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

TitleACBP4 and ACBP5, novel Arabidopsis acyl-CoA-binding proteins with kelch motifs that bind oleoyl-CoA
Authors
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
Citation
Plant Molecular Biology, 2004, v. 55 n. 2, p. 297-309 How to Cite?
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.
Persistent Identifierhttp://hdl.handle.net/10722/68685
ISSN
2013 Impact Factor: 4.072
ISI Accession Number ID
References

 

Author Affiliations
  1. South China Institute of Botany Chinese Academy of Sciences
  2. The University of Hong Kong
DC FieldValueLanguage
dc.contributor.authorLeung, KCen_HK
dc.contributor.authorLi, HYen_HK
dc.contributor.authorMishra, Gen_HK
dc.contributor.authorChye, MLen_HK
dc.date.accessioned2010-09-06T06:06:45Z-
dc.date.available2010-09-06T06:06:45Z-
dc.date.issued2004en_HK
dc.identifier.citationPlant Molecular Biology, 2004, v. 55 n. 2, p. 297-309en_HK
dc.identifier.issn0167-4412en_HK
dc.identifier.urihttp://hdl.handle.net/10722/68685-
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.en_HK
dc.languageengen_HK
dc.publisherSpringer Verlag Dordrecht. The Journal's web site is located at http://springerlink.metapress.com/openurl.asp?genre=journal&issn=0167-4412en_HK
dc.relation.ispartofPlant Molecular Biologyen_HK
dc.subject(His) 6-tagged recombinant proteinsen_HK
dc.subjectACBP gene familyen_HK
dc.subjectAcyl-CoA-binding domainen_HK
dc.subjectLipid metabolismen_HK
dc.subjectLipid transferen_HK
dc.subjectSite-directed mutagenesisen_HK
dc.titleACBP4 and ACBP5, novel Arabidopsis acyl-CoA-binding proteins with kelch motifs that bind oleoyl-CoAen_HK
dc.typeArticleen_HK
dc.identifier.openurlhttp://library.hku.hk:4550/resserv?sid=HKU:IR&issn=0167-4412&volume=55&spage=297&epage=309&date=2004&atitle=ACBP4+and+ACBP5,+novel+Arabidopsis+acyl-CoA+binding+proteins,+with+kelch+motifs+that+bind+oleoyl-CoAen_HK
dc.identifier.emailChye, ML: mlchye@hkucc.hku.hken_HK
dc.identifier.authorityChye, ML=rp00687en_HK
dc.description.naturelink_to_subscribed_fulltext-
dc.identifier.doi10.1007/s11103-004-0642-zen_HK
dc.identifier.pmid15604682en_HK
dc.identifier.scopuseid_2-s2.0-12544254338en_HK
dc.identifier.hkuros96630en_HK
dc.relation.referenceshttp://www.scopus.com/mlt/select.url?eid=2-s2.0-12544254338&selection=ref&src=s&origin=recordpageen_HK
dc.identifier.volume55en_HK
dc.identifier.issue2en_HK
dc.identifier.spage297en_HK
dc.identifier.epage309en_HK
dc.identifier.isiWOS:000225690100011-
dc.publisher.placeNetherlandsen_HK
dc.identifier.scopusauthoridLeung, KC=7401860725en_HK
dc.identifier.scopusauthoridLi, HY=22953303900en_HK
dc.identifier.scopusauthoridMishra, G=12809587900en_HK
dc.identifier.scopusauthoridChye, ML=7003905460en_HK

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