cleavage of acyl CoA by ABC subfamily D transporters in peroxisomes: mechanism and functional roles

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


Cells of animals, plants and fungi are organised into compartments which have different functions, much like the rooms in a house. One such compartment is the peroxisome. The main role of peroxisomes is to release energy and molecular building blocks from stored fat and oil, a process called beta-oxidation. Beta-oxidation also plays other roles in making molecules that are important signals within and between cells and peroxisomes share several other jobs with different compartments in cells. For this to work efficiently, many different types of molecules have to move in and out of peroxisomes in an organised fashion. Thus, peroxisomes are sometimes called "organelles at the crossroads".
This project aims to study a family of proteins which transport molecules into peroxisomes so that they can be processed by beta-oxidation. These proteins are called ATP Binding Cassette transporters from subfamily D, or ABCD transporters. We have been studying how these transporters work at a detailed level and also more globally, to determine how they help to keep the different chemical reactions of the cell in check. By investigating ABCD transporters, we have learned that beta-oxidation is important for seed germination and establishment, fertility, senescence, root growth and wound responses in plants and this tells us that the transporters accept quite a wide range of different molecules. Others have shown that ABCD transporters are important for fat breakdown in humans and when they do not work properly, can cause diseases. Recently, we have found that ABCD transporters are rather unusual because they accept a molecule such as a fatty acid which is joined to another molecule called Coenzyme A (CoA) but then chop off ("cleave") the CoA part as the fatty acid is transported into the peroxisome. CoA is an important chemical in cells because it helps different chemical reactions to happen, thus the levels in different compartments need to be carefully regulated. Once inside the peroxisome, the fatty acid cannot enter the beta-oxidation process until it is "activated"- by joining it to another CoA molecule. At first glance, this seems inefficient but we think that it is important for controlling which molecules can enter the peroxisome and be processed by beta-oxidation (this is known as "metabolic channelling"). Re-joining of the CoA molecule to fatty acids inside the peroxisome requires proteins called acyl activating enzymes (AAEs). We have shown that ABCD transporters in plants are physically and functionally linked to the AAEs which join fatty acids to CoA but we think that they could also interact with different AAEs which join other kinds of molecules to CoA. The availability of different AAEs and their interaction with the transporter provides a potential check-point to control which molecules are allowed to be processed by beta-oxidation.
To understand this process better and to make the knowledge more widely useful, we now need to know more detail about how the transporters work, including how the CoA cleavage fits into the transport process and whether it works equally well for different molecules which can be imported. We also want to find out where the cleaved CoA ends up (outside the peroxisome or inside?). Taken together, this information will tell us how peroxisomes balance their resources between different competing functions and how metabolism is controlled.

Technical Summary

Peroxisomes play essential roles in lipid catabolism and synthesis of bioactive lipid-derived molecules. They also participate in a range of metabolic pathways which are shared with other organelles. Thus transport of solutes across the peroxisomal membrane is a key checkpoint in metabolic control. Import of substrates for peroxisomal beta-oxidation is mediated by ABC subfamily D transporters, but their mechanisms have proved contentious.In supporting BBSRC-funded work, we have demonstrated both a functional and physical interaction between the Arabidopsis ABC transporter protein CTS and the peroxisomal long chain fatty acyl CoA synthetases LACS6 and LACS7. CTS, when expressed in insect cell membranes, possesses an intrinsic ATP-stimulated thioesterase activity towards long chain acyl CoAs. This activity is reduced in a mutant that is defective in mobilisation of stored fatty acids in vivo, and is unable to complement a yeast strain defective in the functionally equivalent ABC transporter Pxa1p/Pxa2p, providing evidence for the physiological relevance of thioesterase activity. Together, our findings provide strong experimental support for the hypothesis that acyl CoAs are accepted by ABCD transporters, cleaved during transport and reactivated by peroxisomal acyl CoA synthetases. Soluble thioesterases are not ATP stimulated suggesting that the intrinsic ABCD thioesterase activity may be linked to the transport cycle. Combining transport biochemistry and plant physiology, this proposal seeks to dissect the relationship between substrate transport and the ATPase and thioesterase activities of CTS, to determine the structural basis for thioesterase activity and to explore the role of the transport/cleavage mechanism and interaction with diverse acyl activating enzymes in metabolic control. This project will provide new mechanistic insight into the function of an important group of ABC transporters and impact on our understanding of the control of peroxisomal metabolism.

Planned Impact

This proposal is focused on fundamental research, dissecting a novel transport mechanism, relating it to downstream enzymatic steps and setting this in the context of metabolic regulation in the plant. As such, this research is expected to benefit researchers in the first instance. The project will also generate novel resources, including recombinant proteins and antisera to acyl activating proteins which will be made available to the plant science community upon request or in the latter case, can be distributed through Agrisera or similar company. The paucity of commercially available CoA esters is a bottleneck in current peroxisome research, therefore compounds synthesised in the project will be shared with other researchers where practical in terms of cost and quantity. Transgenic Arabidopsis lines with altered ability to process different substrates via beta-oxidation will be offered to relevant members of the research community to enable experimentation that is beyond the scope of this proposal.
The knowledge base and resources (protocols, substrates, antisera, transgenic lines) developed can benefit projects across the range of BBSRC's strategic priorities but especially 'food security', 'bioenergy and industrial biotechnology' and potentially "synthetic biology". Although this project employs the model plant, Arabidopsis thaliana, knowledge and techniques are applicable to other plant species including crops and to fungi and mammals. Academic beneficiaries include not only plant scientists and researchers with an interest in peroxisomes but also the membrane transport community and those modelling and manipulating metabolic pathways. Knowledge and resources generated in this project are also of potential interest to plant breeders, either via an improved understanding of plant metabolism and/or through potential transgenic routes to crop improvement, for example varieties with altered oil content. As the mechanism of acyl CoA cleavage may well be shared with other ABCD proteins including the medically important ALDP, knowledge and techniques developed in this project with the plant homologue might have significance for medical bioscientists and clinicians. Our collaborative link with the group at the Amsterdam Medical Centre which includes clinicians and diagnostics professionals as well as scientists working on fundamental underpinning science provides an effective route for dissemination and uptake. Routes by which outcomes will be communicated to potential beneficiaries are outlined in "Pathways to Impact". Finally, one of the most important outcomes of this project will be experienced postdoctoral scientists trained in transport biochemistry and state-of-the art lipidomics techniques as well as transferable skills who should be able to make contributions in either academic or commercial settings.


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Description Our project investigated the mechanism of an ABC transporter protein that transports a range of substrates into peroxisomes. We established an improved expression and purification system for this protein and used these improved methods to investigate the relationship between two enzymatic acitivities possessed by this protein namely ATPase and thioesterase. Using mutagenesis we were able to identify residues that were important for thioesterase activity and show that thioesterase activity depends on ATPase but not vice versa. This is now published.We also developed detergent free purification using SMALPs and a detergent based reconstitution.
Exploitation Route This is the basis for a BBSRC funded PhD project which will move towards structural studies of this protein
Sectors Agriculture, Food and Drink,Pharmaceuticals and Medical Biotechnology

Description Myself and the PDRA took part in the Discovery Zone in 2015 on plant transporters 'Food for Plants' part of our SET week activities for KS2 and KS3 school children. We also participated in an outreach activity for local 6th formers which involved talking to them about membrane protein research at Leeds in December 2015. We also participated in the Discovery Zone in 2016 and 2017
Sector Education
Impact Types Cultural

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 Informal UK ABC get together 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Postgraduate students
Results and Impact I organised an informal 1 day meeting on ABC transporters for postgrad and MSc students working in UK research groups and their PIs. PG and Msc students from 5 different groups presented their data and there was extensive discussion of ideas and methodologies between the students and investigators
Year(s) Of Engagement Activity 2020
Description SET week Discovery Zone 2017 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact We run a stall for KS2 and KS3 children as part of SET week called Food for Plants about mineral nutrition and transporter proteins
Year(s) Of Engagement Activity 2017
Description Schools event around membrane protein research 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact As an adjunct to the Biochemical Society sponsored independent meeting membrane proteins from A to Z I was one of the organisers and participants of both the conference and associated event where local 6th formers were invited to attend a public lecture by Prof sir John Walker FRS entitled Energy in Biology. they also undertook tours of the laboratories and talked with PhD students and post docs about membrane proteins and research at University
Year(s) Of Engagement Activity 2015