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Vol. 20, Issue 1, 530-543, January 1, 2009

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Mutations in the Arabidopsis Peroxisomal ABC Transporter COMATOSE Allow Differentiation between Multiple Functions In Planta: Insights from an Allelic Series

Daniela Dietrich^*,,, Heike Schmuths^*,,, Carine De Marcos Lousa^||, Jocelyn M. Baldwin^¶, Stephen A. Baldwin^¶, Alison Baker^||, Frederica L. Theodoulou^#,@, and Michael J. Holdsworth^*,@

*Department of Plant and Crop Sciences, School of Biosciences, University of Nottingham, Sutton Bonington Campus, Loughborough, LE12 5RD, United Kingdom; ^||Centre for Plant Sciences, University of Leeds; Leeds, LS2 9JT, United Kingdom; ^¶Institute of Membrane and Systems Biology, University of Leeds, Leeds, LS2 9JT, United Kingdom; and ^#Biological Chemistry Department, Rothamsted Research, Harpenden, AL5 2JQ, United Kingdom

Submitted July 18, 2008; Revised September 29, 2008; Accepted October 30, 2008
Monitoring Editor: Reid Gilmore

COMATOSE (CTS), the Arabidopsis homologue of human Adrenoleukodystrophy protein (ALDP), is required for import of substrates for peroxisomal β-oxidation. A new allelic series and a homology model based on the bacterial ABC transporter, Sav1866, provide novel insights into structure-function relations of ABC subfamily D proteins. In contrast to ALDP, where the majority of mutations result in protein absence from the peroxisomal membrane, all CTS mutants produced stable protein. Mutation of conserved residues in the Walker A and B motifs in CTS nucleotide-binding domain (NBD) 1 resulted in a null phenotype but had little effect in NBD2, indicating that the NBDs are functionally distinct in vivo. Two alleles containing mutations in NBD1 outside the Walker motifs (E617K and C631Y) exhibited resistance to auxin precursors 2,4-dichlorophenoxybutyric acid (2,4-DB) and indole butyric acid (IBA) but were wild type in all other tests. The homology model predicted that the transmission interfaces are domain-swapped in CTS, and the differential effects of mutations in the conserved "EAA motif" of coupling helix 2 supported this prediction, consistent with distinct roles for each NBD. Our findings demonstrate that CTS functions can be separated by mutagenesis and the structural model provides a framework for interpretation of phenotypic data.

These authors contributed equally to this work.

Present addresses: Centre for Plant Integrative Biology, University of Nottingham, Sutton Bonington Campus, Loughborough, LE12 5RD, United Kingdom;

Saaten-Union Resistenzlabor GmbH, Betriebsstätte Biopark Gatersleben, Am Schwabeplan 6, 06466 Gatersleben, Germany.

^@ Joint senior author.

Address correspondence to: Frederica L. Theodoulou (freddie.theodoulou{at}bbsrc.ac.uk)