title: Organelle pH in the Arabidopsis endomembrane system.
abstract: 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.
authors.affiliation: School of Life Sciences, Centre for Cell & Developmental Biology and State Key Laboratory of Agrobiotechnology, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China.
authors.forename: Jinbo
authors.initials: J
authors.lastname: Shen
authors.forename: Yonglun
authors.initials: Y
authors.lastname: Zeng
authors.forename: Xiaohong
authors.initials: X
authors.lastname: Zhuang
authors.forename: Lei
authors.initials: L
authors.lastname: Sun
authors.forename: Xiaoqiang
authors.initials: X
authors.lastname: Yao
authors.forename: Peter
authors.initials: P
authors.lastname: Pimpl
authors.forename: Liwen
authors.initials: L
authors.lastname: Jiang
mesh_terms: D000818:Animals; D017360:Arabidopsis / Q000187:drug effects / Q000378:metabolism*; D015374:Biosensing Techniques; D002138:Calibration; D002451:Cell Compartmentation / Q000187:drug effects; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism; D003208:Concanavalin A / Q000494:pharmacology; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006863:Hydrogen-Ion Concentration / Q000187:drug effects; D007425:Intracellular Membranes / Q000187:drug effects / Q000378:metabolism*; D008322:Mammals / Q000378:metabolism; D015388:Organelles / Q000187:drug effects / Q000378:metabolism*; D055571:Secretory Pathway / Q000187:drug effects; D012995:Solubility
chemical_list: C485187:PHluorin; D003208:Concanavalin A; D049452:Green Fluorescent Proteins
keywords: Arabidopsis; ER; Golgi; MVB; TGN; endomembrane system; nucleus; pH; vacuole.