Organelle pH in the Arabidopsis Endomembrane System
Organelle pH in the Arabidopsis Endomembrane System
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拟南芥内膜系统中的细胞器 pH
DOI：
10.1093/mp/sst079
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发表时间：
2013-09-01
期刊：
影响因子：
27.5
通讯作者：
Jiang, Liwen
中科院分区：
文献类型：
--
作者：
Shen, Jinbo;Zeng, Yonglun;Jiang, Liwen
利用两个phfluorin衍生的pH传感器和拟南芥原生质体的瞬时表达为工具，生成了拟南芥内膜系统细胞器的细胞器特异性pH图谱。细胞内区室的pH值对细胞的生存能力至关重要。尽管其相关性，但对这些隔室的pH值知之甚少。为了在体内测量pH值，我们首先将溶解度修饰的绿色荧光蛋白（GFP） （smGFP）的改进溶解度特性与拟南芥中优化表达的pHluorins和密码子的pH传感能力结合起来，生成了两个pH传感器。pHluorin（植物溶解度修饰的黄道型pHluorin）随着pH值的降低逐渐失去荧光，在pH值为6.2时荧光消失，而PRpHluorin（植物溶解度修饰的ratiomatric pHluorin）是一种双激发传感器，可以进行精确测量。通过进一步将特定的分类信号融合到PEpHluorin和PRpHluorin中，产生了室特异性传感器。我们的研究结果表明，细胞质和细胞核的pH值相似（pH值为7.3和7.2），而过氧化物酶体、线粒体基质和质体基质的pH值为碱性。分泌途径的隔间显示逐渐酸化，从内质网（ER）的pH值为7.1到液泡的pH值为5.2。令人惊讶的是，反式高尔基网络（TGN）和多泡体（MVB）的pH值非常相似，分别为6.3和6.2。concanamycin A （ConcA）抑制液泡型H-ATPase （V-ATPase）导致TGN和液泡pH急剧升高。总的来说，PEpHluorin和PRpHluorin分别是体内pH可视化和定量的优秀pH传感器。
Using two pHluorins-derived pH sensors and transient expression of Arabidopsis protoplasts as tools, an organelle-specific pH map was generated for organelles of the Arabidopsis endomemebrane system.The pH of intracellular compartments is essential for the viability of cells. Despite its relevance, little is known about the pH of these compartments. To measure pH in vivo, we have first generated two pH sensors by combining the improved-solubility feature of solubility-modified green fluorescent protein (GFP) (smGFP) with the pH-sensing capability of the pHluorins and codon optimized for expression in Arabidopsis. PEpHluorin (plant-solubility-modified ecliptic pHluorin) gradually loses fluorescence as pH is lowered with fluorescence vanishing at pH 6.2 and PRpHluorin (plant-solubility-modified ratiomatric pHluorin), a dual-excitation sensor, allowing for precise measurements. Compartment-specific sensors were generated by further fusing specific sorting signals to PEpHluorin and PRpHluorin. Our results show that the pH of cytosol and nucleus is similar (pH 7.3 and 7.2), while peroxisomes, mitochondrial matrix, and plastidial stroma have alkaline pH. Compartments of the secretory pathway reveal a gradual acidification, spanning from pH 7.1 in the endoplasmic reticulum (ER) to pH 5.2 in the vacuole. Surprisingly, pH in the trans-Golgi network (TGN) and multivesicular body (MVB) is, with pH 6.3 and 6.2, quite similar. The inhibition of vacuolar-type H-ATPase (V-ATPase) with concanamycin A (ConcA) caused drastic increase in pH in TGN and vacuole. Overall, the PEpHluorin and PRpHluorin are excellent pH sensors for visualization and quantification of pH in vivo, respectively.