title: Binding of chloroplast signal recognition particle to a thylakoid membrane protein substrate in aqueous solution and delineation of the cpSRP43-substrate interaction domain.
abstract: A cpSRP [chloroplast SRP (signal recognition particle)] comprising cpSRP54 and cpSRP43 subunits mediates the insertion of light-harvesting proteins into the thylakoid membrane. We dissected its interaction with a full-length membrane protein substrate in aqueous solution by insertion of site-specific photo-activatable cross-linkers into in vitro-synthesized Lhcb1 (major light-harvesting chlorophyll-binding protein of photosystem II). We show that Lhcb1 residues 166-176 cross-link specifically to the cpSRP43 subunit. Some cross-link positions within Lhcb1 are in the 'L18' peptide required for targeting of cpSRP substrates, whereas other cross-linking positions define a new targeting signal in the third transmembrane span. Lhcb1 was not found to cross-link to cpSRP54 at any position, and cross-linking to cpSRP43 is unaffected by the absence of cpSRP54. cpSRP43 thus effectively binds substrates autonomously, and its ability to independently bind an extended 20+-residue substrate region highlights a major difference with other SRP types where the SRP54 subunit binds to hydrophobic target sequences. The results also show that cpSRP43 can bind to a hydrophobic, three-membrane span, substrate in aqueous solution, presumably reflecting a role for cpSRP in the chloroplast stroma. This mode of action, and the specificity of the cpSRP43-substrate interaction, may be associated with cpSRP's unique post-translational mode of action.
authors.affiliation: School of Life Sciences, University of Warwick, Coventry CV4 7AL, U.K.
authors.forename: Peter
authors.initials: P
authors.lastname: Cain
authors.forename: Iris
authors.initials: I
authors.lastname: Holdermann
authors.forename: Irmgard
authors.initials: I
authors.lastname: Sinning
authors.forename: Arthur E
authors.initials: AE
authors.lastname: Johnson
authors.forename: Colin
authors.initials: C
authors.lastname: Robinson
mesh_terms: D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D002736:Chloroplasts / Q000378:metabolism*; D045342:Light-Harvesting Protein Complexes / Q000737:chemistry / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D018271:Signal Recognition Particle / Q000737:chemistry* / Q000378:metabolism*; D012996:Solutions / Q000378:metabolism; D020524:Thylakoids / Q000378:metabolism*
chemical_list: D029681:Arabidopsis Proteins; D045342:Light-Harvesting Protein Complexes; D008565:Membrane Proteins; D010940:Plant Proteins; D018271:Signal Recognition Particle; D012996:Solutions