A Study Of LFNR-Tic62 Affinity Enhancement By LIR1

The LIR1(Light Induced Rice1) gene encodes a 13-kDa unstructured, chloroplast targeted protein containing two almost identical motifs of unknown function. The LIR1 gene is present in the genomes of vascular plants, mosses, liverworts and algae, but not in cyan bacteria. It has been proved that light could promote the LIR1 protein degradation, whose rate related to the light intensity, and the protein accumulated in the dark(Yang 2014). Co-immunoprecipitation, pull-down assays, and yeast two-hybrid analysis showed that the LIR1 protein interacts with leaf-type ferredoxin-NADP+ oxidoreductase (LFNR). LIR1 and LFNR formed high molecular weight thylakoid protein complexes with the Tic62 and Trol proteins, previously shown to anchor LFNR to the membrane(Yang 2014). Loss of LIR1 resulted in a marked decrease in the accumulation of LFNR-containing thylakoid protein complexes, suggesting that LIR1 plays an important role in the LFNR anchoring in the thylakoid membrane(Hu 2015).Baesd on above knowledge, we further investigated the interaction mechanism of LIR1,LFNR and OsTic62 in this thesis. To determine whether LIR1 strengthens the binding between LFNR and Tic62 or directly increases the attachment of Tic62 to the thylakoid membrane, we incubated the isolated Atlirl thylakoids with purified recombinant protein GST or GST-OsLIR1 under different pH. Intriguingly, the presence of GST-OsLIRl markedly shifted the distribution of OsLFNR towards the membrane fraction, while no such effect was observed for OsTic62 indicating that LIR1 can not influence the attachment of Tic62 to the thylakoid membrane. Next, protein binding affinity assays, GST pull-down assays, and yeast two-hybrid analysis gave further evidence that LIR1 protein does not directly bind to Tic62, but rather, it interacts with both LFNR isforms, which increases the affinity of LFNRs for Tic62.