Cleavage of acyl-CoA by peroxisomal ABC subfamily D transporters in peroxisomes: mechanism and functional roles
Lead Research Organisation:
Rothamsted Research
Department Name: Biointeractions and Crop Protection
Abstract
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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.
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.
Publications
Baker A
(2015)
Peroxisomal ABC transporters: functions and mechanism.
in Biochemical Society transactions
Carrier DJ
(2019)
Mutagenesis separates ATPase and thioesterase activities of the peroxisomal ABC transporter, Comatose.
in Scientific reports
Theodoulou FL
(2016)
How to move an amphipathic molecule across a lipid bilayer: different mechanisms for different ABC transporters?
in Biochemical Society transactions
Theodoulou FL
(2015)
ABC transporter research: going strong 40 years on.
in Biochemical Society transactions
Theodoulou FL
(2014)
Coenzyme A and its derivatives: renaissance of a textbook classic.
in Biochemical Society transactions
Van Roermund CWT
(2021)
The Saccharomyces cerevisiae ABC subfamily D transporter Pxa1/Pxa2p co-imports CoASH into the peroxisome.
in FEBS letters
Description | We have found that a member of the family of transport proteins that we are studying is responsible for importing Coenzyme A (a type of metabolic currency) into subcellular compartments known as peroxisomes. This is important because it will help us to understand the control of metabolism. This project was designed to study how molecules are imported into cellular compartments called peroxisomes, how this transport process is regulated and what that means in the context of the whole organism's physiology. We have prepared protein samples for five peroxisomal enzymes (called AAEs) to test how they interact with the transporter to control metabolism and have produced and characterised antibodies to the five AAEs so now we are able to detect them in plant cells and test whether they interact with the transport protein in different situations. We also carried out genetic experiments to test the roles of the AAEs in intact plants and produced plants with transporters with altered amino acid sequences to work out details of the transport mechanism. We have produced mutant (altered) versions of our transporter protein in yeast and tested the effect of the changes on its function: this enabled us to propose a detailed transporter mechanism and to link it with plant physiology. Finally, through work in yeast, we demonstrated that ABC transporters not only import fatty acids but also an important molecule called coenzyme A into peroxisomes. This plays a key role in balancing the coenzyme A budget within cells. |
Exploitation Route | Metabolic modellers would be interested in our findings because they have important implications for the control of metabolism. The AAE antibodies will be a useful resource for other scientists. We plan to make them available to the community via a company such as Agrisera, once they have been fully characterised and published. |
Sectors | Agriculture Food and Drink Pharmaceuticals and Medical Biotechnology |
Description | The project has resulted in training of a scientist who now works in the pharmaceutical industry. |
First Year Of Impact | 2016 |
Sector | Pharmaceuticals and Medical Biotechnology |
Impact Types | Economic |
Description | Analysis of peroxisomal ABC transporters in yeast |
Organisation | Academic Medical Center |
Country | Netherlands |
Sector | Academic/University |
PI Contribution | My research team provided substantial intellectual contributions and co-wrote manuscripts. |
Collaborator Contribution | The collaborators provided expertise in yeast peroxisome isolation and beta-oxidation measurements. |
Impact | The collaboration brought significant added value through the ability to perform experiments with isolated yeast peroxisomes and resulted in two joint publications. |
Start Year | 2014 |