Functional characterization of recombinant chloroplast signal recognition particle.
The signal recognition particle (SRP) is a ubiquitous system for the targeting of membrane and secreted proteins. The chloroplast SRP (cpSRP) is unique among SRPs in that it possesses no RNA and is functional in post-translational as well as co-translational targeting. We have expressed and purified the two components of the Arabidopsis thaliana chloroplast signal recognition particle (cpSRP) involved in post-translational transport: cpSRP54 and the chloroplast-specific protein, cpSRP43. Recombinant cpSRP supports the efficient in vitro insertion of pea preLhcb1 into isolated thylakoid membranes. Recombinant cpSRP is a stable heterodimer with a molecular mass of approximately 100 kDa as determined by analytical ultracentrifugation, gel filtration analysis, and dynamic light scattering. The interactions of the components of the recombinant heterodimer and pea preLhcb1 were probed using an immobilized peptide library (pepscan) approach. These data confirm two previously reported interactions with the L18 region and the third transmembrane helix of Lhcb1 and suggest that the interface of the cpSRP43 and cpSRP54 proteins is involved in substrate binding. Additionally, cpSRP components are shown to recognize peptides from the cleavable, N-terminal chloroplast transit peptide of preLhcb1. The interaction of cpSRP43 with cpSRP54 was probed in a similar experiment with a peptide library representing cpSPR54. The C terminus of cpSRP54 is essential for the formation of the stable cpSRP complex and cpSPR43 interacts with distinct regions of the M domain of cpSRP54.
Groves
M R
MR
Structural Biology Programme, EMBL, Meyerhofstrasse 1, 69117 Heidelberg, Germany.
Mant
A
A
Kuhn
A
A
Koch
J
J
Dübel
S
S
Robinson
C
C
Sinning
I
I
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000378:metabolism; D015153:Blotting, Western; D060365:Chloroplast Proteins; D002736:Chloroplasts / Q000737:chemistry* / Q000378:metabolism; D002850:Chromatography, Gel; D019281:Dimerization; D008027:Light; D008969:Molecular Sequence Data; D010455:Peptides; D045322:Photosynthetic Reaction Center Complex Proteins / Q000737:chemistry; D011485:Protein Binding; D014176:Protein Biosynthesis; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D012313:RNA / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D012542:Scattering, Radiation; D018271:Signal Recognition Particle / Q000737:chemistry* / Q000378:metabolism; D020524:Thylakoids / Q000737:chemistry; D014461:Ultracentrifugation
D060365:Chloroplast Proteins; D010455:Peptides; D045322:Photosynthetic Reaction Center Complex Proteins; D011994:Recombinant Proteins; C069268:SRP54 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D018271:Signal Recognition Particle; D012313:RNA