氯离子通道蛋白1的缺失突变体CLIC1(Δ49-51)与Sedlin蛋白相互作用的研究

Interaction between deletion mutant CLIC1(Δ49-51) and Sedlin

目的探究胞内氯离子通道蛋白1(CLIC1)的缺失突变体CLIC1(Δ49-51)在哺乳动物细胞中的表达、定位改变,及与Sedlin蛋白在体内、体外相互作用的影响。方法构建CLIC1的缺失突变体的真核表达质粒pc DNA3. 1-CLIC1(Δ49-51)-FLAG;免疫荧光观察CLIC1(Δ49-51)与Sedlin在COS7细胞中的共定位;在HEK 293T细胞中进行免疫共沉淀和GST pulldown实验,研究CLIC1(Δ49-51)与Sedlin的相互作用。结果 CLIC1(Δ49-51)在COS7细胞中主要定位于细胞质,小部分定位于细胞核。相对于野生型CLIC1的细胞核定位,缺失突变体的细胞内定位发生明显改变并且与Sedlin蛋白没有共定位。Western blot结果显示CLIC1(Δ49-51)能在HEK 293T细胞中有效表达;免疫共沉淀实验结果表明其与Sedlin在体内没有相互作用; GST pulldown结果表明其与Sedlin在体外没有相互作用。结论 CLIC1蛋白的第49~51位氨基酸序列KRR对CLIC1蛋白在细胞内的正确定位发挥重要作用;其缺失突变后影响了CLIC1在细胞内的定位、表达及CLIC1与Sedlin的相互作用。

Objective To study the expression and localization of CLIC1(Δ49-51),an intracellular chloride ion channel 1(CLIC1)deletion mutant,and its effect on the interaction with Sedlin in mammalian cells.Methods The eukaryotic expression plasmid pcDNA3.1-CLIC1(Δ49-51)-FLAG was constructed based on the wild type CLIC1 expression plasmid pcDNA3.1-CLIC1-FLAG.Immunofluorescence and nuclear separation experiment were performed to detect the expression and colocalization of CLIC1(Δ49-51)with Sedlin in COS7 cells;co-immunoprecipitation and GST pulldown experiments in HEK 293T cells were conducted to investigate the interaction of CLIC1(Δ49-51)with Sedlin in vivo and in vitro,respectively.Results CLIC1(Δ49-51)mainly localized in the cytoplasm of COS7 cells and much less in the nucleus.Compared with wild-type CLIC1,its intracellular localization changed significantly,and it did not colocalize with Sedlin.The result of Western blot showed that CLIC1(Δ49-51)could be efficiently expressed in HEK 293T and COS7 cells.Co-immunoprecipitation and GST pulldown assays showed that it had no interaction with Sedlin in vivo and in vitro,respectively.Conclusion KRR motif at position 49-51 of CLIC1 plays an important role in the correct localization of CLIC1 protein to the cell membrane.Its deletion mutation affects the localization and expression of CLIC1 in cells and may further affect its interaction with Sedlin.