title: Interaction studies between the chloroplast signal recognition particle subunit cpSRP43 and the full-length translocase Alb3 reveal a membrane-embedded binding region in Alb3 protein.
abstract: Posttranslational targeting of the light-harvesting chlorophyll a,b-binding proteins depends on the function of the chloroplast signal recognition particle, its receptor cpFtsY, and the translocase Alb3. The thylakoid membrane protein Alb3 of Arabidopsis chloroplasts belongs to the evolutionarily conserved YidC/Oxa1/Alb3 protein family; the members of this family facilitate the insertion, folding, and assembly of membrane proteins in bacteria, mitochondria, and chloroplasts. Here, we analyzed the interaction sites of full-length Alb3 with the cpSRP pathway component cpSRP43 by using in vitro and in vivo studies. Bimolecular fluorescence complementation and Alb3 proteoliposome studies showed that the interaction of cpSRP43 is dependent on a binding domain in the C terminus of Alb3 as well as an additional membrane-embedded binding site in the fifth transmembrane domain (TMD5) of Alb3. The C-terminal binding domain was mapped to residues 374-388, and the binding domain within TMD5 was mapped to residues 314-318 located close to the luminal end of TMD5. A direct binding between cpSRP43 and these binding motifs was shown by pepspot analysis. Further studies using blue-native gel electrophoresis revealed that full-length Alb3 is able to form dimers. This finding and the identification of a membrane-embedded cpSRP43 binding site in Alb3 support a model in which cpSRP43 inserts into a dimeric Alb3 translocation pore during cpSRP-dependent delivery of light-harvesting chlorophyll a,b-binding proteins.
authors.affiliation: Molecular Biology of Plant Organelles, Ruhr-University Bochum, 44780 Bochum, Germany.
authors.forename: Beatrix
authors.initials: B
authors.lastname: Dünschede
authors.forename: Thomas
authors.initials: T
authors.lastname: Bals
authors.forename: Silke
authors.initials: S
authors.lastname: Funke
authors.forename: Danja
authors.initials: D
authors.lastname: Schünemann
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000235:genetics* / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000235:genetics* / Q000378:metabolism; D001665:Binding Sites; D002736:Chloroplasts / Q000378:metabolism*; D019281:Dimerization; D008081:Liposomes / Q000378:metabolism; D008969:Molecular Sequence Data; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D021381:Protein Transport; D017386:Sequence Homology, Amino Acid; D018271:Signal Recognition Particle / Q000235:genetics / Q000378:metabolism*; D020524:Thylakoids / Q000378:metabolism
chemical_list: C106404:ALBINO 3 protein, Arabidopsis; D029681:Arabidopsis Proteins; D008081:Liposomes; D018271:Signal Recognition Particle; C568473:cpSRP43 protein, Arabidopsis