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

TitleArabidopsis ACBP3 is an extracellularly targeted acyl-CoA-binding protein
Authors
Keywords(His)6-tagged recombinant protein
Acyl-CoA-binding domain
Lipid metabolism
Protein targeting
Site-directed mutagenesis
Issue Date2006
PublisherSpringer Verlag. The Journal's web site is located at http://link.springer.de/link/service/journals/00425
Citation
Planta, 2006, v. 223 n. 5, p. 871-881 How to Cite?
AbstractCytosolic 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 [ 14C]arachidonyl-CoA with high affinity in comparison to [ 14C]palmitoyl-CoA and [14C]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 [14C]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. © Springer-Verlag 2005.
Persistent Identifierhttp://hdl.handle.net/10722/68422
ISSN
2015 Impact Factor: 3.239
2015 SCImago Journal Rankings: 1.470
ISI Accession Number ID
References

 

DC FieldValueLanguage
dc.contributor.authorLeung, KCen_HK
dc.contributor.authorLi, HYen_HK
dc.contributor.authorXiao, Sen_HK
dc.contributor.authorTse, MHen_HK
dc.contributor.authorChye, MLen_HK
dc.date.accessioned2010-09-06T06:04:29Z-
dc.date.available2010-09-06T06:04:29Z-
dc.date.issued2006en_HK
dc.identifier.citationPlanta, 2006, v. 223 n. 5, p. 871-881en_HK
dc.identifier.issn0032-0935en_HK
dc.identifier.urihttp://hdl.handle.net/10722/68422-
dc.description.abstractCytosolic 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 [ 14C]arachidonyl-CoA with high affinity in comparison to [ 14C]palmitoyl-CoA and [14C]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 [14C]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. © Springer-Verlag 2005.en_HK
dc.languageengen_HK
dc.publisherSpringer Verlag. The Journal's web site is located at http://link.springer.de/link/service/journals/00425en_HK
dc.relation.ispartofPlantaen_HK
dc.subject(His)6-tagged recombinant proteinen_HK
dc.subjectAcyl-CoA-binding domainen_HK
dc.subjectLipid metabolismen_HK
dc.subjectProtein targetingen_HK
dc.subjectSite-directed mutagenesisen_HK
dc.titleArabidopsis ACBP3 is an extracellularly targeted acyl-CoA-binding proteinen_HK
dc.typeArticleen_HK
dc.identifier.openurlhttp://library.hku.hk:4550/resserv?sid=HKU:IR&issn=0032-0935&volume=223&spage=871&epage=881&date=2006&atitle=Arabidopsis+ACBP3+is+an+extracellularly-targeted+acyl-CoA+binding+proteinen_HK
dc.identifier.emailXiao, S: xiaoshi@graduate.hku.hken_HK
dc.identifier.emailChye, ML: mlchye@hkucc.hku.hken_HK
dc.identifier.authorityXiao, S=rp00817en_HK
dc.identifier.authorityChye, ML=rp00687en_HK
dc.description.naturelink_to_subscribed_fulltext-
dc.identifier.doi10.1007/s00425-005-0139-2en_HK
dc.identifier.pmid16231156-
dc.identifier.scopuseid_2-s2.0-33645226641en_HK
dc.identifier.hkuros116571en_HK
dc.relation.referenceshttp://www.scopus.com/mlt/select.url?eid=2-s2.0-33645226641&selection=ref&src=s&origin=recordpageen_HK
dc.identifier.volume223en_HK
dc.identifier.issue5en_HK
dc.identifier.spage871en_HK
dc.identifier.epage881en_HK
dc.identifier.isiWOS:000236357500001-
dc.publisher.placeGermanyen_HK
dc.identifier.scopusauthoridLeung, KC=7401860725en_HK
dc.identifier.scopusauthoridLi, HY=22953303900en_HK
dc.identifier.scopusauthoridXiao, S=7402022635en_HK
dc.identifier.scopusauthoridTse, MH=12791679900en_HK
dc.identifier.scopusauthoridChye, ML=7003905460en_HK

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