IntAct European Bioinformatics Institute IntAct intact http://www.ebi.ac.uk/ http://www.ebi.ac.uk/intact/query/${ac} EBI-[0-9]+|IA:[0-9]+ INTerAction database (IntAct) provides an open source database and toolkit for the storage, presentation and analysis of molecular interactions. http://www.ebi.ac.uk/intact European Bioinformatics Institute; Wellcome Trust Genome Campus; Hinxton, Cambridge; CB10 1SD; United Kingdom http://www.ebi.ac.uk/intact/ The thylakoid membrane protein ALB3 associates with the cpSecY-translocase in Arabidopsis thaliana. The thylakoid membrane protein ALB3 associates with the cpSecY-translocase in Arabidopsis thaliana. Biochem. J. (0264-6021) 2002 Klostermann E., Droste Gen Helling I., Carde JP., Schunemann D. Thylakoid membranes were prepared from Arabidopsis thaliana (ecotype Columbia) seedlings and solubilized with 1% digitonin prior to blue native gel electrophoresis. schuenemann@bio3.rwth.aachen.de 16-Oct-2008: Contacted by JYOTI. orchard arath-leaf Arabidopsis thaliana leaf leaf leaf electron microscopy electron microscopy Electron crystallography Electron cryomicroscopy Electron crystallography Electron cryomicroscopy immunostaining immunostaining Klostermann E., Droste Gen Helling I., Carde JP., Schunemann D. Thylakoid membranes were prepared from Arabidopsis thaliana (ecotype Columbia) seedlings and solubilized with 1% digitonin prior to blue native gel electrophoresis. schuenemann@bio3.rwth.aachen.de Accepted 2008-OCT-15 by ORCHARD. 16-Oct-2008: Contacted by JYOTI. Biochem. J. (0264-6021) 2002 orchard The thylakoid membrane protein ALB3 associates with the cpSecY-translocase in Arabidopsis thaliana. The thylakoid membrane protein ALB3 associates with the cpSecY-translocase in Arabidopsis thaliana. Biochem. J. (0264-6021) 2002 Klostermann E., Droste Gen Helling I., Carde JP., Schunemann D. Thylakoid membranes were prepared from Arabidopsis thaliana (ecotype Columbia) seedlings and solubilized with 1% digitonin prior to blue native gel electrophoresis. schuenemann@bio3.rwth.aachen.de 16-Oct-2008: Contacted by JYOTI. orchard arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress molecular sieving molecular sieving Gel Filtration Size Exclusion Chromatography Size Exclusion Chromatography Gel Filtration Sizing column western blot western blot Immuno blot Immuno blot Klostermann E., Droste Gen Helling I., Carde JP., Schunemann D. Thylakoid membranes were prepared from Arabidopsis thaliana (ecotype Columbia) seedlings and solubilized with 1% digitonin prior to blue native gel electrophoresis. schuenemann@bio3.rwth.aachen.de Accepted 2008-OCT-15 by ORCHARD. 16-Oct-2008: Contacted by JYOTI. Biochem. J. (0264-6021) 2002 orchard The thylakoid membrane protein ALB3 associates with the cpSecY-translocase in Arabidopsis thaliana. The thylakoid membrane protein ALB3 associates with the cpSecY-translocase in Arabidopsis thaliana. Biochem. J. (0264-6021) 2002 Klostermann E., Droste Gen Helling I., Carde JP., Schunemann D. Thylakoid membranes were prepared from Arabidopsis thaliana (ecotype Columbia) seedlings and solubilized with 1% digitonin prior to blue native gel electrophoresis. schuenemann@bio3.rwth.aachen.de 16-Oct-2008: Contacted by JYOTI. orchard arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress bn-page blue native page bn-page western blot western blot Immuno blot Immuno blot Klostermann E., Droste Gen Helling I., Carde JP., Schunemann D. Thylakoid membranes were prepared from Arabidopsis thaliana (ecotype Columbia) seedlings and solubilized with 1% digitonin prior to blue native gel electrophoresis. schuenemann@bio3.rwth.aachen.de Accepted 2008-OCT-15 by ORCHARD. 16-Oct-2008: Contacted by JYOTI. Biochem. J. (0264-6021) 2002 orchard The thylakoid membrane protein ALB3 associates with the cpSecY-translocase in Arabidopsis thaliana. The thylakoid membrane protein ALB3 associates with the cpSecY-translocase in Arabidopsis thaliana. Biochem. J. (0264-6021) 2002 Klostermann E., Droste Gen Helling I., Carde JP., Schunemann D. Thylakoid membranes were prepared from Arabidopsis thaliana (ecotype Columbia) seedlings and solubilized with 1% digitonin prior to blue native gel electrophoresis. schuenemann@bio3.rwth.aachen.de 16-Oct-2008: Contacted by JYOTI. orchard arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress crosslink cross-linking study crosslink western blot western blot Immuno blot Immuno blot Klostermann E., Droste Gen Helling I., Carde JP., Schunemann D. Thylakoid membranes were prepared from Arabidopsis thaliana (ecotype Columbia) seedlings and solubilized with 1% digitonin prior to blue native gel electrophoresis. schuenemann@bio3.rwth.aachen.de Accepted 2008-OCT-15 by ORCHARD. 16-Oct-2008: Contacted by JYOTI. Biochem. J. (0264-6021) 2002 orchard The thylakoid membrane protein ALB3 associates with the cpSecY-translocase in Arabidopsis thaliana. The thylakoid membrane protein ALB3 associates with the cpSecY-translocase in Arabidopsis thaliana. Biochem. J. (0264-6021) 2002 Klostermann E., Droste Gen Helling I., Carde JP., Schunemann D. Thylakoid membranes were prepared from Arabidopsis thaliana (ecotype Columbia) seedlings and solubilized with 1% digitonin prior to blue native gel electrophoresis. schuenemann@bio3.rwth.aachen.de 16-Oct-2008: Contacted by JYOTI. orchard arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress anti bait coip anti bait coimmunoprecipitation anti bait coip western blot western blot Immuno blot Immuno blot Klostermann E., Droste Gen Helling I., Carde JP., Schunemann D. Thylakoid membranes were prepared from Arabidopsis thaliana (ecotype Columbia) seedlings and solubilized with 1% digitonin prior to blue native gel electrophoresis. schuenemann@bio3.rwth.aachen.de Accepted 2008-OCT-15 by ORCHARD. 16-Oct-2008: Contacted by JYOTI. Biochem. J. (0264-6021) 2002 orchard scy1_arath Preprotein translocase subunit SCY1, chloroplastic SCY1 CpSecY SECY MSF3.9 At2g18710 protein protein arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress MITVSEVSSYSSSSSNFASLSRLNHKSSSRLRSSSLYKGSFFSVSTKTRRNTCKAKSWNLGLVINSRSSEASVFDPLGINPDETSGLSSIWESFVSLLSPSFESSSGNRRDKPSSGRGVAAAIEDSSIDFGDFFKGPLPGKFLKLLGFLALSRLGIYIPLGGVNREAFVGNLDQNSILSTLDTFSGGGIGRLGICSLGIVPFINAQIVFQLLAQVYPKLQDLQKKEGEAGRKKILQYTRYASVGFAIVQAIGQVFYLRPYVNDFSTEWVVSSVTLLTLGSVLTTYIGERISDLKLGNGTSLLIFTSIISYLPASFGRTTAEALQEGNYTGLGTIVVSFLLLVLGIVYVQEAERKIPLNYASRYTSKAGGLQKSAYLPFKVNSAGVMPIIFSTSSLALPATLARFTGISALKNVAFALTPGGSFYLPTNILLIAFFNYYYTFLQLDPDDVSEQLKRQGASIPLVRPGKSTALFIKTVLGRISVLGSAFLAVLAAGPAVVEQITHLTAFRGFAGTSVLILVGCATDTARKVQAEIISQKYKNIEFYELDKYDP 526854797A3B8059 alb3_arath Inner membrane protein ALBINO3, chloroplastic ALB3 F8N16.9 At2g28800 protein protein arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress MARVLVSSPSSFFGSPLIKPSSSLRHSGVGGGGTAQFLPYRSNNNKLFTTSTTVRFSLNEIPPFHGLDSSVDIGAIFTRAESLLYTIADAAVVGADSVVTTDSSAVQKSGGWFGFISDAMELVLKILKDGLSAVHVPYAYGFAIILLTIIVKAATYPLTKQQVESTLAMQNLQPKIKAIQQRYAGNQERIQLETSRLYKQAGVNPLAGCLPTLATIPVWIGLYQALSNVANEGLFTEGFFWIPSLGGPTSIAARQSGSGISWLFPFVDGHPPLGWYDTVAYLVLPVLLIASQYVSMEIMKPPQTDDPAQKNTLLVFKFLPLMIGYFALSVPSGLSIYWLTNNVLSTAQQVYLRKLGGAKPNMDENASKIISAGRAKRSIAQPDDAGERFRQLKEQEKRSKKNKAVAKDTVELVEESQSESEEGSDDEEEEAREGALASSTTSKPLPEVGQRRSKRSKRKRTV 2BBF7AF5BA2E6DDE secy-alb3-1 2 7 unspecified role unspecified role neutral component neutral component 6 unspecified role unspecified role neutral component neutral component physical association physical association Figure 1 As shown in Figure 1(A), most ALB3 and cpSecY eluted from the gel �ltration column as higher-molecularmass species of approx. 200 kDa. This molecular mass is close to that ofC180 kDa determined previously for the cpSecY complex Note that ALB3 and cpSecY eluted from the gel filtration column as higher-molecularmass species of approx. 200 kDa when thylakoids were solubilized using 1.5% digitonin. ALB3 and cpSecY eluted from the gel filtration column as higher-molecular mass species of approx. 180 kDa when the thylakoid membranes were solubilized under harsher conditions using dodecyl b-dmaltosideas detergent secy-alb3-2 3 6 unspecified role unspecified role neutral component neutral component 7 unspecified role unspecified role neutral component neutral component physical association physical association Figure 2 Thylakoid membrane proteins were solubilized in digitonin and subjected to blue native gel electrophoresis, and the distributions of ALB3 and cpSecY within the gel were detected by Western blot analysis. The two proteins showed a very similar fractionation pattern ( Note that ALB3 and cpSecY were found to be components of a higher-molecular-mass complex of approx. 160 kDa, and no monomeric forms of ALB3 or cpSecY were detected. However, blue native gel electrophoresis revealed that ALB3 and cpSecY were also present in a variety of oligomeric complexes with molecular masses ranging from 120 to 700 kDa. secy-alb3-3 5 6 unspecified role unspecified role prey prey 7 unspecified role unspecified role bait bait physical association physical association Figure 3 Co-immunoprecipitation of ALB3 and cpSecY alb3-secy 5 6 unspecified role unspecified role bait bait 7 unspecified role unspecified role prey prey physical association physical association Figure 3 Co-immunoprecipitation of ALB3 and cpSecY secy-alb3-4 1 7 unspecified role unspecified role neutral component neutral component 6 unspecified role unspecified role neutral component neutral component colocalization colocalization Figure 4 Electron microscopy of double-immunogold-labelled ultrathin sections of chloroplasts from Arabidopsis leaves was performed to verify further an interaction ofALB3 with the cpSecY complex. ALB3 and cpSecY were visualized using secondary antibodies conjugated to 5 nm and 10 nm gold particles respectively. In all cross-reactions of different sections analysed, the gold particles were attached to the thylakoid membrane and showed a similar labelling pattern Note that almost all observed gold particles corresponding to cpSecY (10 nm) co-localized with the gold particles corresponding to ALB3 (5 nm), whereas a signi�cant number of free gold particles for ALB3 were detected. These observations provide further evidence that at least a portion of ALB3 interacts with the cpSecY complex. secy-alb3-5 4 6 unspecified role unspecified role neutral component neutral component 7 unspecified role unspecified role neutral component neutral component direct interaction direct interaction Figure 5 It was found that DSS cross-linked ALB3 to the cpSecY complex, as both proteins were present in the immunoprecipitated crosslinked product