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.
Ozawa
Takeaki
T
Department of Chemistry, School of Science, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
Sako
Yusuke
Y
Sato
Moritoshi
M
Kitamura
Toshio
T
Umezawa
Yoshio
Y
D000595:Amino Acid Sequence; D002478:Cells, Cultured; D020869:Gene Expression Profiling / Q000379:methods*; D015723:Gene Library; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins; D008928:Mitochondria / Q000737:chemistry / Q000235:genetics / Q000502:physiology; D024101:Mitochondrial Proteins / Q000032:analysis / Q000737:chemistry* / Q000145:classification / Q000235:genetics*; D008970:Molecular Weight; D020543:Proteome / Q000737:chemistry / Q000235:genetics* / Q000502:physiology; D040901:Proteomics / Q000379:methods; D011994:Recombinant Proteins; D020539:Sequence Analysis, Protein; D013050:Spectrometry, Fluorescence / Q000379:methods
D008164:Luminescent Proteins; D024101:Mitochondrial Proteins; D020543:Proteome; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins