In Vivo Intracellular pH Measurements in Tobacco andArabidopsisReveal an Unexpected pH Gradient in the Endomembrane System

Abstract

The pH homeostasis of endomembranes is essential for cellular functions. In order to provide direct pH measurements in the endomembrane system lumen, we targeted genetically encoded ratiometric pH sensors to the cytosol, the endoplasmic reticulum, and the trans-Golgi, or the compartments labeled by the vacuolar sorting receptor (VSR), which includes the transGolgi network and prevacuoles. Using noninvasive live-cell imaging to measure pH, we show that a gradual acidification from the endoplasmic reticulum to the lytic vacuole exists, in both tobacco (Nicotiana tabacum) epidermal (DpH 21.5) and Arabidopsis thaliana root cells (DpH 22.1). The average pH in VSR compartments was intermediate between that of the transGolgi and the vacuole. Combining pH measurements with in vivo colocalization experiments, we found that the trans-Golgi network had an acidic pH of 6.1, while the prevacuole and late prevacuole were both more alkaline, with pH of 6.6 and 7.1, respectively. We also showed that endosomal pH, and subsequently vacuolar trafficking of soluble proteins, requires both vacuolar-type H + ATPase-dependent acidification as well as proton efflux mediated at least by the activity of endosomal sodium/proton NHX-type antiporters.

Crystal Structure of Plant Legumain Reveals a Unique Two-Chain State with pH-Dependent Activity Regulation

“…1). A two-chain state would be a perfect candidate, since it can survive the neutral pH compartments (Reguera et al, 2015;Martinière et al, 2013;Otegui and Spitzer, 2008) like cytoplasm, the late endosome/prevacuolar compartment (PVC) or the nucleus (Fig. 7).…”

Job Sharing in the Endomembrane System: Vacuolar Acidification Requires the Combined Activity of V-ATPase and V-PPase

“…An alternative explanation would be that vesicular trafficking from the TGN/EE to the vacuole delivers a substantial amount of protons and/or organic acids to the vacuolar lumen. The TGN/EE is the most acidic compartment of the plant endomembrane system (Martinière et al, 2013;Shen et al, 2013), and using a pH-sensor with suitable resolution at low pH, we have recently shown that in Arabidopsis roots pH in the TGN/EE is indeed lower than the vacuolar pH (Luo et al, 2015). At least three independent, parallel trafficking pathways exist from the TGN/EE to the vacuole (Pedrazzini et al, 2013;Rojas-Pierce, 2013), and it remains to be determined if the combined flux of protons delivered to the vacuolar lumen would be sufficient to maintain a proton gradient even in the absence of tonoplast-localized proton pumps.…”

AtNHX5 and AtNHX6: Roles in protein transport

“…[17][18][19][20] In the exocytic or endocytic pathways, the organelles become more acidic along the process of maturation. 17,18,21 In plants, the acidic pH of the organelles is maintained by the proton pumps V-ATPases and pyrophosphatase. 20,21 Studies have shown that AtNHX5 and AtNHX6 may act as a H C -leak pathway to counter the luminal acidification.…”

“…17,18,21 In plants, the acidic pH of the organelles is maintained by the proton pumps V-ATPases and pyrophosphatase. 20,21 Studies have shown that AtNHX5 and AtNHX6 may act as a H C -leak pathway to counter the luminal acidification. 21 Reguera et al found that the endosomal pH homeostasis maintained by AtNHX5 and AtNHX6 is critical for the transport of seed storage proteins.…”

“…20,21 Studies have shown that AtNHX5 and AtNHX6 may act as a H C -leak pathway to counter the luminal acidification. 21 Reguera et al found that the endosomal pH homeostasis maintained by AtNHX5 and AtNHX6 is critical for the transport of seed storage proteins. 5 Consistently, Wu et al found that the exchange activity of AtNHX5 and AtNHX6 was crucial for protein transport, indicating the importance of pH homeostasis regulated by AtNHX5 and AtNHX6 in protein transport.…”