Functional analysis of the protein-interacting domains of chloroplast SRP43.
The chloroplast signal recognition particle (cpSRP) consists of an evolutionarily conserved 54-kDa subunit (cpSRP54) and a dimer of a unique 43-kDa subunit (cpSRP43). cpSRP binds light-harvesting chlorophyll proteins (LHCPs) to form a cpSRP/LHCP transit complex, which targets LHCP to the thylakoid membrane. Previous studies showed that transit complex formation is mediated through the binding of the L18 domain of LHCP to cpSRP43. cpSRP43 is characterized by a four-ankyrin repeat domain at the N terminus and two chromodomains at the C terminus. In the present study we used the yeast two-hybrid system and in vitro binding assays to analyze the function of different domains of cpSRP43 in protein complex formation. We report here that the first ankyrin repeat binds to the 18-amino acid domain on LHCP that binds to cpSRP43, whereas the third and fourth ankyrin repeats are involved in the dimerization of cpSRP43. We show further that the interaction of cpSRP43 with cpSRP54 is mediated via binding of the methionine-rich domain of cpSRP54 to the C-terminally located chromodomains of cpSRP43. Both chromodomains contain essential elements for binding cpSRP54, indicating that the closely spaced chromodomains together create a single binding site for cpSRP54. In addition, our data demonstrate that the interaction of cpSRP54 with the chromodomains of cpSRP43 is enhanced indirectly by the dimerization motif of cpSRP43.
Jonas-Straube
E
E
Institut für Biologie III der Rheinisch-Westfälische Technische Hochschule Aachen, Worringerweg 1, 52074 Aachen, Germany.
Hutin
C
C
Hoffman
N E
NE
Schünemann
D
D
D017487:Ankyrins; D001665:Binding Sites; D060365:Chloroplast Proteins; D002736:Chloroplasts / Q000378:metabolism*; D019281:Dimerization; D045342:Light-Harvesting Protein Complexes; D008715:Methionine / Q000378:metabolism; D008958:Models, Molecular; D045322:Photosynthetic Reaction Center Complex Proteins / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D012091:Repetitive Sequences, Nucleic Acid; D029701:Saccharomyces cerevisiae Proteins; D012689:Sequence Homology, Nucleic Acid; D018271:Signal Recognition Particle / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013329:Structure-Activity Relationship
D017487:Ankyrins; D060365:Chloroplast Proteins; D045342:Light-Harvesting Protein Complexes; D045322:Photosynthetic Reaction Center Complex Proteins; C069268:SRP54 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D018271:Signal Recognition Particle; D008715:Methionine