Chemical Resources Laboratory, Tokyo Institute of Technology, Yokohama 226-8503, Japan; Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency (JST), Saitama 332-0012, Japan.
Biochem Biophys Res Commun. 2013 Sep 20;439(2):264-9. doi: 10.1016/j.bbrc.2013.08.045. Epub 2013 Aug 22.
The target of rapamycin (TOR) is serine/threonine protein kinase that is highly conserved among eukaryotes and can be inactivated by the antibiotic rapamycin through the formation of a ternary complex composed of rapamycin and two proteins, TOR and FKBP12. Differing from fungi and animals, plant FKBP12 proteins are unable to form the ternary complex, and thus plant TORs are insensitive to rapamycin. This has led to a poor understanding of TOR functions in plants. As a first step toward the understanding of TOR function in a rapamycin-insensitive unicellular red alga, Cyanidioschyzon merolae, we constructed a rapamycin-susceptible strain in which the Saccharomyces cerevisiae FKBP12 protein (ScFKBP12) was expressed. Treatment with rapamycin resulted in growth inhibition and decreased polysome formation in this strain. Binding of ScFKBP12 with C. merolae TOR in the presence of rapamycin was demonstrated in vivo and in vitro by pull-down experiments. Moreover, in vitro kinase assay showed that inhibition of C. merolae TOR kinase activity was dependent on ScFKBP12 and rapamycin.
雷帕霉素靶蛋白（TOR）是一种丝氨酸/苏氨酸蛋白激酶，在真核生物中高度保守，可通过雷帕霉素与两种蛋白 TOR 和 FKBP12 形成三元复合物而失活。与真菌和动物不同，植物 FKBP12 蛋白无法形成三元复合物，因此植物 TOR 对雷帕霉素不敏感。这导致人们对 TOR 在植物中的功能知之甚少。为了初步了解雷帕霉素不敏感的单细胞红藻 Cyanidioschyzon merolae 中的 TOR 功能，我们构建了一个对雷帕霉素敏感的菌株，该菌株中表达了酿酒酵母 FKBP12 蛋白（ScFKBP12）。雷帕霉素处理导致该菌株生长抑制和多核糖体形成减少。通过下拉实验在体内和体外证明了雷帕霉素存在时 ScFKBP12 与 C. merolae TOR 的结合。此外，体外激酶测定表明 C. merolae TOR 激酶活性的抑制依赖于 ScFKBP12 和雷帕霉素。
