Mito-FUNCAT-FACS reveals cellular heterogeneity in mitochondrial translation
- Yusuke Kimura1,2,5,
- Hironori Saito1,2,5,
- Tatsuya Osaki3,
- Yasuhiro Ikegami3,
- Taisei Wakigawa1,2,
- Yoshiho Ikeuchi3,4 and
- Shintaro Iwasaki1,2
- 1Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa, Chiba 277-8561, Japan
- 2RNA Systems Biochemistry Laboratory, RIKEN Cluster for Pioneering Research, Wako, Saitama 351-0198, Japan
- 3Institute of Industrial Science, The University of Tokyo, Meguro-ku, Tokyo 153-8505, Japan
- 4Institute for AI and Beyond, The University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan
- Corresponding author: shintaro.iwasaki{at}riken.jp
-
↵5 These authors contributed equally to this work.
Abstract
Mitochondria possess their own genome that encodes components of oxidative phosphorylation (OXPHOS) complexes, and mitochondrial ribosomes within the organelle translate the mRNAs expressed from the mitochondrial genome. Given the differential OXPHOS activity observed in diverse cell types, cell growth conditions, and other circumstances, cellular heterogeneity in mitochondrial translation can be expected. Although individual protein products translated in mitochondria have been monitored, the lack of techniques that address the variation in overall mitochondrial protein synthesis in cell populations poses analytic challenges. Here, we adapted mitochondrial-specific fluorescent noncanonical amino acid tagging (FUNCAT) for use with fluorescence-activated cell sorting (FACS) and developed mito-FUNCAT-FACS. The click chemistry-compatible methionine analog L-homopropargylglycine (HPG) enabled the metabolic labeling of newly synthesized proteins. In the presence of cytosolic translation inhibitors, HPG was selectively incorporated into mitochondrial nascent proteins and conjugated to fluorophores via the click reaction (mito-FUNCAT). The application of in situ mito-FUNCAT to flow cytometry allowed us to separate changes in net mitochondrial translation activity from those of the organelle mass and detect variations in mitochondrial translation in cancer cells. Our approach provides a useful methodology for examining mitochondrial protein synthesis in individual cells.
Keywords
- Received January 2, 2022.
- Accepted February 12, 2022.
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