Arabidopsis ACBP3 is an extracellularly targeted acyl-CoA-binding protein |
| |
Authors: | Ka-Chun Leung Hong-Ye Li Shi Xiao Muk-Hei Tse Mee-Len Chye |
| |
Institution: | (1) Department of Botany, The University of Hong Kong, Pokfulam Road, Hong Kong, China;(2) South China Botanical Garden, Chinese Academy of Sciences, Guangzhou, 510650, China |
| |
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. |
| |
Keywords: | Acyl-CoA-binding domain (His)6-tagged recombinant protein Lipid metabolism Protein targeting Site-directed mutagenesis |
本文献已被 PubMed SpringerLink 等数据库收录! |
|