The Study On The Interaction Of Scaffold Protein RACK1 And Its Mutants With Chloride Intracellular Channel Protein 1(CLIC1)

Objective To investigate the co-localization and interaction with RACK1, its mutant proteins and chloride ion channel protein1(CLIC1), we constructed the eukaryotic plasmids of RACK1 mutants gene with the FLAG-tagged, prokaryotic plasmids of CLIC1 and RACK1 mutants gene by molecular cloning technology. Using a series of experimental studies including Yeast two-hybrid, transient transfection, immuno-fluorescence assay, co-immunoprecipitation assay and GST-pulldown assay to research the association with them in vivo and vitro. Methods Recombinant plasmids were amplified by PCR with the template pc DNA3.1-RACK1. p GBKT7/p GADT7-RACK1, p GBKT7/p GADT7-RACK1-N(1-13 8), p GBKT7/p GADT7-RACK1-C(130-317) were constructed by molecular cloning methods, respectively. AH109 cells co-transformed RACK1 or its mutants with CLIC1 were screened on the SD3-(-Leu/-His/-Trp) medium, then determined the activities of X-α-gal of the clone to demonstrate the interaction of them. Constructed Eukaryotic plasmids pc DNA3.1-RACK1-N(1-138)-FLAG, pc DNA3.1-RACK1-C(130-317)-FLAG, and prokayotic plasmids p GEX-5X-3-RACK1-N(1-138), p GEX-5X-3-RACK1-C(130-317), p GEX-5X-3-CLIC1. The distributions of expression products in COS7 cells were observed by laser confocal microscope. Cytoplasmic proteins and nucleoproteins were respectively extracted from the HEK 293 T or COS7 cells transfected with plasmids containing RACK1 or its mutants sequences, then the proteins were detected by Western blotting analysis. Employing co-immunoprecipitation analyses, determined whether there were a interaction between CLIC1 and RACK1 or its mutants. GST-CLIC1, GST-RACK1, GST-RACK1-N and GST-RACK1-C were purified from BL21 cells transformed with relevant plasmids, and then GST-pulldown experiment was performed. Results The enzyme digestion and sequencing identification results show that all the recombinant plasmids were constructed successfully. Yeast two-hybrid assays showed the yeast cells that co-transformed the CLIC1 and RACK1 or its mutant(RACK1-N) could grow normally in SD3- medium, and the most clones turned blue in the X-α-galactosidase activity assay, which proved the existence of potential interaction between them. Western blot results of separated the cytoplasmic proteins and nucleo-proteins indicated that all of them located in the cytoplasm and nuclei, cytoplasm mainly, we observed the wild type RACK1 proteins located in the cytoplasm and nuclear membrane by confocal fluorescence microscopy as well as the mutant proteins, there were a certain amount of localization in the nuclei also, and co-localization with CLIC1, located in the cytoplasm, nucleus and nuclear membrane. The co-immunoprecipitation analysis indicated that the wild type RACK1 proteins and its mutant proteins interacted with CLIC1 protein. Purifying the fusion proteins of GST-CLIC1, GST-RACK1, GST-RACK1-N, GST-RACK1-C under the best conditions were successful. The GST-pulldown analysis showed that the wild type RACK1 and its mutant proteins could interact with CLIC1 protein also. Conclusion Yeast two-hybrid assays shows RACK1 or its mutant proteins interact with CLIC1, potentially. The products of RACK1 or its mutant appears co-localize with CLIC1, co-immunoprecipitation assay and GST-pulldown assay indicate the interaction of them in vivo and vitro.