Multicolor BiFC analysis of competition among G protein beta and gamma subunit interactions.
Multicolor BiFC analysis of competition among G protein beta and gamma subunit interactions.
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G 蛋白 β 和 γ 亚基相互作用之间竞争的多色 BiFC 分析。
DOI：
10.1016/j.ymeth.2008.06.008
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发表时间：
2008
期刊：
影响因子：
--
通讯作者：
Berlot,CatherineH
中科院分区：
文献类型：
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作者：
Hynes,ThomasR;Yost,Evan;Mervine,Stacy;Berlot,CatherineH
我们应用双光子荧光显微镜研究了活细胞中G蛋白β和γ亚基的缔合偏好。细胞共表达β和γ亚基的多种同种型，其中大多数可以形成复合物。尽管许多βγ复合物在重组系统中检测时表现出相似的性质，但体内敲除实验表明，单个同种型具有独特的功能。BiFC通过比较荧光βγ复合物的量与其调节效应蛋白的能力，使βγ复合物的形成与完整细胞中的功能相关成为可能。特异性βγ复合物在体内的相对优势尚不清楚。为了解决这一问题，BiFBIFC可以通过同时观察两种荧光复合物来确定β和γ亚基的缔合偏好，这两种荧光复合物是在β或γ亚基分别与黄色荧光蛋白（YFP-N）和青色荧光蛋白（CFP-N）的氨基末端片段融合，与有限量的共同γ或β亚基（CFP-C）融合时形成的。多色BiFC还可以确定相互作用蛋白质在复合物亚细胞靶向中的作用，研究在分离条件下不稳定的蛋白质复合物的形成，确定共表达蛋白质在调节相互作用蛋白质的缔合偏好中的作用，并可视化影响多个蛋白质复合物的动态事件。这些方法可以应用于研究活细胞中各种蛋白质复合物的组装和功能。
We have applied multicolor BiFC to study the association preferences of G protein β and γ subunits in living cells. Cells co-express multiple isoforms of β and γ subunits, most of which can form complexes. Although many βγ complexes exhibit similar properties when assayed in reconstituted systems, knockout experiments in vivo suggest that individual isoforms have unique functions. BiFC makes it possible to correlate βγ complex formation with functionality in intact cells by comparing the amounts of fluorescent βγ complexes with their abilities to modulate effector proteins. The relative predominance of specific βγ complexes in vivo is not known. To address this issue, multicolor BiFC can determine the association preferences of β and γ subunits by simultaneously visualizing the two fluorescent complexes formed when β or γ subunits fused to amino terminal fragments of yellow fluorescent protein (YFP-N) and cyan fluorescent protein (CFP-N) compete to interact with limiting amounts of a common γ or β subunit, respectively, fused to a carboxyl terminal fragment of CFP (CFP-C). Multicolor BiFC also makes it possible to determine the roles of interacting proteins in the subcellular targeting of complexes, study the formation of protein complexes that are unstable under isolation conditions, determine the roles of co-expressed proteins in regulating the association preferences of interacting proteins, and visualize dynamic events affecting multiple protein complexes. These approaches can be applied to studying the assembly and functions of a wide variety of protein complexes in the context of a living cell.