Arabidopsis ACBP3 is an extracellularly targeted acyl-CoA-binding protein.
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 [(14)C]arachidonyl-CoA with high affinity in comparison to [(14)C]palmitoyl-CoA and [(14)C]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 [(14)C]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.
Leung
Ka-Chun
KC
Department of Botany, The University of Hong Kong, Pokfulam Road, Hong Kong, China.
Li
Hong-Ye
HY
Xiao
Shi
S
Tse
Muk-Hei
MH
Chye
Mee-Len
ML
D000214:Acyl Coenzyme A / Q000378:metabolism; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D017360:Arabidopsis / Q000737:chemistry / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000378:metabolism*; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli; D005110:Extracellular Space / Q000378:metabolism; D006639:Histidine; D008969:Molecular Sequence Data; D009842:Oligopeptides; D019697:Onions; D010940:Plant Proteins / Q000737:chemistry / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000378:metabolism; D040681:Structural Homology, Protein; D014026:Nicotiana
C513211:ACBP1 protein, Arabidopsis; C513212:ACBP3 protein, Arabidopsis; D000214:Acyl Coenzyme A; D029681:Arabidopsis Proteins; D002352:Carrier Proteins; C471213:His-His-His-His-His-His; D009842:Oligopeptides; D010940:Plant Proteins; D011994:Recombinant Proteins; C026563:arachidonyl-coenzyme A; D006639:Histidine