Arabidopsis and C. elegans GTG/GPHRs have a conserved role across kingdoms as anion channels

Abstract

The G-protein coupled receptor type G proteins/Golgi pH regulator proteins (GTG/GPHRs) are a highly conserved family of eukaryotic membrane proteins, but contrasting roles have been reported for these proteins in different organisms. So far, three possible functions have been described: anion channels responsible for Golgi pH regulation in animal cells; plasma membrane abscisic acid receptors in Arabidopsis; and plasma membrane and ER localised cold receptors in rice. In this study, a role for plant (Arabidopsis thaliana) and nematode (Caenorhabditis elegans) GTG/GPHRs as anion channels has been examined and insights into key residues important for protein function have been explored. Electrophysiological recordings of the mammalian cell line HEK-293 expressing GFP tagged At GTG1 and Ce GTG1 revealed a conserved role for these proteins as anion channels. Currents were DIDS-sensitive and anion specific, with a high permeability for chloride. These currents were not regulated by changes in pH or by the addition of ABA. Furthermore, recombinant At GTG1 and Ce GTG1 expressed using a cell-free synthesis kit and incorporated into a planar lipid bilayer displayed single-channel events that were anion specific and inhibited by DIDS. Recombinant At GTG1 is highly selective for chloride, but is also permeable to nitrate. The multiple open states of At GTG1 in asymmetrical salt gradient conditions could indicate complex gating mechanisms for this channel in vivo.

The GTG/GPHR family have regions of high homology throughout the protein sequences. One residue, a tyrosine at position 401 is completely conserved in all 179 analysed GTG/GPHR sequences. To explore the role of this residue in GTG/GPHR function, mutant GTG/GPHRs from Arabidopsis and C. elegans were produced. The mutant proteins were expressed in Arabidopsis gtg1 gtg2 mutant plants, as well as in HEK-293 cells for electrophysiological recordings. Arabidopsis gtg1 gtg2 double knockout mutants have decreased root length and fresh weight defects when grown on 0 % sucrose media. Interestingly, when At GTG1 Y401 is mutated to the hydrophobic residues phenylalanine or leucine, it can functionally complement these mutant phenotypes. At GTG1^Y401L expressing HEK-293 cells also have currents that are comparable to non-mutated At GTG1-expressing cells. However, At GTG1^Y401S can only partially rescue fresh weight and root length phenotypes of the gtg1 gtg2 mutant, with confocal microscopy revealing that this is not due to differences in intracellular localisation. Electrophysiological recordings have also shown that At GTG1^Y401S-expressing HEK-293 cells have significantly lower currents in comparison to At GTG1-expressing cells. The results in this thesis, together with further studies in our lab are consistent with the GTG/GPHRs family having a conserved role across kingdoms as anion channelsthat regulate ER and Golgi luminal pH.

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ORCID for Matthew Terry: orcid.org/0000-0001-5002-2708

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