Binding of chloroplast signal recognition particle to a thylakoid membrane protein substrate in aqueous solution and delineation of the cpSRP43-substrate interaction domain

Peter Cain , Iris Holdermann , Irmgard Sinning , Arthur E Johnson , Colin Robinson (1)

1 University of Warwick [Coventry]

A chloroplast signal recognition particle (cpSRP) 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 photoactivatable crosslinkers into in vitro-synthesised Lhcb1. We show that Lhcb1 residues 166-176 crosslink specifically to the cpSRP43 subunit. Some crosslink positions within Lhcb1 are in the 'L18' peptide required for targeting of cpSRP substrates, while other crosslinking positions define a new targeting signal in the third transmembrane span. Lchb1 was not found to crosslink to cpSRP54 at any position, and crosslinking 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 to other SRP types where the SRP54 subunit binds to hydrophobic target sequences. The data also show that cpSRP43 can bind to a hydrophobic, 3-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 uniquely post-translational mode of action.

Mots clés: Life Sciences

Peter Cain, Iris Holdermann, Irmgard Sinning, Arthur E Johnson, Colin Robinson. Binding of chloroplast signal recognition particle to a thylakoid membrane protein substrate in aqueous solution and delineation of the cpSRP43-substrate interaction domain. Biochemical Journal, 2011, 437 (1), pp.149-155. ⟨10.1042/BJ20110270⟩. ⟨hal-00600762⟩