Crespo José L, Díaz-Troya Sandra, Florencio Francisco J
Instituto de Bioquímica Vegetal y Fotosíntesis, Consejo Superior de Investigaciones Científicas, Universidad de Sevilla, Centro de Investigaciones Científicas Isla de la Cartuja, 41092 Seville, Spain.
Plant Physiol. 2005 Dec;139(4):1736-49. doi: 10.1104/pp.105.070847. Epub 2005 Nov 18.
The macrolide rapamycin specifically binds the 12-kD FK506-binding protein (FKBP12), and this complex potently inhibits the target of rapamycin (TOR) kinase. The identification of TOR in Arabidopsis (Arabidopsis thaliana) revealed that TOR is conserved in photosynthetic eukaryotes. However, research on TOR signaling in plants has been hampered by the natural resistance of plants to rapamycin. Here, we report TOR inactivation by rapamycin treatment in a photosynthetic organism. We identified and characterized TOR and FKBP12 homologs in the unicellular green alga Chlamydomonas reinhardtii. Whereas growth of wild-type Chlamydomonas cells is sensitive to rapamycin, cells lacking FKBP12 are fully resistant to the drug, indicating that this protein mediates rapamycin action to inhibit cell growth. Unlike its plant homolog, Chlamydomonas FKBP12 exhibits high affinity to rapamycin in vivo, which was increased by mutation of conserved residues in the drug-binding pocket. Furthermore, pull-down assays demonstrated that TOR binds FKBP12 in the presence of rapamycin. Finally, rapamycin treatment resulted in a pronounced increase of vacuole size that resembled autophagic-like processes. Thus, our findings suggest that Chlamydomonas cell growth is positively controlled by a conserved TOR kinase and establish this unicellular alga as a useful model system for studying TOR signaling in photosynthetic eukaryotes.
大环内酯类药物雷帕霉素特异性结合12-kD的FK506结合蛋白（FKBP12），并且这种复合物能有效抑制雷帕霉素靶蛋白（TOR）激酶。在拟南芥（Arabidopsis thaliana）中对TOR的鉴定表明，TOR在光合真核生物中是保守的。然而，植物对雷帕霉素的天然抗性阻碍了对植物中TOR信号传导的研究。在此，我们报道了在光合生物中通过雷帕霉素处理使TOR失活。我们在单细胞绿藻莱茵衣藻（Chlamydomonas reinhardtii）中鉴定并表征了TOR和FKBP12的同源物。野生型衣藻细胞的生长对雷帕霉素敏感，而缺乏FKBP12的细胞对该药物完全抗性，这表明该蛋白介导雷帕霉素抑制细胞生长的作用。与植物同源物不同，衣藻FKBP12在体内对雷帕霉素表现出高亲和力，药物结合口袋中保守残基的突变增加了这种亲和力。此外，下拉实验表明在雷帕霉素存在的情况下TOR与FKBP12结合。最后，雷帕霉素处理导致液泡大小显著增加，类似于自噬样过程。因此，我们的研究结果表明衣藻细胞生长受到保守的TOR激酶的正向调控，并将这种单细胞藻类确立为研究光合真核生物中TOR信号传导的有用模型系统。
