tailieunhanh - Báo cáo Y học: Tag-mediated isolation of yeast mitochondrial ribosome and mass spectrometric identification of its new components

Mitochondrial ribosomal proteins (mrps) of the budding yeast, Saccharomyces cerevisiae, have been extensively characterized genetically and biochemically. However, the list of thegenes encoding individualmrps is still not complete and quite a fewof the mrps are only predicted from their similarity to bacterial ribosomal proteins. We have con-structed a yeast strain in which one of the small subunit proteins, termed Mrp4, was tagged with S-peptide and used for affinity purification of mitochondrial ribosome. . | Eur. J. Biochem. 269 5203-5214 2002 FEBS 2002 doi Tag-mediated isolation of yeast mitochondrial ribosome and mass spectrometric identification of its new components Xiang Gan1 Madoka Kitakawa2 Ken-ichi Yoshino3 Noriko Oshiro3 Kazuyoshi Yonezawa3 and Katsumi Isono1 2 1Graduate School of Science and Technology department of Biology Faculty of Science and 3Biosignal Research Center Kobe University Japan Mitochondrial ribosomal proteins mrps of the budding yeast Saccharomyces cerevisiae have been extensively characterized genetically and biochemically. However the list of the genes encoding individual mrps is still not complete and quite a few of the mips are only predicted ram therr similarity to bacterial ribosomal proteins. We have constructed a yeast strain in which one of the small subunit proteins termed Mrp4 was tagged with S-peptide and used for affinity purification of mitochondrial ribosome. Mass spectrometric analysis of the isolated proteins detected most of the small subunit mrps which were previously identified or predicted and about half of the large subunit mrps. In addition several proteins of unknown function were identified. To confirm their identity further we added tags to these proteins and analyzed their localization in subcellular fractions. Thus we have newly established Ymr158w MrpS8 Ypl013c MrpS16 Ymr188c MrpS17 and Ygr165w MrrpS 35 as small subunit mips and ImgI Img2. Ydr116c MrpL1 Ynl177c MrpL22 Ynr022c MrpL50 and Ypr100w MipL5l as large uul un t mips. Keywords mitochondrial ribosomal proteins Saccharomy-ces cerevisiae tag-assisted purification mass-spectrometry. The mitochondrial genome codes for only a small number of proteins that are translated on the mitochondrial ribosome mitoribosome . Previous studies showed that the mitoribosome contains more proteins than its bacterial counterpart 1-5 . This may indicate that some of the mitochondrial ribosomal proteins mrps have been recruited to compensate the .