difference in m/z of adjacent singly charged y ions corresponding to the labeled amino acid residue mass. For the isobaric amino acids leucine and isoleucine, both possibilities are shown as L/I.

Fig. 3.

Sequence of human MRP-S5 (P82675) and its alignment with homologs from C. elegans (Q93425), E. coli (P02356), and yeast (P33759, YBR251W or YBR1704). (↓) Shows the signal peptide cleavage site predicted by MitoProtII and, additionally, represents the predicted peptide signal cleavage sites in Figures 4, 5, and 6 ▶ ▶ ▶.

Fig. 4.

Sequence of the human MRP-S27 (Q92552) and its alignment with homologs from C. elegans (CE16258) and F. rubripes (AAC60297).

Fig. 5.

Sequence of the human MRP-S9 and its alignment with homologs from C. elegans (P34388), D. melanogaster (CG2957), yeast (P38120, YBR146W or YBR1123), and E. coli (P02363).

Fig. 6.

Sequence of the human MRP-S28 (P82673) and its alignment with homologs from D. melanogaster (CG2101), C. elegans (CAB61063), and yeast (NP_010460 or YDR175Cp).