Are GTGs a new class of plant anion channels regulating pH in the endomembrane system?

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Membrane proteins play important physiological roles in all organisms with fundamental functions including transport, signalling, and bioenergetics. This project focusses on a unique membrane protein class that is highly conserved in eukaryotes: the G protein coupled receptor type-G proteins/Golgi pH regulator (GTG/GPHR) family. The proposal builds on our recent breakthroughs using Arabidopsis and C.elegans demonstrating that plant and animal GTGs are critical for growth and fertility. GTG proteins are required for Golgi function, cell wall synthesis and light-regulated growth, all crucial processes in plant growth and development and therefore critical to the global priorities of food security and bioenergy. This project will define the function of this novel membrane protein class and specifically test the hypothesis that they function as anion channels regulating Golgi pH in plants.
Firstly, to demonstrate whether there is conservation of function across kingdoms, we will determine if the Arabidopsis GTG1 gene can complement a Chinese hamster ovary GTG/GPHR-mutant cell line which shows defects in Golgi acidification or the C. elegans gtg1gtg2 mutant. We have already rescued the seedling phenotypes of Arabidopsis gtg1gtg2 with C. elegans GTG1 and we will test if CeGTG2 also rescues. Secondly, we will use patch clamp technology to determine whether the GTGs show channel activity following reconstitution into giant unilamellar vesicles, and in addition determine the biophysical properties of these putative channels. Thirdly, it has been proposed that GTGs could function as pH regulators in the Golgi and this project will develop imaging methodology to determine their role in regulating Golgi acidification in plants. Finally, to address the importance of Galpha interaction in the function of GTGs we will determine whether a Galpha-GTG interaction can be observed in planta and the extent to which the phenotype of the gtg1gtg2 double mutant is dependent on Galpha.

This project will seek to determine the function of GTGs in the plant cell secretory pathway. The role of these proteins has been disputed but we now hypothesise that they function in pH regulation within the endomembrane system. This hypothesis will be tested at Oxford Brookes using confocal microscopy techniques that enable determination of pH differences between compartments that are separated by membranes such as the Golgi bodies and endoplasmic reticulum.

Intellectual property: In the event of any exploitable IP being generated during the course of the project the Research and Business Development Office at Oxford Brookes will ensure a timely protection of IP and will direct any exploitation.

Outreach: The Brookes plant cell biology group are actively involved in science outreach programmes, including organising events for the Oxfordshire Science Festival, hosting school teachers in the laboratory, organising equipment loan schemes for Schools, presenting School talks, writing articles for various blogs and using social media to disseminate educational videos and plant cell biology breakthroughs. Outcomes from this project will, when appropriate, be disseminated via these activities.

Training: The Postdoctoral Research Associate on this project should start with a good understanding of plant cell biology and molecular biology but will learn advanced imaging techniques such as ratio imaging and bimolecular fluorescence complementation by working with the Co-I at Oxford Brookes University