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ACBP4 and ACBP5, novel Arabidopsis acyl-CoA-binding proteins with kelch motifs that bind oleoyl-CoA

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Abstract

In plants, fatty acids synthesized in the chloroplasts are exported as acyl-CoA esters to the endoplasmic reticulum (ER). Cytosolic 10-kDa acyl-CoA-binding proteins (ACBPs), prevalent in eukaryotes, are involved in the storage and intracellular transport of acyl-CoAs. We have previously characterized Arabidopsis thaliana cDNAs encoding membrane-associated ACBPs with ankyrin repeats, designated ACBP1 and ACBP2, which show conservation to cytosolic ACBPs at the acyl-CoA-binding domain. Analysis of the Arabidopsis genome has revealed the presence of three more genes encoding putative proteins with acyl-CoA-binding domains, designated ACBP3, ACBP4 and ACBP5. Homologues of ACBP1 to ACBP5 have not been reported in any other organism. We show by reverse-transcriptase polymerase chain reaction (RT-PCR) analysis that ACBP3, ACBP4 and ACBP5 are expressed in all plant organs, like ACBP1 and ACBP2. ACBP4 and ACBP5 that share 81.4 identity and which contain kelch motifs were further investigated. To demonstrate their function in binding acyl-CoA, we have expressed them as (His)<inf>6</inf>-tagged recombinant proteins in Escherichia coli for in vitro binding assays. Both (His)_6-ACBP4 and (His)_6-ACBP5 bind [14C]oleoyl-CoA with high affinity, [14C]palmitoyl-CoA with lower affinity and did not bind [14C]arachidonyl-CoA. Eight mutant forms of each protein with single amino acid substitutions within the acyl-CoA-binding domain were produced and analyzed. On binding assays, all mutants were impaired in oleoyl-CoA binding. Hence, these novel ACBPs with kelch motifs have functional acyl-CoA-binding domains that bind oleoyl-CoA. Their predicted cytosol localization suggests that they could maintain an oleoyl-CoA pool in the cytosol or transport oleoyl-CoA from the plastids to the ER in plant lipid metabolism.

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  1. Department of Botany, The University of Hong Kong, Pokfulam Road, China

    Ka-Chun Leung, Girish Mishra & Mee-Len Chye

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  1. Ka-Chun Leung
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Leung, KC., Li, HY., Mishra, G. et al. ACBP4 and ACBP5, novel Arabidopsis acyl-CoA-binding proteins with kelch motifs that bind oleoyl-CoA. Plant Mol Biol 55, 297–309 (2004). https://doi.org/10.1007/s11103-004-0642-z

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