The Interaction And Function Of FKBP25 And CLIC1 Protein In Eukaryotic Eells

Objective This study aims to investigate the expression and localization of FKBP25 and its deletion mutants FKBP25(1-42 aa), FKBP25(1-110 aa), FKBP25(43-224 aa), FKBP25(43-110 aa), FKBP25(111-224 aa) in eukaryotic cells, and the colocalization of FKBP25 and its mutants with CLIC1 in mammalian cells. Yeast twohybrid, GST pulldown, indirect immunofluorescence and co-immunoprecipitation were used to demonstrate the interaction of FKBP25 and CLIC1 in vitro and in vivo.Methods Firstly, The full-length sequence of wild-type FKBP25 and its deletion mutants FKBP25(1-42 aa), FKBP25(1-110 aa), FKBP25(43-224 aa), FKBP25(43-110aa), FKBP25(111-224 aa) were amplified, and then inserted into the yeast expression vectors p GADT7 and p GBKT7, mammalian expression vectors p CDNA3.1 tagged with FLAG and HA, p CDGFP vector, and prokaryotic expression vecto p GEX-5X-3respectively. The constructs were confirmed by specific restriction enzymes digestion and sequencing. Secondly, the co-transformation of p GADT7-FKBP25 and p GBKT7-CLIC1 or p GADT7-CLIC1 and p GBKT7-FKBP25 into yeast AH109 strain was performed on SD/-Leu/-Trp/-His/3AT/X-α-gal solid medium. Thirdly, these plasmids were transfected into HEK 293 T cells respectively and the expression was checked by Western blot; their localization and co-localization with FLAG-CLIC1 in COS7 cells were detected by fluorescence microscopy. Forthy, the GST fusion protein of FKBP25 or its deletion mutants were incubated with the lysate of GFP-CLIC1 respectively, then GST pulldown assays were performed. Lastly, co-immunoprecipitation of overexpressed or endogenous expressed FKBP25 and CLIC1 was performed in HEK293 T cells.Results The expression plasmids of FKBP25 and a series of its deletion mutants were constructed successfully, which can be effectively expressed in HEK 293 T and COS7 cells. The localization of GFP-FKBP25 was mainly in cytoplasm and its mutants were in both nucleus and cytoplasm. The co-localization of its mutants with FLAGCLLIC1 were much less than that of GFP-FKBP25 itself, and there were some differences in distribution. GST pulldown assay showed that FKBP25, FKBP25(43-224aa), FKBP25(111-224 aa) were associated with CLIC1 respectively in vitro. Fruthmore,endogenous FKBP25 protein and CLIC1 protein were colocalized in the nuclei of COS7 cells with indirect immunofluorescence assay. Co-immunoprecipitation assay showed that endogenous expressed FKBP25 and CLIC1 interacted in HEK 293 T cells.Conclusion Yeast two-hybridization, GST pulldown, indirect immunofluorescence and co-immuneprecipitation showed that FKBP25 protein could interact with CLIC1 protein in vitro and in vivo. These suggest that FKBP25 may play critical roles in mammalian cells, combined with our previous data.