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

Abstract

COMATOSE (CTS), the Arabidopsis homologue of human Adrenoleukodystrophy protein (ALDP), is required for import of substrates for peroxisomal ␤-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. INTRODUCTIONATP-binding cassette (ABC) transporters are ubiquitous, integral membrane proteins that mediate vectorial transport of a diverse range of molecules across membranes and consequently are involved in a wide variety of biological processes (Higgins, 1992;Rea, 2007). All ABC transporters share the same basic architecture, comprising two transmembrane domains (TMDs), each composed of several ␣-helices that are involved in substrate recognition and translocation across the lipid bilayer, and two nucleotide-binding domains (NBDs) that bind and hydrolyze ATP, providing the driving force for transport. In prokaryotes, these domains are usually present on separate polypeptides, whereas in eukaryotes, fusion of a TMD and NBD module to form a "half transporter," or indeed fusion of all modules to form a "full transporter" is more common. The past decade has seen the determination of high-resolution crystal structures for several isolated NBDs and, more recently, complete ABC transporters (reviewed in .The NBDs characteristically contain several highly conserved features common to nucleotide hydrolases, including the Walker A motif (GxxGxGKS/T, where x is any amino acid), forming the P-loop, which binds to the ␣-and ␤-phosphates of nucleotides, and the Walker B motif (⌽⌽⌽⌽DE, where ⌽ is hydrophobic), thought to coordinate the Mg 2ϩ ion or polarize the attacking water molecule (Walker et al., 1982;Hung et al., 1998;Schneider and Hunke, 1998;Hopfner et al., 2000). ABC transporters additionally contain the signature motif (LSGGQ), which contacts the ␥-phosphate of ATP; the Q-loop (or "lid") containing a glutamine residue, which interacts with ␥-phosphate through a water molecule; and the swi...

The Arabidopsis Peroxisomal ABC Transporter, Comatose, Complements the Saccharomyces cerevisiae pxa1 pxa2Δ Mutant for Metabolism of Long-chain Fatty Acids and Exhibits Fatty Acyl-CoA-stimulated ATPase Activity

“…The K487A mutant was generated by site-directed mutagenesis, as described in Dietrich et al (28). To create an in-frame CFP fusion with a 10-amino acid spacer between the C terminus of CTS and CFP, the native stop codon of CTS was removed by site-directed mutagenesis using the QuikChange II site-directed mutagenesis kit (Stratagene, La Jolla, CA), with primer pair CTSstopF (GAACAGACAACAG-AGTCAGAATTCGATATCAAGC) and CTSstopR (CTTGT-CTGTTGTCTCAGTCTTAAGCTATAGTTCG), according to the manufacturer's instructions.…”

“…Analysis of cts null mutants has demonstrated that CTS plays key roles in a number of developmental and physiological processes, including germination, seedling establishment, fertility, root growth, and dark-induced senescence (16 -18, 20 -27). The different roles of CTS in planta are separable by mutagenesis (28) and can be related to different biochemical roles, specifically the ability to metabolize distinct substrates such as fatty acids and hormone precursors via ␤-oxidation. Taken together, these findings strongly suggest that CTS is a transporter with broad acyl chain specificity, which mediates uptake of substrates for ␤-oxidation into the peroxisome.…”

Individual Maize Chromosomes in the C3 Plant Oat Can Increase Bundle Sheath Cell Size and Vein Density

“…Nile red stains positively for lipids in plants Pighin et al, 2004;Schmidt and Herman, 2008;Dietrich et al, 2009), fluorescing red in the presence of polar lipids and yellow when staining esterified cholesterol and triacylglycerols (Diaz et al, 2008). This approach showed polar lipids in the PBS of maize, but in the oat parental line, only the MS and some of the veinal cells stained (Fig.…”

Highlights of recent progress in plant lipid research

“…These data suggest that the two nucleotide binding domains are functionally distinct and that the substrate selectivity can be altered [11]. Lousa et al (O34) also showed that CTS forms a high molecular weight complex suggesting that the protein might interact with other components involved in fatty acid catabolism.…”

“…This protein is a homologue of human ALDP, which has been implicated in the disease X-linked Adrenoleukodystrophy [11]. It is probable that CTS imports either free fatty acids or acyl-CoAs into the peroxisome, but its precise substrate specificity is not known.…”