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Monitoring mitochondrial translation by pulse SILAC

Item Type:Preprint
Title:Monitoring mitochondrial translation by pulse SILAC
Creators Name:Imami, K. and Selbach, M. and Ishihama, Y.
Abstract:Mitochondrial ribosomes are specialized to translate the 13 membrane proteins encoded in the mitochondrial genome, but it is challenging to quantify mitochondrial translation products due to their hydrophobic nature. Here, we introduce a proteomic method that combines biochemical isolation of mitochondria with pulse stable isotope labeling by amino acids in cell culture (pSILAC). Our method provides the highest protein coverage (quantifying 12 out of the 13 inner-membrane proteins; average 2-fold improvement over previous studies) with the shortest measurement time. We applied this method to uncover the global picture of (post)translational regulation of both mitochondrial- and nuclear-encoded proteins involved in the assembly of protein complexes that mediate oxidative phosphorylation (OXPHOS). The results allow us to infer the assembly order of complex components and/or partners, as exemplified by complex III. This method should be applicable to study mitochondrial translation programs in many contexts, including oxidative stress and mitochondrial disease.
Keywords:Proteomics, Pulse SILAC, Translation, Mitochondria, OXPHOS, Protein Complex
Source:bioRxiv
Publisher:Cold Spring Harbor Laboratory Press
Article Number:2021.01.31.428997
Date:1 February 2021
Official Publication:https://doi.org/10.1101/2021.01.31.428997

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