Shibasaki Tadao, Seino Susumu
Methods Enzymol. 2005;403:408-19. doi: 10.1016/S0076-6879(05)03036-3.
Rab, monomeric small Ras-like GTPase, regulates intracellular membrane trafficking in eukaryotic cells. Rab3 is involved in the exocytotic process in a variety of secretory cells including neuronal, neuroendocrine, endocrine, and exocrine cells. Noc2, originally identified as a molecule homologous to Rabphilin-3, is a putative effector of Rab3. Noc2 interacts with the active (GTP-bound) form of Rab3 and regulates hormone secretion in neuroendocrine and endocrine cells and enzyme release in exocrine cells. This chapter describes two kinds of interaction assay by which the association of Noc2 with Rab3 is analyzed: a yeast two-hybrid assay to detect the interaction of Noc2 with the active form of Rab3 in intact cells and a pull-down assay using GST-fused Noc2 protein to ascertain the physical interaction of Noc2 and Rab3 in vitro. Thus, the Noc2 knockout mouse is a useful model for studying the functional consequences of disruption of the interaction.
Rab，一种单体小Ras样GTP酶，调节真核细胞内的膜运输。Rab3参与包括神经元、神经内分泌、内分泌和外分泌细胞在内的多种分泌细胞的胞吐过程。Noc2最初被鉴定为与Rabphilin-3同源的分子，是Rab3的一种假定效应器。Noc2与Rab3的活性（GTP结合）形式相互作用，并调节神经内分泌和内分泌细胞中的激素分泌以及外分泌细胞中的酶释放。本章描述了两种用于分析Noc2与Rab3结合的相互作用检测方法：一种酵母双杂交检测法，用于检测完整细胞中Noc2与Rab3活性形式的相互作用；另一种下拉检测法，使用GST融合的Noc2蛋白来确定Noc2与Rab3在体外的物理相互作用。因此，Noc2基因敲除小鼠是研究这种相互作用破坏的功能后果的有用模型。
