Award details

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

Reference	BBS/E/C/00004916
Principal Investigator / Supervisor	Professor Frederica Theodoulou
Co-Investigators / Co-Supervisors
Institution	Rothamsted Research
Department	Rothamsted Research Department
Funding type	Research
Value (£)	86,659
Status	Completed
Type	Institute Project
Start date	01/06/2008
End date	31/05/2011
Duration	36 months

Abstract

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.

Summary

unavailable

Committee	Closed Committee - Biochemistry & Cell Biology (BCB)
Research Topics	Plant Science
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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