Abstract
Four different classes of mammalian mitochondrial ribosomal proteins were identified and characterized. Mature proteins were purified from bovine liver and subjected to N-terminal or matrix-assisted laser-desorption mass spectroscopic amino acid sequencing after tryptic in-gel digestion and high pressure liquid chromatography separation of the resulting peptides. Peptide sequences obtained were used to virtually screen expressed sequence tag data bases from human, mouse, and rat. Consensus cDNAs were assembled in silico from various expressed sequence tag sequences identified. Deduced mammalian protein sequences were characterized and compared with ribosomal protein sequences of Escherichia coli and yeast mitochondria. Significant sequence similarities to ribosomal proteins of other sources were detected for three out of four different mammalian protein classes determined. However, the sequence conservation between mitochondrial ribosomal proteins of mammalian and yeast origin is much less than the sequence conservation between cytoplasmic ribosomal proteins of the same species. In particular, this is shown for the mammalian counterparts of the E. coli EcoL2 ribosomal protein (MRP-L14), that do not conserve the specific and functional highly important His(229) residue of E. coli and the corresponding yeast mitochondrial Rml2p.
Highlights
Four different classes of mammalian mitochondrial ribosomal proteins were identified and characterized
The mature Mitochondrial ribosomal proteins (MRPs)-L5human after cleavage of the mitochondrial import signal peptide (MISP) has a calculated MM of 36,583 Da. This value is in good correspondence to the MM of the mature MRP-L5bovine of 35.5 kDa (Table I), based on the proposal that mammalian MRPs are quite similar in their properties like MM, pI, and amino acid sequence (8).[2]
Characterization and Evolution of Mammalian MRPs— Mammalian MRPs have been characterized on the molecular level only by chance in the past (4 –7)
Summary
Analysis of Bovine MRPs—Isolation and purification of bovine MRPs has been described.[2] After two-dimensional PAGE and Coomassie Blue staining, individual spots of MRPs were cut from the gel. Individual proteins were termed in accordance to the established two-dimensional map of bovine MRPs (2). Proteins were subjected to in-gel digestion by trypsin according to the method of Otto et al (14). Resulting peptides were isolated and concentrated, purified by reverse phase HPLC, and subjected to Edman sequencing (14) or tandem mass spectroscopic analysis with a Q-Tof (Micromass, Manchester, UK) equipped with a nanoflow Z-spray ion source. Computing—Virtual screening of public EST data bases was performed using the blast program of Altschul et al (15) and the NCBI server. For screening of short peptides the advanced blast program was performed using the modified options “expected”: 1000 and “other options”: Ϫe2.
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