Genetically encoded optical probe for detecting release of proteins from mitochondria toward cytosol in living cells and mammals.
We developed a genetically encoded bioluminescence indicator for monitoring the release of proteins from the mitochondria in living cells. The principle of this method is based on reconstitution of split Renilla reniformis luciferase (Rluc) fragments by protein splicing with an Ssp DnaE intein. A target mitochondrial protein connected with an N-terminal fragment of Rluc and an N-terminal fragment of DnaE is expressed in mammalian cells. If the target protein is released from the mitochondria toward the cytosol upon stimulation with a specific chemical, the N-terminal Rluc meets the C-terminal Rluc connected with C-terminal DnaE in the cytosol, and thereby, the full-length Rluc is reconstituted by protein splicing. The extent of release of the target fusion protein is evaluated by measuring activities of the reconstituted Rluc. To test the feasibility of this method, here we monitored the release of Smac/DIABLO protein from mitochondria during apoptosis in living cells and mice. The present method allowed high-throughput screening of an apoptosis-inducing reagent, staurosporine, and imaging of the Smac/DIABLO release in cells and in living mice. This rapid analysis can be used for screening and assaying chemicals that would increase or inhibit the release of mitochondrial proteins in living cells and animals.
Kanno
Akira
A
Department of Chemistry, School of Science, The University of Tokyo, 7-3-1 Hongo Bunkyo-ku, Tokyo 113-0033, Japan.
Ozawa
Takeaki
T
Umezawa
Yoshio
Y
D000818:Animals; D045744:Cell Line, Tumor; D003600:Cytosol / Q000378:metabolism*; D017930:Genes, Reporter / Q000235:genetics*; D006801:Humans; D051379:Mice; D008928:Mitochondria / Q000378:metabolism*; D024101:Mitochondrial Proteins / Q000235:genetics* / Q000378:metabolism*; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
D024101:Mitochondrial Proteins; D011993:Recombinant Fusion Proteins