A luminescent Nanoluc-GFP fusion protein enables readout of cellular pH in photosynthetic organisms.
A luminescent Nanoluc-GFP fusion protein enables readout of cellular pH in photosynthetic organisms.
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DOI：
10.1074/jbc.ra120.016847
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发表时间：
2021-01
期刊：
影响因子：
--
通讯作者：
Sugiura K
中科院分区：
文献类型：
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作者：
Nakamura S;Fu N;Kondo K;Wakabayashi KI;Hisabori T;Sugiura K
pH值是决定重要细胞活动（如光合性能）的最关键生理参数之一。已经报道了能够在动物细胞中原位测量pH的荧光传感器蛋白。然而，这些蛋白质需要激发激光进行pH测量，这可能会影响光合性能并诱导叶绿素的自发荧光。因此，不可能测量植物中细胞内或细胞器内的pH变化。为了克服这个问题，我们开发了一种发光pH传感器，通过融合发光蛋白Nanoluc到一个独特设计的pH敏感的GFP变体蛋白。在该系统中，激发激光器是不必要的，因为融合的GFP变体通过来自Nanoluc的生物发光共振能量转移报告发光信号。来自传感器蛋白的两个发光峰的比率相对于pH在7.0至8.5的范围内近似线性。我们将这种传感器蛋白命名为“发光pH指示蛋白”（Luphin）。我们将Luphin应用于在波动的光照条件下的光合生物的原位pH测量，使我们能够成功地观察到与光合电子转移相关的蓝细菌集胞藻属PCC 6803的胞质pH变化。详细分析的机制，所观察到的估计pH值的变化，在这方面的研究表明，光合电子传递抑制减少质体醌池在光照条件下。这些结果表明，Luphin可以作为一个有用的工具，以进一步阐明整个光合生物的pH依赖性过程。
pH is one of the most critical physiological parameters determining vital cellular activities, such as photosynthetic performance. Fluorescent sensor proteins capable of measuring in situ pH in animal cells have been reported. However, these proteins require an excitation laser for pH measurement that may affect photosynthetic performance and induce autofluorescence from chlorophyll. As a result, it is not possible to measure the intracellular or intraorganelle pH changes in plants. To overcome this problem, we developed a luminescent pH sensor by fusing the luminescent protein Nanoluc to a uniquely designed pH-sensitive GFP variant protein. In this system, an excitation laser is unnecessary because the fused GFP variant reports on the luminescent signal by bioluminescence resonance energy transfer from Nanoluc. The ratio of two luminescent peaks from the sensor protein was approximately linear with respect to pH in the range of 7.0 to 8.5. We designated this sensor protein as “luminescent pH indicator protein” (Luphin). We applied Luphin to the in situ pH measurement of a photosynthetic organism under fluctuating light conditions, allowing us to successfully observe the cytosolic pH changes associated with photosynthetic electron transfer in the cyanobacterium Synechocystis sp. PCC 6803. Detailed analyses of the mechanisms of the observed estimated pH changes in the cytosol in this alga suggested that the photosynthetic electron transfer is suppressed by the reduced plastoquinone pool under light conditions. These results indicate that Luphin may serve as a helpful tool to further illuminate pH-dependent processes throughout the photosynthetic organisms.