ACBP4 and ACBP5, novel Arabidopsis acyl-CoA-binding proteins with kelch motifs that bind oleoyl-CoA.
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.
Leung
Ka-Chun
KC
Department of Botany, The University of Hong Kong, Pokfulam Road, Hong Kong, China.
Li
Hong-Ye
HY
Mishra
Girish
G
Chye
Mee-Len
ML
D000214:Acyl Coenzyme A / Q000378:metabolism*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D026261:Diazepam Binding Inhibitor / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D005091:Exons; D018506:Gene Expression Regulation, Plant; D017343:Genes, Plant / Q000235:genetics; D006639:Histidine / Q000235:genetics; D007438:Introns; D008969:Molecular Sequence Data; D005810:Multigene Family / Q000235:genetics; D009154:Mutation; D010802:Phylogeny; D011485:Protein Binding; D018749:RNA, Plant / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid
C495420:ACBP4 protein, Arabidopsis; C495421:ACBP5 protein, Arabidopsis; D000214:Acyl Coenzyme A; D029681:Arabidopsis Proteins; D002352:Carrier Proteins; D026261:Diazepam Binding Inhibitor; D018749:RNA, Plant; D011994:Recombinant Proteins; C017585:oleoyl-coenzyme A; D006639:Histidine