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Arabidopsis ACBP3 is an extracellularly targeted acyl-CoA-binding protein

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Abstract

Cytosolic 10-kDa acyl-CoA-binding proteins (ACBPs) function in the storage and intracellular transport of acyl-CoA esters in eukaryotes. Fatty acids synthesized de novo in plant chloroplasts are exported as oleoyl-CoA and palmitoyl-CoA esters. In Arabidopsis, other than the 10-kDa ACBP, there exists five larger ACBPs (ACBP1 to ACBP5) of which homologues have not been characterized in other organisms. To investigate the significance of this gene family, we have attempted to subcellularly localize them and compare their acyl-CoA-binding affinities. We have previously shown that Arabidopsis ACBP1 and ACBP2 are membrane-associated proteins while ACBP4 and ACBP5 contain kelch motifs. Here, to localize ACBP3, we have expressed ACBP3-red fluorescent protein (DsRed2) from the CaMV 35S promoter. ACBP3-DsRed was localized extracellularly in transiently expressed tobacco BY-2 cells and onion epidermal cells. The function of the acyl-CoA-binding domain in ACBP3 was investigated by in vitro binding assays using (His)6-ACBP3, which was observed to bind [14C]arachidonyl-CoA with high affinity in comparison to [14C]palmitoyl-CoA and [14C]oleoyl-CoA. To identify the residues functional in binding, five mutants with single amino acid substitutions in the acyl-CoA-binding domain of (His)6-ACBP3 and (His)6-ACBP1 (which also binds [14C]arachidonyl-CoA) were generated by site-directed mutagenesis. Binding assays with arachidonyl-CoA revealed that replacement of a conserved R residue (R150A in ACBP1 and R284A in ACBP3), disrupted binding. In contrast, other substitutions in ACBP1 (Y126A, K130A, K152A and Y171A) and in ACBP3 (F260A, K264A, K286A and Y305A) did not affect arachidonyl-CoA binding, unlike their equivalents in (His)6-ACBP2, (His)6-ACBP4 and (His)6-ACBP5, which had altered binding to palmitoyl-CoA or oleoyl-CoA.

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Abbreviations

ACBP:

Acyl-Coenzyme A-binding protein

BY-2:

Tobacco Bright Yellow-2

RT-PCR:

Reverse transcription-polymerase chain reaction

SDS-PAGE:

SDS-polyacrylamide gel electrophoresis

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Acknowledgements

We thank Dr. W.K. Yip for provision of the liquid scintillation counter. This work was supported by the Research Grants Council of the Hong Kong Special Administrative Region, China (HKU7232/00 M, HKU7314/04 M and HKU7504/05 M) and the University of Hong Kong (CRCG 10204478). KCL, MHT and SX were supported by postgraduate studentships from the University of Hong Kong.

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Authors and Affiliations

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

    Ka-Chun Leung, Hong-Ye Li, Shi Xiao, Muk-Hei Tse & Mee-Len Chye

  2. South China Botanical Garden, Chinese Academy of Sciences, Guangzhou, 510650, China

    Hong-Ye Li

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  1. Ka-Chun Leung
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  2. Hong-Ye Li
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  3. Shi Xiao
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  4. Muk-Hei Tse
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  5. Mee-Len Chye
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Correspondence to Mee-Len Chye.

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Leung, KC., Li, HY., Xiao, S. et al. Arabidopsis ACBP3 is an extracellularly targeted acyl-CoA-binding protein. Planta 223, 871–881 (2006). https://doi.org/10.1007/s00425-005-0139-2

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