Article: ACBP4 and ACBP5, novel Arabidopsis acyl-CoA-binding proteins with kelch motifs that bind oleoyl-CoA

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TitleACBP4 and ACBP5, novel Arabidopsis acyl-CoA-binding proteins with kelch motifs that bind oleoyl-CoA
AuthorsLeung, KC2
Li, HY1 2
Mishra, G2
Chye, ML2
Keywords(His) 6-tagged recombinant proteins
ACBP gene family
Acyl-CoA-binding domain
Lipid metabolism
Lipid transfer
Site-directed mutagenesis
Issue Date2004
PublisherSpringer Verlag Dordrecht. The Journal's web site is located at http://springerlink.metapress.com/openurl.asp?genre=journal&issn=0167-4412
CitationPlant Molecular Biology, 2004, v. 55 n. 2, p. 297-309 [How to Cite?]
DOI: http://dx.doi.org/10.1007/s11103-004-0642-z
AbstractIn 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.
ISSN0167-4412
2011 Impact Factor: 4.15
2011 SCImago Journal Rankings: 0.401
DOIhttp://dx.doi.org/10.1007/s11103-004-0642-z
ISI Accession Number IDWOS:000225690100011
ReferencesReferences in Scopus
DC Field
Value
dc.contributor.authorLeung, KC
dc.contributor.authorLi, HY
dc.contributor.authorMishra, G
dc.contributor.authorChye, ML
dc.date.accessioned2010-09-06T06:06:45Z
dc.date.available2010-09-06T06:06:45Z
dc.date.issued2004
dc.description.abstractIn 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.
dc.description.natureLink_to_subscribed_fulltext
dc.identifier.citationPlant Molecular Biology, 2004, v. 55 n. 2, p. 297-309 [How to Cite?]
DOI: http://dx.doi.org/10.1007/s11103-004-0642-z
dc.identifier.doihttp://dx.doi.org/10.1007/s11103-004-0642-z
dc.identifier.epage309
dc.identifier.hkuros96630
dc.identifier.isiWOS:000225690100011
dc.identifier.issn0167-4412
2011 Impact Factor: 4.15
2011 SCImago Journal Rankings: 0.401
dc.identifier.issue2
dc.identifier.openurl
dc.identifier.pmid15604682
dc.identifier.scopuseid_2-s2.0-12544254338
dc.identifier.spage297
dc.identifier.urihttp://hdl.handle.net/10722/68685
dc.identifier.volume55
dc.languageeng
dc.publisherSpringer Verlag Dordrecht. The Journal's web site is located at http://springerlink.metapress.com/openurl.asp?genre=journal&issn=0167-4412
dc.publisher.placeNetherlands
dc.relation.ispartofPlant Molecular Biology
dc.relation.referencesReferences in Scopus
dc.subject(His) 6-tagged recombinant proteins
dc.subjectACBP gene family
dc.subjectAcyl-CoA-binding domain
dc.subjectLipid metabolism
dc.subjectLipid transfer
dc.subjectSite-directed mutagenesis
dc.titleACBP4 and ACBP5, novel Arabidopsis acyl-CoA-binding proteins with kelch motifs that bind oleoyl-CoA
dc.typeArticle
Author Affiliations
  1. South China Institute of Botany Chinese Academy of Sciences
  2. The University of Hong Kong