Purification and functional characterisation of COMATOSE a peroxisomal ABC transporter from Arabidopsis thaliana

Lead Research Organisation: University of Leeds
Department Name: Ctr for Plant Sciences

Abstract

Cells of higher organisms such as plants and animals are divided into compartments that contain specific biological functions. Each compartment is surrounded by a membrane which acts as a barrier to movement of most biological molecules. In order for the activities of different compartments to proceed, the controlled transport of molecules across membranes has to occur. In most cases transport is mediated by proteins (transporters) within the membrane and often expenditure of energy is required to move the transported molecules against a concentration gradient. ABC transporters are a large class of membrane proteins which transport a wide range of biological molecules using energy released by breakdown of ATP, the principal 'energy currency' of living organisms. This proposal is concerned with one specific ABC transporter found in the membrane of the cellular compartment known as the peroxisome. Peroxisomes carry out a range of different biological functions but the one of particular interest to this project is beta-oxidation. In this pathway fat-like molecules are chopped up by sequential removal of 2 carbon atoms at a time. Different molecules may be subjected to beta-oxidation for different purposes - to provide energy, to remove unwanted molecules or to generate molecules with different biological activities, such as hormones. Peroxisome membranes of plants, animals and yeast contain similar ABC transporter proteins. There is quite good evidence in the case of yeast and plants that these proteins are involved in transporting fats into peroxisomes to be broken down. This may also be the case in animals, but the evidence is not so clear. What is clear is that plants and animals which have defective peroxisomal ABC transporters are very sick. Children born with a defective peroxisome ABC transporter called ALDP may develop a severe neurological disorder called Childhood Cerebral X-linked Adrenoleukodystrophy which results in progressive disability and death, and for which there is no cure and limited ameliorative treatment. Plant seeds with a defect in the equivalent ABC transporter (called CTS) can't germinate unless special tricks are used, have some hormone deficiencies and are less fertile than wild-type plants. We don't know to what extent the plant, yeast and human peroxisomal ABC transporters do the same things, but since they probably share a common evolutionary origin there is a good chance some functions are conserved. We will seek to investigate this by finding out if the plant transporter can function in place of the yeast transporter and if the human and yeast transporters can function in place of the plant transporter. We will also find out more about the precise functions of the plant transporter by investigating which molecules it can transport and what parts of the protein are necessary for this activity. By learning about the activity of this plant protein we would hope to understand how we could manipulate it to produce improved crop varieties. Understanding the similarities and differences between the plant, yeast and human transporters might also yield new insights into what the human transporter is doing which could be of benefit to understanding the pathology of X-linked Adrenoleukodystrophy.

Technical Summary

COMATOSE (CTS) is a peroxisomal ABC transporter protein of Arabidopsis which shares significant sequence identity with mammalian peroxisomal ABC transporters, one of which, ALDP, is defective in the genetic disorder X-linked Adrenoleukodystrophy. Genetic and physiological evidence point to CTS being a broad specificity transporter, which functions to integrate metabolic and developmental responses at several stages of the plant life cycle. However, this evidence is indirect, and to fully understand and exploit CTS requires the ability to study the transport capability of this protein biochemically and quantitatively. We have expressed CTS in a functional form in S. cerevisiae and will use this system to assess the ability of CTS to utilise different substrates via substrate stimulation of ATPase activity and substrate-induced conformational changes, measured by altered sensitivity of CTS to proteolysis. We will test the ability of CTS to complement the S. cerevisiae pxa1/pxa2 double mutant which lacks endogenous peroxisomal ABC transporters. IF CTS complements the mutant, this will indicate functional similarity and permit the use of a loss-of function growth assay to isolate novel mutants within the transmembrane domains (TMDs) that may influence substrate recognition. If CTS does not complement, we will identify TMD residues for site-directed mutagenesis using a homology model of CTS based on an ABC transporter of known structure and test the phenotypic effects in planta. We will test the ability of the yeast and mammalian peroxisomal ABC transporters to complement different aspects of the cts mutant phenotype in planta. Finally, we will use a heterologous expression system (baculovirus or a yeast-based, depending upon results of expression trials) to produce functional protein for purification and reconstitution, thereby allowing results from endogenous or heterologous systems to be analysed by quantitative transport assays in a defined and controlled system.

Publications

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Baker A (2014) The life of the peroxisome: from birth to death. in Current opinion in plant biology

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De Marcos Lousa C (2013) Intrinsic acyl-CoA thioesterase activity of a peroxisomal ATP binding cassette transporter is required for transport and metabolism of fatty acids. in Proceedings of the National Academy of Sciences of the United States of America

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Hu J (2012) Plant peroxisomes: biogenesis and function. in The Plant cell

 
Description We expressed the peroxisomal ABC transporter COMATOSE in insect cells and also in yeast. This allowed us to investigate its function in more detail than was possible in the native plant. We could show that the protein had two distinct biochemical activities, ATPase and thioesterase. Thioesterase activity was unexpected but provided an answer to a question that had been outstanding in the literature for a decade, are CoA esters or free fatty acids transported. Our model is that CoA esters are the preferred substrate but are cleaved by the thioesterase activity in CTS. We also found that CTS associates with peroxisomal acyl coA synthetases which would add the CoA back on the other side of the membrane
Exploitation Route This has led to a new grant BB/L001012/1 to unravel details of this novel mechanism.
the expression systems and experimental protocols are relevant to the study of the human homologue ALDP which is defective in the human disorder X-linked adrenoleukodystrophy which our collaborators at AMC work on
Sectors Agriculture, Food and Drink,Pharmaceuticals and Medical Biotechnology

 
Description The information gained about this important class of ABC transporters is being used by our collaborators at AMC to inform research into X-linked adrenoleukodystrophy
First Year Of Impact 2008
Sector Healthcare,Pharmaceuticals and Medical Biotechnology
Impact Types Societal

 
Description Japan Partnering award
Amount £36,000 (GBP)
Funding ID 474346 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 04/2008 
End 03/2011
 
Description Responsive mode
Amount £767,505 (GBP)
Funding ID BB/L001012/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 03/2014 
End 07/2017
 
Description Amsterdam Medical Centre 
Organisation VU University Medical Center
Country Netherlands 
Sector Academic/University 
PI Contribution Producing constructs and biochemical characterisation of CTS transporters and mutants expressed in yeast Expression of CTS in insect cells developing expression, purification, reconstitution and ATPase and thioesterase assays
Collaborator Contribution Providing plasmids and strains. Carrying out beta oxidation measurements, constructs to mislocalise LACS6 and 7 in yeast
Impact 10.1073/pnas.1218034110 10.1042/BJ20110249 10.1074/jbc.M110.151225
Start Year 2008
 
Description Rothamsted Research 
Organisation Rothamsted Research
Country United Kingdom 
Sector Academic/University 
PI Contribution Screening of TILLING mutants in CTS NBD1 Expression of CTS in insect cells development of purification, reconstitution and enzyme assay methodology Expression of CTS in yeast and biochemical characterisation Confocal microscopy of CTS localisation in planta QPCR antibody production and western blotting protein-protein interaction studies by co-IP 2D gels and sucrose gradients
Collaborator Contribution Characterisation of TILLING mutants Plant physiology Transgenic plant production pull down experiments (CTS-PEX19) construct production
Impact 10.1042/BJ20130078 10.1073/pnas.1218034110 10.1016/j.febslet.2012.05.065 10.1042/BJ20110249 10.1074/jbc.M110.151225 10.1016/j.bbabio.2010.04.154 10.1091/mbc.E08-07-0745 10.1104/pp.107.099903 10.1093/jxb/erl045 10.1016/j.febslet.2005.12.095 10.1104/pp.105.059352
 
Description Inaugural Irene Manton lacture 2015 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Other audiences
Results and Impact I was invited by the local Athena Swan organisers to give the inaugural Irene Manton Lecture to celebrate women in science in our Faculty. I gave my lectures on 'The ins and outs of plant cells' on 8th May 2015
Year(s) Of Engagement Activity 2015
 
Description Talk to Leeds Soroptimist International group 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact I was invited to give a talk to the Leeds Soroptimists on the subject of Women in Science. I received the following feedback from 2 of the committee members:

I have had wonderful feedback from Club members about your presentation and would like to pass them on to you.-
A well prepared, interesting and accomplished talk. Stimulating and positive as to the current situation with women considering Science today. Best speaker yet and appreciation as to how friendly and interactive you were as evident by the very many questions afterwards.
Well done and much appreciated and many thanks for your time and effort. THANK YOU.

I want to say a big thank you for your presentation. We really enjoyed your style which made it fun and interesting, whilst also being really clear about some of the barriers that women still face, particularly in the sciences. We are all keen to try and encourage girls and women to make the most of their potential in whatever is their chosen field. We have taken some useful messages away from your presentation that we can use in our everyday lives, whether at work or with children, grandchildren etc. Thank you again
Year(s) Of Engagement Activity 2013