Functional characterization of recombinant chloroplast signal recognition particle

The signal recognition particle (SRP) is a ubiquitous system for the target ing of membrane and secreted proteins. The chloroplast SRP (cpSRP) is uniqu e among SRPs in that it possesses no RNA and is functional in post-translat ional as well as co-translational targeting. We have expressed and purified the two components of the Arabidopsis thaliana chloroplast signal recognit ion particle (cpSRP) involved in post-translational transport: cpSRP54 and the chloroplast-specific protein, cpSRP43. Recombinant cpSRP supports the e fficient arc vitro insertion of pea preLhcb1 into isolated thylakoid membra nes. Recombinant cpSRP is a stable heterodimer with a molecular mass of sim ilar to 100 kDa as determined by analytical ultracentrifugation, gel filtra tion analysis, and dynamic light scattering. The interactions of the compon ents of the recombinant heterodimer and pea preLhcb1 were probed using an i mmobilized peptide library (pepscan) approach. These data confirm two previ ously reported interactions with the L18 region and the third transmembrane helix of Lhcb1 and suggest that the interface of the cpSRP43 and epSRP54 p roteins is involved in substrate binding. Additionally, cpSRP components ar e shown to recognize peptides from the cleavable, N-terminal chloroplast tr ansit peptide of preLhcb1. The interaction of cpSRP43 with epSRP54 was prob ed in a similar experiment with a peptide library representing cpSPR54. The C terminus of cpSRP54 is essential for the formation of the stable cpSRP c omplex and cpSPR43 interacts with distinct regions of the M domain of cpSRP 54.