A genetic approach to identifying mitochondrial proteins


The control of intricate networks within eukaryotic cells relies on differential compartmentalization of proteins. We have developed a method that allows rapid identification of novel proteins compartmentalized in mitochondria by screening large-scale cDNA libraries. The principle is based on reconstitution of split-enhanced green fluorescent protein (EGFP) by protein splicing of DnaE derived from Synechocystis sp. PCC6803. The cDNA libraries are expressed in mammalian cells following infection with retrovirus. If a test protein contains a functional mitochondrial targeting signal (MTS), it translocates into the mitochondrial matrix, where EGFP is then formed by protein splicing. The cells harboring this reconstituted EGFP are screened rapidly by fluorescence-activated cell sorting, and the cDNAs are isolated and identified from the cells. The analysis of 258 cDNAs revealed various MTSs, among which we identified new transcripts corresponding to mitochondrial proteins. This method should provide a means to map proteins distributed within intracellular organelles in a broad range of different tissues and disease states.

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Figure 1: Schematic of RING-MITO technology.
Figure 2: Selective and sensitive detection of mitochondrial proteins.
Figure 3: Selection of fluorescent cells by FACS.
Figure 4: Flow cytometry profiles and fluorescent images of representative cloned cells.
Figure 5: (A) Expression of EGFP-tagged cDNA clones (clones no. 16, 33, and 59) and their localization to mitochondria.


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This work has been supported by Core Research for Evolutional Science and Technology of Japan Science and Technology and by grants to Y.U. from the Ministry of Education, Science and Culture, Japan.

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Correspondence to Yoshio Umezawa.

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The authors declare no competing financial interests.

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Ozawa, T., Sako, Y., Sato, M. et al. A genetic approach to identifying mitochondrial proteins. Nat Biotechnol 21, 287–293 (2003). https://doi.org/10.1038/nbt791

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