Rap1GAP二聚体的形成对其在细胞内定位的影响

目的观察Rap1GAP二聚体的形成对其在细胞内定位的影响。方法对绿色荧光蛋白标记的Rap1GAP野生体编码第90和99位氨基酸的核苷酸进行定点突变,制备绿色荧光蛋白标记的Rap1GAP二聚体缺失突变体M90和M99。将Rap1GAP野生体及二聚体缺失突变体单独或者分别与活化型Gα共转染HEK293细胞,在荧光显微镜下观察野生体和二聚体缺失突变体在细胞内的分布情况。结果野生型Rap1GAP与二聚体缺失突变体Rap1GAP分别单独转染HEK293细胞时,野生型和突变体Rap1GAP均弥漫分布胞质内;当野生型与突变体Rap1GAP分别与活化型Gα共转染HEK293细胞时,野生型和突变体Rap1GAP均主要分布在细胞膜上。结论Rap1GAP二聚体的形成对其在细胞内的定位没有影响。

Influence of Formation of Rap1GAP Dimer on Intracellular Localization of Rap1GAP

Objective To observe the influence of formation of Rap1GAP dimer on the intracellular localization of Rap1GAP.Methods Induce the site-directed mutations of nucleotides encoding the amino acids at sites 90 and 99 of green fluorescent protein-labeled wild Rap1GAP to obtain mutants M90 and M99 with Rap1GAP dimer deleted respectively.HEK293 cells were transfected with wild Rap1GAP,mutant M90,mutant M99,wild Rap1GAP+Gα,mutant M90+Gα and M99+Gα respectively.The distributions of wild Rap1GAP and mutants were observed by fluorescent microscopy.Results When HEK293 cells were transfected with wild Rap1GAP,mutant M90 or mutant M99,both wild Rap1GAP and mutants were diffused in cytoplasm.However,when the cells were co-transfected with wild Rap1GAP+Gα,M90+Gα or M99+Gα,both wild Rap1GAP and mutants were mainly distributed on cell membrane.Conclusion The formation of Rap1GAP dimer showed no significant influence on the intracellular localization of Rap1GAP.