title: pH in the trans-Golgi network/early endosome of Arabidopsis thaliana: Suppliers and consumers
creator: Scholl, Stefan
subject: 570
subject: 570 Life sciences
description: 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.
date: 2018
type: Dissertation
type: info:eu-repo/semantics/doctoralThesis
type: NonPeerReviewed
format: application/pdf
identifier: https://archiv.ub.uni-heidelberg.de/volltextserverhttps://archiv.ub.uni-heidelberg.de/volltextserver/23248/1/Dissertation%20Stefan%20Scholl%202017.pdf
identifier: DOI:10.11588/heidok.00023248
identifier: urn:nbn:de:bsz:16-heidok-232487
identifier:   Scholl, Stefan  (2018) pH in the trans-Golgi network/early endosome of Arabidopsis thaliana: Suppliers and consumers.  [Dissertation]     
relation: https://archiv.ub.uni-heidelberg.de/volltextserver/23248/
rights: info:eu-repo/semantics/openAccess
rights: http://archiv.ub.uni-heidelberg.de/volltextserver/help/license_urhg.html
language: eng