TY  - GEN
TI  - pH in the trans-Golgi network/early endosome of Arabidopsis thaliana: Suppliers and consumers
Y1  - 2018///
UR  - https://archiv.ub.uni-heidelberg.de/volltextserver/23248/
ID  - heidok23248
N2  - The trans-Golgi network (TGN)/ early endosome (EE) is the main sorting station for proteins
travelling along the secretory- or endocytic route. Acidification by the TGN/EE-localised
vacuolar-type proton (H+)-ATPase (V-ATPase) is essential for functional protein trafficking as
well as compartment structure. Inhibition of this V-ATPase ceases protein trafficking through the
TGN/EE and inhibits cell elongation. To sustain TGN/EE functions, luminal pH has to be kept on
a homeostatic level. A mechanism to maintain V-ATPase-mediated acidification is to balance the
bulk positive charges in the TGN/EE lumen accumulated by H+ pumping via parallel anion
import. However, previous measurements of V-ATPase-dependent acidification in the TGN/EE
were mainly done in transient expression systems and used sensors suited for rather alkaline pH
conditions. Furthermore, TGN/EE-localised anion transporter from the ClC-family which have
been proposed to serve in luminal charge balance are largely uncharacterised. For that purpose,
in vivo TGN/EE pH measurements using the genetically encoded pH sensor pHusion linked to
the TGN/EE resident protein SYP61 were established. We determined that the steady state pH
of 5,6 in the TGN/EE in Arabidopsis roots is controlled by the combined activity of the V-ATPase
and H+-coupled antiporters. Moreover the role of ClCd and ClCf in supporting the TGN/EElocalised
V-ATPase in acidification was investigated. Whereas knock-out of both TGN/EElocalised
ClCs caused male gametophyte lethality, we identified via inducible knock-downs an
essential role of both transporters in cell elongation. However, neither TGN/EE pH nor TGN/EEmediated
trafficking was altered upon reduced reduction of ClCf in the clcd background.
Curiously, trans-Golgi pH was more acidic than WT due to a partial mislocalisation of the
TGN/EE-localised V-ATPases to the Golgi stack. Finally, a computational modelling approach
suggested that ClCd functions as Cl-/H+ antiporter whereas clear predictions on ClCf functions
could not been made. We established a method to measure V-ATPase-mediated TGN/EE
acidification in vivo in Arabidopsis thaliana. Furthermore, we showed that the TGN/EE is
acidified by the V-ATPase and co-determined by TGN/EE-localised transporters. Besides VATPase
activity, anion import in the TGN/EE mediated by two ClC members ClCd and ClCf is
essential for cell elongation. By that we revealed a previously unknown role of luminal anion
homeostasis in the TGN/EE.
A1  - Scholl, Stefan
AV  - public
ER  -