Is there a conserved function for the GTG/GPHR family of membrane proteins?

• Main Author: Wong, Nancy
• Other Authors: Williams, Lorraine ; Terry, Matthew ; Smyth, Neil
• Published: University of Southampton 2014
• Subjects: 570; QH301 Biology
• Online Access: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.632611

The G protein-coupled receptor (GPCR)-type G protein/Golgi pH regulator (GTG/GPHR) proteins are a conserved family of membrane proteins in eukaryotes, but are yet to be fully characterised. So far, two possible functions have been described: anion channels for Golgi pH regulation in animals or plasma membrane abscisic acid receptors in plant signalling. Here, the role GTG/GPHRs has been explored using plant (Arabidopsis thaliana), animal (Caenorhabditis elegans) and fungal (Saccharomyces cerevisiae) models. There are two Arabidopsis thaliana GTG/GPHR genes, At GTG1 and At GTG2. Knocking out both in the gtg1-2 gtg2-2 and gtg1-3 gtg2-3 mutants results in shorter root and hypocotyl growth under certain conditions, and expression of either At GTG can restore these defects (Jaffé et al., 2012). In this thesis, these growth defects are confirmed in another gtg1 gtg2 double mutant (gtg1-1 gtg2-1). GTG/GPHRs have a conserved glycine in their domain of unknown function, DUF3735. Mutating this glycine (G166) to leucine in At GTG1 negates its rescue of gtg1-3 gtg2-3 defects, indicating its importance for function. There are also two C. elegans (Cel-) GTG/GPHR genes, Cel-gphr-1 and Cel-gphr-2. In C. elegans, GFP-tagged Cel-GPHR-1 shows an intracellular punctate pattern consistent with Golgi localisation. Backcrossed single mutants (Cel-gphr-1(ok1579) and Cel-gphr-2(tm4228)) and a double mutant (Cel-gphr-1(ok1579) gphr-2(tm4228)) are generated in this project. Single mutants display similar characteristics to wild-type C. elegans whereas the double mutant, lacking both gphr genes, shows abnormal egg-laying, egg development and hatching, as well as larval arrest and reduced pharyngeal pumping. When expressed in Arabidopsis, Cel-GPHR-1 shows a similar localisation pattern to the endogenous At GTGs and rescues the root and hypocotyl defects observed in Arabidopsis gtg1-3 gtg2-3. Localisation studies also show that both Arabidopsis and C. elegans GTG/GPHRs are Golgi/ER localised in S. cerevisiae following heterologous expression. All results are consistent with the GTG/GPHRs having a conserved function across kingdoms.