University of Cambridge, Cambridge Institute for Medical Research, Cambridge CB2 0XY, UK.
Dev Cell. 2010 Feb 16;18(2):324-31. doi: 10.1016/j.devcel.2009.12.015.
We have developed a method for rapidly inactivating proteins with rapamycin-induced heterodimerization. Cells were stably transfected with siRNA-resistant, FKBP-tagged subunits of the adaptor protein (AP) complexes of clathrin-coated vesicles (CCVs), together with an FKBP and rapamycin-binding domain-containing construct with a mitochondrial targeting signal. Knocking down the endogenous subunit with siRNA, and then adding rapamycin, caused the APs to be rerouted to mitochondria within seconds. Rerouting AP-2 to mitochondria effectively abolished clathrin-mediated endocytosis of transferrin. In cells with rerouted AP-1, endocytosed cation-independent mannose 6-phosphate receptor (CIMPR) accumulated in a peripheral compartment, and isolated CCVs had reduced levels of CIMPR, but normal levels of the lysosomal hydrolase DNase II. Both observations support a role for AP-1 in retrograde trafficking. This type of approach, which we call a "knocksideways," should be widely applicable as a means of inactivating proteins with a time scale of seconds or minutes rather than days.
我们开发了一种利用雷帕霉素诱导的异二聚体快速失活蛋白质的方法。细胞稳定转染了抗 siRNA 的网格蛋白包被小泡（CCV）衔接蛋白（AP）复合物的 FKBP 标记亚基，以及含有线粒体靶向信号的 FKBP 和雷帕霉素结合结构域的构建体。用 siRNA 敲低内源性亚基，然后加入雷帕霉素，可在几秒钟内使 AP 被重新定向到线粒体。将 AP-2 重新导向线粒体可有效抑制转铁蛋白的网格蛋白介导的内吞作用。在重新定向的 AP-1 细胞中，内吞的阳离子非依赖性甘露糖 6-磷酸受体（CIMPR）积累在周围隔室中，并且分离的 CCV 具有降低的 CIMPR 水平，但溶酶体水解酶 DNase II 的水平正常。这两个观察结果都支持 AP-1 在逆行运输中的作用。这种方法，我们称之为“knocksideways”，应该可以广泛应用于在几秒钟或几分钟而不是几天内失活蛋白质的方法。
Zotero 插件
