小鼠OSTβ的K70Q突变对OSTα-OSTβ膜转位的影响

Effect of K70Q mutation of OSTβ on membrane translocation of OSTα-OSTβ

(1)目的探讨小鼠有机溶质转运体β亚单位(organic solute transporter beta,OSTβ)的点突变OSTβ(K70Q)对OSTα-OSTβ膜定位的影响。(2)方法提取小鼠回肠RNA,克隆OSTα和OSTβ的编码序列,测序检测OSTβ(K70Q)突变体,进而构建OSTα、OSTβ和OSTβ(K70Q)的双分子荧光互补BiFC表达载体及绿色荧光蛋白GFP融合蛋白,转染HEK-293细胞,于共聚焦显微镜下观察OSTβ(K70Q)对OSTα与OSTβ的相互作用和膜定位的影响。(3)结果突变不影响OSTα和OSTβ(K70Q)的相互作用,但阻碍了OSTα-OSTβ(K70Q)复合体的膜转位,并且可通过显性负效应干扰正常OSTα-OSTβ复合体的膜转位。(4)结论发现了1个小鼠OSTβ的点突变OSTβ(K70Q),突变不影响OSTα和OSTβ(K70Q)的相互作用,但阻碍了OSTα-OSTβ(K70Q)复合体的膜转位,并且可通过显性负效应干扰正常OSTα-OSTβ复合体的膜转位。

Objective To investigate the effect of a K70Q mutation in mouse β subunit of organic solute transporter（OSTβ ）on the membrane localization of OSTα-OSTβ .Methods Mouse ileum RNA was extracted to clone the coding sequence for OSTα and OSTβ .OSTβ （K70Q）mutant was detected by sequencing.Then,bimolecular fluorescence complementation（BiFC）vectors and green fluorescent protein（GFP）fusion proteins for OSTα,OSTβ and OSTβ （K70Q）were constructed and transfected into HEK-293 cells respectively.The confocal microscope was used to observe the effect of the OSTβ （K70Q）mutant on the interaction and membrane translocation of OSTα-OSTβ .ResultsThe mutation did not affect the interaction of OSTαwith OSTβ （K70Q）,but blocked the membrane translocation of the OSTα-OSTβ （K70Q）complex,and interfered with the membrane translocation of the normal OSTα-OSTβ complex through a dominant negative effect.Conclusion This study found a point mutation in the mouse OSTβ ,termed OSTβ （K70Q）.The mutation did not affect the interaction of OSTα with OSTβ （K70Q）,but hindered the membrane translocation of the OSTα-OSTβ （K70Q）complex,and also interfered with the membrane translocation of the normal OSTα-OSTβ complex through a dominant negative effect.