Rehm A, Ploegh H L
Center for Cancer Research, Department of Biology, Massachusetts Institute of Technology, Cambridge 02139, USA.
J Cell Biol. 1997 Apr 21;137(2):305-17. doi: 10.1083/jcb.137.2.305.
The assembly in living cells of heterotrimeric guanine nucleotide binding proteins from their constituent alpha, beta, and gamma subunits is a complex process, compounded by the multiplicity of the genes that encode them, and the diversity of receptors and effectors with which they interact. Monoclonal anti-beta antibodies (ARC5 and ARC9), raised against immunoaffinity purified beta gamma complexes, recognize beta subunits when not associated with gamma and can thus be used to monitor assembly of beta gamma complexes. Complex formation starts immediately after synthesis and is complete within 30 min. Assembly occurs predominantly in the cytosol, and association of beta gamma complexes with the plasma membrane fraction starts between 15-30 min of chase. Three pools of beta subunits can be distinguished based on their association with gamma subunits, their localization, and their detergent solubility. Association of beta and alpha subunits with detergent-insoluble domains occurs within 1 min of chase, and increases to reach a plateau of near complete detergent resistance within 30 min of chase. Brefeldin A treatment does not interfere with delivery of beta gamma subunits to detergent-insoluble domains, suggesting that assembly of G protein subunits with their receptors occurs distally from the BFA-imposed block of intracellular membrane trafficking and may occur directly at the plasma membrane.
异源三聚体鸟嘌呤核苷酸结合蛋白在活细胞中由其组成的α、β和γ亚基组装而成，这是一个复杂的过程，编码它们的基因具有多样性，与之相互作用的受体和效应器也具有多样性，使得该过程更加复杂。针对免疫亲和纯化的βγ复合物产生的单克隆抗β抗体（ARC5和ARC9），在β亚基不与γ亚基结合时能识别β亚基，因此可用于监测βγ复合物的组装。复合物的形成在合成后立即开始，并在30分钟内完成。组装主要发生在细胞质中，βγ复合物与质膜部分的结合在追踪15 - 30分钟之间开始。根据β亚基与γ亚基的结合情况、定位以及去污剂溶解性，可以区分出三个β亚基池。β亚基和α亚基与去污剂不溶性结构域的结合在追踪1分钟内发生，并在追踪30分钟内增加至接近完全抗去污剂的平台期。布雷菲德菌素A处理不干扰βγ亚基向去污剂不溶性结构域的递送，这表明G蛋白亚基与其受体的组装发生在布雷菲德菌素A阻断细胞内膜运输的远端，可能直接发生在质膜上。
