Organelle pH in the Arabidopsis Endomembrane System

• Published in: Molecular plant Vol. 6; no. 5; pp. 1419 - 1437
• Main Authors: Shen, Jinbo, Zeng, Yonglun, Zhuang, Xiaohong, Sun, Lei, Yao, Xiaoqiang, Pimpl, Peter, Jiang, Liwen
• Format: Journal Article
• Language: English
• Published: England Elsevier Inc 01.09.2013; Cell Press
• Subjects: Animals; Arabidopsis; Arabidopsis - drug effects; Arabidopsis - metabolism; Biosensing Techniques; Calibration; Cell Compartmentation - drug effects; Cell Nucleus - drug effects; Cell Nucleus - metabolism; Concanavalin A - pharmacology; endomembrane system; Golgi; Green Fluorescent Proteins - metabolism; Hydrogen-Ion Concentration - drug effects; Intracellular Membranes - drug effects; Intracellular Membranes - metabolism; Mammals - metabolism; MVB; nucleus; Organelles - drug effects; Organelles - metabolism; pH传感器; pH值; Secretory Pathway - drug effects; Sensors; Solubility; TGN; V-ATP酶; vacuole; 内膜系统; 拟南芥; 细胞器; 绿色荧光蛋白; 过氧化物酶体; vacuole; Arabidopsis; MVB; pH; nucleus; Golgi; TGN; ER; endomembrane system
• ISSN: 1674-2052; 1752-9867
• DOI: 10.1093/mp/sst079

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 capabil- ity 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 mul- tivesicular 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.