The Preliminary Study On The Interaction Of CLICs And Its Mutants With Sedlin

Objective To investigate the interaction of the intracellular chloride channel protein(CLICs) and its mutants with delayed epiphyseal development spine disease protein(Sedlin), we constructed the eukaryotic plasmids of CLICs and its mutants with the FLAG-tagged by molecular cloning technology. Using transient transfection, immunofluorescence, immunoblotting and GST pulldown experiments, we study co-localization and interaction of CLICs or their mutants with Sedlin to explore biological functions of CLIC family.Methods The recombinant plasmids of CLICs were constructed with the template pc DNA3-CLIC(1-5)-FLAG, all of the point mutants(pc DNA3-CLIC1(C24A)-FLAG, pc DNA3-CLIC1(C59A)-FLAG,pc DNA3-CLIC1(C24A-C59A)-FLAG,pc DNA3- CLIC1(△49-51)-FLAG,pc DNA3-CLIC2(C30A)-FLAG,pc DNA3-CLIC3(C22A)-FLAG,pc DN A3-CLIC4(C35A)-FLAG and pc DNA3-CLIC5(C32A)-FLAG) were constructed respectively by molecular cloning technology. Each eukaryotic expression plasmids were transiently transfected into COS7 cells to observe its cellular localization, then CLICs or its mutants and p CDGFP-Sedlin were transiently co-transfected into COS7 cells to observe the co-localization of proteins by laser confocal microscopy; the full-length of CLICs and their mutants were transfected into HEK 293 T cells respectively, protein expression was detected by Western blotting; p GEX-5X-3-SEDL was transformed into competent BL21 cells to prepare fusion protein, and the interaction of CLICs or its mutants with Sedlin by GST pulldown in vitro was detected responsively.Results Restriction endonuclease analysis and sequencing revealed the successful construction of all recombinant plasmids; Western blotting showed CLIC1 and its three point mutants can be expressed in HEK 293 T cells, CLIC2, CLIC3 and CLIC4 can also be expressed; immunofluorescence experiments showed that CLIC1 was mainly localized in the nucleus, some in cytoplasm; CLIC1 mutants distributed in the cytoplasm, being nucleus-free; CLIC1 deletion mutants localized mainly in the cytoplasm, slightly in nucleus; CLIC2 localized both in the nucleus and cytoplasm; CLIC2 point mutants changed the localization significantly and showed a small amount of punctate distribution; CLIC3 and CLIC4 mainly localized in the cytoplasm, small amount in the nucleus; CLIC5 and its point mutants localized only in the cytoplasm; CLIC3 and CLIC4 point mutants localized in the cytoplasm, slightly in the nucleus; CLIC1 and CLIC2 co-localized with Sedlin in the cytoplasm and nucleus; CLIC1 point mutants colocalized with Sedlin only in the cytoplasm,but its point mutant not; Both CLIC3 and its point mutant co-localized with Sedlin in the cytoplasm. GST pulldown assay showed that all of the wild-type of CLICs interact with Sedlin in vitro, while all the mutants of CLICs do not.Conclusion CLIC1 and its point mutants, CLIC2, CLIC3 and its point mutant co-localize with Sedlin; CLIC1 and its three point mutants, CLIC(2-4) can all be effectively expressed in mammalian cells. What is more, there is interaction between CLIC(1-5) and Sedlin respectively, but not point mutants of CLIC1. The study is critical for further functional research of CLIC family in mammalian cells.