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

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.

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.