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

Membrane proteins play important physiological roles in all organisms with fundamental functions including transport, signalling, and bioenergetics. This project will focus on a unique membrane protein class which is highly conserved in eukaryotes: the G protein coupled receptor type-G proteins/Golgi pH regulator (GTG/GPHR) family. Contrasting theories exist for the roles of these proteins and the physiological function of this family remains enigmatic. One model suggests the Arabidopsis GTG/GPHRs are plasma membrane receptors for the plant hormone, abscisic acid (ABA; Pandey et al, 2009, Cell 136, 136-148). In contrast, a second group used patch clamp technology, which can be used to measure current carried across membranes by proteins called ion channels, and found that the mammalian GTG/GPHR possesses voltage-dependent anion-channel activity and is critical in regulating Golgi acidification (Maeda et al 2008 Nat Cell Biol, 10, 1135-1145). They made these finding using a Chinese hamster ovary cell line which had a mutated GTG/GPHR, and showed defects in Golgi function. The Golgi is part of the endomembrane system and is a critical organelle in eukaryotes required for packaging and sorting of molecules to be delivered to other parts of the cell and for secretion. This vital process in eukaryotic cell biology is thought to be dependent on a pH gradient along the endomembrane pathway.

The proposal builds on our recent breakthroughs using the model plant, Arabidopsis, and model animal, C.elegans, demonstrating that plant and animal GTGs are critical for growth and fertility. We have shown in Arabidopsis that GTG proteins are required for Golgi function, cell wall synthesis and light-regulated growth (Jaffé et al, 2012, Plant Cell 24, 3649-68). This was carried out using mutants that we have isolated independently and in which we observe normal responses to ABA treatments. This and the fact that we find them localised to the Golgi questions their role as plasma membrane ABA receptors. In addition, we have produced the first whole animal model (C.elegans) where both GTGs are mutated and this mutant also shows defects in fertility and growth. Transformation of C. elegans GTG1 into plant gtg1gtg2 mutants shows that its expression restores normal root and hypocotyl (seedling stem) growth. As the animal protein restores these defects in plants we propose a common function for plant and animal GTGs.

This project will define the function of this novel membrane protein class, further investigating conservation of function and specifically testing the hypothesis that they function as anion channels regulating Golgi pH in plants.

To demonstrate whether there is conservation of function across kingdoms, we will determine if the Arabidopsis GTG1 gene can restore the defects in two animal mutant systems which lack GTG function. The first will be the mammalian Chinese hamster ovary GTG/GPHR-mutant cell line which shows defects in protein secretion due to poor Golgi acidification; the second will be the C. elegans gtg1gtg2 mutant. To determine directly whether plant GTGs have channel activity we will use patch clamp technology to demonstrate anion transport activity following purification and insertion of AtGTG1 into giant unilamellar vesicles. This system will be used to determine biophysical and pharmacological properties of these putative channels and structure/function analysis. We will develop systems for assessing Golgi/ER pH in Arabidopsis using a range of pH probes and test whether plant GTGs can function as pH regulators allowing acidification of these endomembrane compartments. Finally, a regulatory protein called Galpha has been shown to interact with AtGTG1 in yeast. To address the importance of this 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 gtg1 gtg2 double mutant is dependent on Galpha.

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. Plants are central both to agriculture and to conservation of the natural environment. As well as food, they offer resources for construction, natural fibre, and fuel, and contribute to mitigation of climate change. Given these key roles, it is becoming increasingly recognised that we need to understand better the underlying factors that control plant growth, development and reproduction. This underpins future strategies to enhance plant productivity under both stress and non-stress conditions. This project will provide key information about mechanisms underlying plant growth and fertility. Our research will have both academic impact, and will provide indicators that may be useful in future crop development and management.

From an academic perspective, the topic of Golgi/ER function in relation to both root and shoot growth, as well as the impact on fertility of reduced pollen tube growth is of fundamental importance, but we still have only a poor understanding of the underlying processes. Regulation of intracellular pH is essential in all organisms and defective acidification of endomembrane compartments can have serious consequences on vesicular trafficking, glycosylation and protein sorting. Establishing whether GTGs act as anion channels in plants regulating this process will therefore have major impact. Moreover, the GTG proteins that are the subject of this proposal are not similar to any other known group of membrane proteins and may therefore define a completely new category of membrane transporter in eukaryotes. As GTGs are highly conserved in eukaryotic species, information we obtain in this project will be of fundamental importance and relevance to researchers in many fields of eukaryotic biology.

Training
The staff employed on this project will receive a broad training in molecular, cellular and biochemical methods that will be applicable to many research environments both academically and in the industrial sector, including medical and bioenergy research. In addition, they will learn advanced imaging techniques such as ratio imaging and bimolecular fluorescence complementation by working with the Co-PI at Oxford Brookes University.

Intellectual property
In terms of economic/societal impact, our work on the role of GTGs in plant growth and development will impact on our understanding of the basic mechanism underlying plant growth, an essential factor in productivity of food and bioenergy crops. Although the research proposed is fundamental in nature we will look to exploit any opportunities for knowledge transfer to the commercial sector through established routes set up by the Universities such as Southampton's Research and Innovation Services, or Brookes' Research and Business Development Office, as well as by participation in the National Institute of Agricultural Botany (NIAB) Innovation Farm, where we will be able to engage businesses, innovators and stakeholders in the agricultural and horticultural sectors, and ongoing links with commercial partners (see "Pathways to Impact" for details).

Outreach
Finally, we will also be extremely active in developing outreach opportunities both to Schools, where we will convey the concepts and importance of our research, and through the NIAB Innovation farm. The Brookes plant cell biology group is 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.