Nature Biotechnology
21, 287 - 293 (2003)
Published online: 10 February 2003; | doi:10.1038/nbt791
A genetic approach to identifying mitochondrial proteinsTakeaki Ozawa1, Yusuke Sako1, Moritoshi Sato1, Toshio Kitamura2
& Yoshio Umezawa11
Department of Chemistry, School of Science, The
University of Tokyo, Hongo, Bunkyo-ku, Tokyo
113-0033, Japan, and Japan Science and Technology
Corporation, Tokyo, Japan. 2
Department of Hematopoietic Factors, Institute of
Medical Science, The University of Tokyo, Shirokanedai, Minato-ku,
Tokyo, 108-8639, Japan.
Correspondence should be addressed to Yoshio Umezawa umezawa@chem.s.u-tokyo.ac.jpThe 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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