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. 2018 Sep 20;19(11):e46193. doi: 10.15252/embr.201846193

Figure 2. Assessment of mitoribosomal misreading in in organello translation.

Figure 2

  1. Autoradiography of in organello mitochondrial translation products derived from HEK293 wild‐type cells in the presence of tobramycin, incorporation of 35S‐Met and 35S‐Cys in MT‐CO1 protein. Following in organello translation proteins were immunoprecipitated and analyzed by autoradiography. Lane 1, 3: controls; lanes 2, 4: in organello translation in the presence of 1,000 μM tobramycin. MT‐CO1 bands were quantified and ratio of 35S‐Cys/35S‐Met calculated, and the 35S‐Cys/35S‐Met ratio in the absence of tobramycin was set as 1 (n = 3), error bars indicate SEM.
  2. Ratio of 35S‐Cys/35S‐Met‐labeled MT‐CO1 and MT‐CO2 proteins synthesized in in organello translation and effect of tobramycin. Following in organello translation in the presence of tobramycin (1,000 μM, 2,000 μM), proteins were immunoprecipitated and analyzed by autoradiography. MT‐CO1 and MT‐CO2 bands were quantified, and ratio of 35S‐Cys/35S‐Met incorporation was calculated (n = 3), error bars indicate SEM; *P < 0.05 (Student's t‐test), ns, not significant.
  3. Ratio of 35S‐Cys/35S‐Met‐labeled MT‐CO1 protein synthesized in in organello translation using mitochondria from HEK293 MRPS5 wild‐type cells and HEK MRPS5 mutant V336Y cells. Following in organello translation, proteins were immunoprecipitated and analyzed by autoradiography. MT‐CO1 bands quantified and ratios calculated. The ratio of the MRPS5 wild‐type was set as 1 (n = 8 clones), error bars indicate SD; ***P < 0.0001 (Student's t‐test).
  4. Autoradiography of in organello mitochondrial translation products derived from HEK293 MRPS5 wild‐type cells in the presence of tobramycin (0–750 μM). Proteins were 35S‐Met‐labeled and loaded either directly on SDS–PAGE (total, top) or following poly‐lysine immunoprecipitation (bottom). * MT‐CO1 and ** MT‐CO1 with poly‐lysine extension.
  5. Quantification of MT‐CO1 proteins in HEK293 MRPS5 wild‐type cells and HEK293 MRPS5 mutant V336Y cells. In organello mitochondrial translation in the presence of 35S‐Met and 750 μM tobramycin. Proteins were loaded either directly on SDS–PAGE (total) or following poly‐lysine immunoprecipitation and analyzed by autoradiography. For total protein samples, the MT‐CO1 band was quantified, for poly‐lysine immunoprecipitated samples, the extended MT‐CO1 band was quantified and the ratio of extended MT‐CO1/total MT‐CO1 was calculated. The ratio of MRPS5 wild‐type was set as 1 (MRPS5 wild‐type, n = 5 clones, MRPS5 V336Y, n = 7 clones), error bars indicate SD; ***P < 0.0001 (Student's t‐test).