Why green fluorescent fusion proteins have not been observed in the vacuoles of higher plants
Why green fluorescent fusion proteins have not been observed in the vacuoles of higher plants
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DOI：
10.1046/j.1365-313x.2003.01822.x
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发表时间：
2003-08-01
期刊：
影响因子：
7.2
通讯作者：
Hara-Nishimura, I
中科院分区：
文献类型：
--
作者：
Tamura, K;Shimada, T;Hara-Nishimura, I
绿色荧光蛋白（GFP）使活细胞中的细胞器和蛋白质转运途径可视化成为可能。然而，尚未在高等植物的任何器官的液泡中观察到GFP荧光。我们发现，在转基因拟南芥植物的液泡中，在黑暗条件下稳定地观察到液泡靶向GFP的荧光，并且在光照条件下荧光迅速消失。空泡状的GFP在光照下，特别是蓝光下，在1h内迅速降解。空泡型H+-ATP酶的抑制剂，康卡那霉素A，和木瓜蛋白酶型半胱氨酸蛋白酶的抑制剂，E-64 d，取消了光依赖性消失的GFP荧光和GFP降解的空泡。体外试验表明，细菌表达的GFP降解的拟南芥培养细胞原生质体提取物在酸性pH值的光。这些结果表明，蓝光诱导GFP的构象变化，并产生的GFP在液泡中容易降解的液泡木瓜蛋白酶型半胱氨酸蛋白酶（s）在酸性pH值下。光依赖性降解解释了未能观察到GFP荧光的植物器官的液泡。我们的研究结果表明，稳定的GFP荧光空泡是通过将植物从光转移到黑暗中，然后用荧光显微镜检查。这可能会消除一个很大的障碍，在研究的液泡靶向机制和器官和阶段特异性分化的内膜系统在植物中。
Green fluorescent protein (GFP) makes it possible for organelles and protein transport pathways to be visualized in living cells. However, GFP fluorescence has not yet been observed in the vacuoles of any organs of higher plants. We found that the fluorescence of a vacuole-targeted GFP was stably observed in the vacuoles of transgenic Arabidopsis plants under dark conditions, and that the fluorescence rapidly disappeared under light conditions. The vacuolar GFP was rapidly degraded within 1 h in the light, especially blue light. An inhibitor of vacuolar type H+-ATPase, concanamycin A, and an inhibitor of papain-type cysteine proteinase, E-64d, abolished both the light-dependent disappearance of GFP fluorescence and GFP degradation in the vacuoles. An in vitro assay showed that bacterially expressed GFP was degraded by extracts of Arabidopsis cultured-cell protoplasts at an acidic pH in the light. These results suggest that blue light induced a conformational change in GFP, and the resulting GFP in the vacuole was easily degraded by vacuolar papain-type cysteine proteinase(s) under the acidic pH. The light-dependent degradation accounts for the failure to observe GFP fluorescence in the vacuoles of plant organs. Our results show that stable GFP-fluoresced vacuoles are achieved by transferring the plants from the light into the dark before inspection with a fluorescent microscope. This might eliminate a large hurdle in studies of the vacuolar-targeting machinery and the organ- and stage-specific differentiation of endomembrane systems in plants.