Single amino acid substitutions at the acyl-CoA-binding domain interrupt (14)[C]palmitoyl-CoA binding of ACBP2, an Arabidopsis acyl-CoA-binding protein with ankyrin repeats

Cytosolic acyl-CoA-binding proteins (ACBPs) are small proteins (ca. 10 kDa) that bind long-chain acyl-CoAs and are involved in the storage and intrace llular transport of acyl-CoAs. Previously, we have characterized an Arabido psis thaliana cDNA encoding a novel membrane-associated ACBP, designated AC BP1, demonstrating the existence of a new form of ACBP in plants (M.-L. Chy e, Plant Mol. Biol. 38 (1998) 827-838). ACBP1 likely participates in interm embrane lipid transport from the ER to the plasma membrane, where it could maintain a membrane-associated acyl pool (Chye et al., Plant J. 18 (1999) 2 05-214). Here we report the isolation of cDNAs encoding ACBP2 (M-r 38 479) that shows conservation in the acyl-CoA-binding domain to previously report ed ACBPs, and contains ankyrin repeats at its carboxy terminus. These repea ts, which likely mediate protein-protein interactions, could constitute a p otential docking site in ACBP2 for an enzyme that uses acyl-CoAs as substra te. In vitro binding assays on recombinant (His)(6)-ACBP2 expressed in Esch erichia coli show that it binds (14)[C]palmitoyl-CoA preferentially to (14) [C]oleoyl-CoA. Analysis of the acyl-CoA-binding domain in ACBP2 was carried out by in vitro mutagenesis. Mutant forms of recombinant (His)(6)-ACBP2 wi th single amino acid substitutions at conserved residues within the acyl-Co A-binding domain were less effective in binding (1)4[C]palmitoyl-CoA. North ern blot analysis showed that the 1.6 kb ACBP2 mRNA, like that of ACBP1, is expressed in all plant organs. Analysis of the ACBP2 promoter revealed tha t, like the ACBP1 promoter, it lacks a TATA box suggesting the possibility of a housekeeping function for ACBP2 in plant lipid metabolism.