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

Lead Research Organisation: University of Southampton
Department Name: School of Biological Sciences

Abstract

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 membane 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.

Technical Summary

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.

Planned Impact

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.

Publications

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Description 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 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. This project aims to 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 (ie a role in acidification of endomembrane compartments). 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. We have now successfully developed a range of molecular tools (constructs and cell lines) to specifically answer these questions. To investigate the role of GTGs as pH regulators of the secretory pathway we have generated a range of constructs which have been expressed in plants. We have now used these to detect pH in various cellular compartments (ER, cis-Golgi, Trans Golgi Network and prevacuolar compartments). We have generated Arabidopsis wild-type and gtg1gtg2 double mutants so that we can monitor what effect knocking out these particular genes has on pH in the secretory pathway. By doing this we have shown that AtGTG can regulate the pH of the ER and cis Golgi.
In addition, in order to understand the role of G proteins in GTG function, we have made constructs suitable for Bimolecular Fluorescence complementation (BiFC). We are using these to detect an in planta interaction of GTGs with G proteins in the plant. Our data indicates there is an interaction in controls (GPA1 and AGB1) but no evidence for a clear interaction wiht GTG1 and GPA1.
We have also tested the importance of Galpha for the gtg1gtg2 mutant phenotype by crossing gtg1gtg2 with the Galpha mutant gpa1-1. The cross has been carried out and we have now isolated a gpa1gtg1gtg2 triple mutant. These have been specifically analysed for light-dependent hypocotyl growth inhibition, inhibition of root growth and silique length. Our evidence to date suggests there is a genetic interaction of the GTGs and GPA1 as we observed an additive effect of knocking out all genes under particular conditions.
GTG proteins are conserved across the full protein sequence. However, there are two major domains in which conservation is especially strong. One of these (named the GTP-binding domain) contains a single conserved tyrosine residue, conserved across all GTGs. Using site-directed mutagenesis, we have mutated this residue conservatively and non-conservatively to test whether this mutated form can restore the seedling and fertility defects observed in the Arabidopsis gtg1gtg2 mutants. We have tested the mutants which has indicated that there may be a small effect on the activity when mutated.
The most significant finding we have made to date is that AtGTG1 and CeGTG1 can function as anion channels. We have successfully expressed these genes in a mammalian cell line and demonstrated channel activity using patch clamp technology. We have tested substrate specificity and inhibitor sensitivity of these channels. Moreover we have expressed AtGTG1 in a cell free system and inserted it into a lipid bilayer and been able to measure anion channel activity. The demonstration that the plant GTGs function as anion channels is a major finding.
Exploitation Route The work will have major implications for our understanding of Golgi (and endoplasmic reticulum, ER) function in plants and in other eukaryotic systems. Our work suggests that the plant GTGs function as a new class of anion channel and information about these proteins will therefore impact all researchers interested in how ions are transported across membranes. More specifically the identity of the anion channels required for efficient endomembrane acidification is largely unknown and certainly in plants there are few clues to how this is achieved. We have shown that the GTG proteins can fulfil this role and that our data will have major implications for the understanding of Golgi and ER function in plants. The major academic beneficiaries will be all researchers in the fields of membrane biology, transport, and endomembrane function. Since GTG proteins also have major effects on plant growth and fertility, researchers interested in the underlying mechanisms will also benefit from this work. In that sense the research is broadly relevant to BBSRC research priorities on bioenergy and crop science. Importantly, we have identified homologues of the GTGs in the major crop species: wheat, barley and rice. A recent report has suggested that the rice GTG may function as a cold receptor and certainly our mechanistic work on GTGs will have important consequences.
Therefore longer-term beneficiaries will include research institutes and industry in the plant breeding/agricultural biotechnology sector, both in the UK and internationally. Our work will also address the role of the GTGs in cell wall synthesis and structure, again fundamental aspects relevant to these research priorities. Explaining in more detail the processes underlying Golgi function and cell wall synthesis and structure is important to those working in plant cell biology.
The increased understanding of animal cell biology from this fundamental work may also have long term implications for medical research. Certainly, further analysis of the animal model that we have produced, which has defects in development and fertility, will be of benefit to those addressing these topics not only in nematodes but more widely.
Sectors Agriculture, Food and Drink,Education,Environment

 
Description The Gerald Kerkut Charitable Trust- studentship scheme
Amount £70,000 (GBP)
Organisation The Gerald Kerkut Charitable Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2014 
End 09/2018
 
Description The Gerald Kerkut Charitible trust studentship scheme
Amount £33,000 (GBP)
Organisation The Gerald Kerkut Charitable Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2016 
End 09/2019
 
Title Arabidopsis containing AtGTG1 and AtGTG2 under an inducible promoter 
Description Arabidopsis gtg1 gtg2 mutants have been generated that contain AtGTG1 and AtGTG2 under an inducible promoter 
Type Of Material Cell line 
Provided To Others? No  
Impact Lines are available that have been transformed with AtGTG1 and AtGTG2 under an inducible promoter for functional studies 
 
Title Arabidopsis expressing pHluorin at different compartments 
Description Arabidopsis expressing pHluorin at different compartments for pH determination (ER, cis Golgi, TGN and MVP/PVC) 
Type Of Material Cell line 
Provided To Others? No  
Impact Lines are available to determine the pH regulation at various compartments in order to determine the role of GTGs 
 
Title Arabidopsis gtg double mutants expressing pHluorin at different compartments 
Description Arabidopsis gtg double mutants expressing pHluorin at different compartments for pH determination (ER, cis Golgi, TGN and MVP/PVC) 
Type Of Material Cell line 
Year Produced 2017 
Provided To Others? No  
Impact Lines are available to determine the pH regulation at various compartments in order to determine the role of GTGs 
 
Title Arabidopsis with mutated GTGs 
Description Arabidopsis expressing mutated GTGs 
Type Of Material Cell line 
Provided To Others? No  
Impact These lines are available for investigating GTG structure function 
 
Title CeGTG2 -expressing Arabidopsis lines 
Description Arabidopis lines expressing C.elegans GTG2 gene 
Type Of Material Cell line 
Provided To Others? No  
Impact Lines are available for assessing the function of CeGTG2 
 
Title HEK cells expressing AtGTGs and CeGTGs 
Description HEK cells transformed with wild-type and mutated AtGTGs and CeGTGs 
Type Of Material Cell line 
Provided To Others? No  
Impact These lines are available for investigating the channel activity of the GTGs using patch clamp technology 
 
Title Lines transformed with AtGTG1 in an RNAi construct 
Description Lines transformed with AtGTG1 in an RNAi construct under a dexamethasone inducible promoter have been generated for down-regulating GTG1 
Type Of Material Cell line 
Provided To Others? No  
Impact these lines are availble for investigating the impact of down-regulating GTG1 
 
Title gpa1gtg1gtg2 triple mutant generated 
Description Arabidopsis triple mutant gpa1gtg1gtg2 generated 
Type Of Material Cell line 
Provided To Others? No  
Impact Arabidopsis lines will be available that are mutated in Galpha, GTG1 and GTG2 for functional analysis 
 
Title tobacco with constructs for BiFC analysis 
Description Tobacco plants expressing GTG1 and Galpha constructs for bimolecular fluorescence complementation (BiFC) 
Type Of Material Cell line 
Provided To Others? No  
Impact Tobacco plants expressing GTG1 and Galpha constructs for bimolecular fluorescence complementation (BiFC) for functional analysis 
 
Description School engagement activity 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact Dr. L.E. Williams worked with undergraduate students to develop an activity to deliver to A Level students (from local colleges like Peter Symonds Sixth
Form College, Winchester). This was related to the use of transgenic crops and various activities were undertaken. A quiz, a debate, a painting exercise to show where nutrients accumulate in crops and a poster to show a scheme for making transgenic plants. The A-level students engaged in all activities and improved their knowledge of the importance of plants in areas related to food security
Year(s) Of Engagement Activity 2017
 
Description school engagement activity 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact Dr. L.E. Williams worked with undergraduate students to develop an activity to deliver to A Level students (from colleges like Peter Symonds Sixth Form College, Winchester). This was related to the use of reporter genes in plants and the operation of transporters. Various activities were undertaken. A quiz, a role playing exercise, making a poster. The A-level students engaged in all activities and improved their knowledge on the importance of plant research, molecular genetics, reporter genes, transporters and food security
Year(s) Of Engagement Activity 2018