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

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.

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.

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.