RRC ID 593
Author Leung KC, Li HY, Mishra G, Chye ML.
Title ACBP4 and ACBP5, novel Arabidopsis acyl-CoA-binding proteins with kelch motifs that bind oleoyl-CoA.
Journal Plant Mol. Biol.
Abstract In plants, fatty acids synthesized in the chloroplasts are exported as acyl-CoA esters to the endoplasmic reticulum (ER). Cytosolic 10-kDa acyl-CoA-binding proteins (ACBPs), prevalent in eukaryotes, are involved in the storage and intracellular transport of acyl-CoAs. We have previously characterized Arabidopsis thaliana cDNAs encoding membrane-associated ACBPs with ankyrin repeats, designated ACBP1 and ACBP2, which show conservation to cytosolic ACBPs at the acyl-CoA-binding domain. Analysis of the Arabidopsis genome has revealed the presence of three more genes encoding putative proteins with acyl-CoA-binding domains, designated ACBP3, ACBP4 and ACBP5. Homologues of ACBP1 to ACBP5 have not been reported in any other organism. We show by reverse-transcriptase polymerase chain reaction (RT-PCR) analysis that ACBP3 , ACBP4 and ACBP5 are expressed in all plant organs, like ACBP1 and ACBP2 . ACBP4 and ACBP5 that share 81.4 identity and which contain kelch motifs were further investigated. To demonstrate their function in binding acyl-CoA, we have expressed them as (His)6-tagged recombinant proteins in Escherichia coli for in vitro binding assays. Both (His)6-ACBP4 and (His)6-ACBP5 bind [14C]oleoyl-CoA with high affinity, [14C]palmitoyl-CoA with lower affinity and did not bind [14C]arachidonyl-CoA. Eight mutant forms of each protein with single amino acid substitutions within the acyl-CoA-binding domain were produced and analyzed. On binding assays, all mutants were impaired in oleoyl-CoA binding. Hence, these novel ACBPs with kelch motifs have functional acyl-CoA-binding domains that bind oleoyl-CoA. Their predicted cytosol localization suggests that they could maintain an oleoyl-CoA pool in the cytosol or transport oleoyl-CoA from the plastids to the ER in plant lipid metabolism.
Volume 55(2)
Pages 297-309
Published 2004-5
DOI 10.1007/s11103-004-0642-z
PII DO00000642
PMID 15604682
MeSH Acyl Coenzyme A / metabolism* Amino Acid Sequence Amino Acid Substitution Arabidopsis Proteins / genetics Arabidopsis Proteins / metabolism* Binding Sites / genetics Carrier Proteins / genetics Carrier Proteins / metabolism* Diazepam Binding Inhibitor / metabolism* Escherichia coli / genetics Exons Gene Expression Regulation, Plant Genes, Plant / genetics Histidine / genetics Introns Molecular Sequence Data Multigene Family / genetics Mutation Phylogeny Protein Binding RNA, Plant / genetics RNA, Plant / metabolism Recombinant Proteins / genetics Recombinant Proteins / isolation & purification Recombinant Proteins / metabolism Reverse Transcriptase Polymerase Chain Reaction Sequence Homology, Amino Acid
IF 3.543
Times Cited 52
Arabidopsis / Cultured plant cells, genes pda08201