Arabidopsis GTG1 functions as an anion channel regulating endomembrane pH

The G-protein coupled receptor type G proteins/Golgi pH regulator proteins (GTG/ GPHRs) are a family of membrane proteins found in eukaryotes with diverse functions proposed. These include functioning as an ABA receptor1 (Arabidopsis), an endomembrane protein involved in growth and development2 (Arabidopsis), a calcium channel for cold perception3 (rice) or as an anion channel regulating Golgi pH4 (Chinese hamster). Here we use model organisms, Arabidopsis thaliana and Caenorhabditis elegans, to reveal a conserved function across kingdoms for these proteins as a novel family of anion channels. Both organisms contain two genes (AtGTG1, AtGTG2, Cel-GPHR-1, Cel-GPHR-2) that act redundantly and knocking out both results in defects in growth and development. We demonstrate a conserved function, with Cel-GPHR-1 rescuing the root and hypocotyl defects observed in the Arabidopsis gtg1gtg2 double mutant. Both AtGTG1 and Cel-GPHR-1 localize to the ER and Golgi when expressed stably in Arabidopsis and transiently in tobacco. Patch-clamp studies reveal that AtGTG1 and Cel-GPHR-1 exhibit anion-specific channel activity when expressed in a mammalian cell line (HEK-293) and the AtGTG1-dependent Cl- current is inhibited by the anion channel blocker 4,4-diisothiocyanatostilbene-2,2-disulfonic acid (DIDS). Furthermore, anion channel activity is confirmed when AtGTG1 is reconstituted in a planar lipid bilayer. By targeting the pH sensor pHluorin to different endomembrane compartments in Arabidopsis, we demonstrate a physiological role for AtGTG1 in regulating ER and Golgi pH. Overall, this study demonstrates a fundamental conserved function of the GTG/GPHRs as anion channels providing counter-ion conductance for regulating endomembrane pH.