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. 2014 Sep 1;25(17):2542–2555. doi: 10.1091/mbc.E14-01-0014

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© 2014 Rorbach et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

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FIGURE 6: — Interaction of MRM2 and MRM3 with the mitochondrial ribosome. (A, B) IP of components of the mitochondrial ribosome using tagged version of MRM3 and MRM3. Mitochondria from HEK293T cells expressing MRM2.FLAG (A) or MRM3.FLAG (B) were lysed, and IP was performed using anti-FLAG antibodies. The lysates (Input) and eluates were analyzed by Western blotting with antibodies as indicated. NDUFB8, antibody against a subunit of mitochondrial complex I. (C, D) IP of endogenous MRM2 and MRM3 using tagged components of the mitoribosome. Mitochondria from HEK293T cells expressing a component of mt-LSU (ICT1.FLAG; C) or a component of mt-SSU (MRPS27.FLAG; D) were lysed, and IP was performed using anti-FLAG antibodies. The lysates (Input) and eluates were analyzed by Western blotting using antibodies as indicated. NDUFB8, antibody against a subunit of mitochondrial complex I. (E, F) Cosedimentation of MRM2 and MRM3 with mitochondrial ribosome in sucrose gradients. Total lysates of HEK293T cells expressing MRM2.FLAG (E) or untransfected HEK293T cells (F) were separated through a 10–30% (wt/vol) isokinetic sucrose gradient and fractionated. The fractions were analyzed by Western blotting using antibodies specific for the FLAG tag (MRM2), endogenous MRM3, components of mt-LSU (MRPL12 or MRPL3), and a component of mt-SSU (MRPS18B). (G) Summary of MRM2 and MRM3 interactions with the mitoribosome. Interpretation of the IP and sucrose gradient experiments.