Proteomic analysis of the mammalian mitochondrial ribosome - Identification of protein components in the 28 S small subunit

The mammalian mitochondrial ribosome (mitoribosome) has a highly protein-ri ch composition with a small sedimentation coefficient of 55 S, consisting o f 39 S large and 28 S small subunits. In the previous study, we analyzed 39 S large subunit proteins from bovine mitoribosome (Suzuki, T., Terasaki, M , Takemoto-Hori, C., Hanada, T., Ueda, T., Wada, A., and Watanabe, VL (2001 ) J. BioL Chem. 276,21724-21736). The results suggested structural compensa tion for the rRNA deficit through proteins of increased molecular mass in t he mitoribosome. We report here the identification of 28 S small subunit pr oteins. Each protein was separated by radical-free high-reducing two-dimens ional polyacrylamide gel electrophoresis and analyzed by liquid chromatogra phy/mass spectrometry/mass spectrometry using electrospray ionization/ion t rap mass spectrometer to identify DNA sequence by expressed sequence tag da ta base searches in silico. Twenty one proteins from the small subunit were identified, including 11 new proteins along with their complete cDNA seque nces from human and mouse. In addition to these proteins, three new protein s were also identified in the 55 S mitoribosome. We have clearly identified a mitochondrial homologue of S12, which is a key regulatory protein of tra nslation fidelity and a candidate for the autosomal dominant deafness gene, DFNA4. The apoptosis-related protein DAP3 was found to be a component of t he small subunit, indicating a new function for the mitoribosome in program med cell death. In summary, we have mapped a total of 55 proteins from the 55 S mitoribosome on the two-dimensional polyacrylamide gels.