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

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 [(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.

Arabidopsis cytosolic acyl-CoA-binding proteins ACBP4, ACBP5 and ACBP6 have overlapping but distinct roles in seed development

“…Their recombinant proteins have been reported to bind acyl-CoA esters in vitro as well as to PC (phosphatidylcholine) [15,18,29,32], while recombinant (r) AtACBP1 also binds to phosphatidic acid, and rAtACBP2 to lysophophatidylcholine (lysoPC) [28,32,36]. The 39.3-kDa AtACBP3 contains a cleavable N-terminal signal peptide and is the only member with an ACB domain localized at the C-terminus [17]. The extracellularly-targeted AtACBP3 [17,20,37] has been shown to bind acyl-CoA esters, PC and PE (phosphatidylethanolamine) [17,20].…”

Overexpression of the Arabidopsis 10-Kilodalton Acyl-Coenzyme A-Binding Protein ACBP6 Enhances Freezing Tolerance

“…These include membrane-associated ACBP1 and ACBP2, which have been subcellularly localized to the ER and plasma membrane (Chye et al, 1999;Li and Chye, 2003), extracellularly targeted ACBP3 (Leung et al, 2006), and Kelch motif-containing ACBP4 and ACBP5 (Leung et al, 2004). Only ACBP6, the smallest member of this family, has well-characterized homologs in other eukaryotes (Hills et al, 1994;Faergeman and Knudsen, 1997).…”

“…Conserved domains that potentially mediate protein-protein interactions occur in the larger Arabidopsis ACBPs: ankyrin repeats (ACBP1 and ACBP2) and Kelch motifs (ACBP4 and ACBP5; Leung et al, 2004;Li and Chye, 2004). The function of the acyl-CoA-binding domain in binding acyl-CoA esters has been established for ACBP1 to ACBP5 using His-tagged recombinant proteins and site-directed mutagenesis (Chye et al, 2000;Leung et al, 2004Leung et al, , 2006. These ACBPs bind differentially to various acyl-CoA esters, implying that they have different cellular functions.…”

Interactions between Arabidopsis acyl-CoA-binding proteins and their protein partners

“…Acyl-CoA-binding proteins (ACBPs) have been reported to bind long-chain acyl-CoA esters (C 12 -C 22 ) through their acyl-CoA-binding domains with high specificities and affinities (Rasmussen et al 1993;Chye 1998;Chye et al 2000;Knudsen et al 2000;Burton et al 2005;Leung et al 2004Leung et al , 2006. The first identified ACBP was a neuropeptide (diazepam-binding inhibitor) from rat brain that inhibits benzodiazepine-binding (Guidotti et al 1983).…”

“…AtACBP3 has an N-terminal signal peptide and a transmembrane domain, and is targeted to the extracellular space ( Fig. 1) (Leung et al 2006). Kelch-motif containing AtACBP4 and AtACBP5 share 81.4 % amino acid identity ( Fig.…”