Mutations in the Arabidopsis peroxisomal ABC transporter COMATOSE allow differentiation between multiple functions in planta: insights from an allelic series.
COMATOSE (CTS), the Arabidopsis homologue of human Adrenoleukodystrophy protein (ALDP), is required for import of substrates for peroxisomal beta-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.
Dietrich
Daniela
D
Department of Plant and Crop Sciences, School of Biosciences, University of Nottingham, Sutton Bonington Campus, Loughborough, United Kingdom.
Schmuths
Heike
H
De Marcos Lousa
Carine
C
Baldwin
Jocelyn M
JM
Baldwin
Stephen A
SA
Baker
Alison
A
Theodoulou
Frederica L
FL
Holdsworth
Michael J
MJ
D018528:ATP-Binding Cassette Transporters / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000251:Adenosine Triphosphatases; D000483:Alleles; D000595:Amino Acid Sequence; D000818:Animals; D017360:Arabidopsis / Q000235:genetics / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain; D050612:Fatty Acid Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007210:Indoleacetic Acids / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D020675:Peroxisomes / Q000378:metabolism*; D010641:Phenotype; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D013395:Sucrose / Q000378:metabolism
D018528:ATP-Binding Cassette Transporters; D029681:Arabidopsis Proteins; D050612:Fatty Acid Transport Proteins; D007210:Indoleacetic Acids; D013395:Sucrose; D000251:Adenosine Triphosphatases; C456843:At4g39850 protein, Arabidopsis