通过基于荧光共振能量转移（FRET）分析的流式细胞术来描绘 Rab6 GTPase 和 Mint3 之间的相互作用。
Characterizing the interaction between the Rab6 GTPase and Mint3 via flow cytometry based FRET analysis.
机构信息
Department of Experimental Tumorbiology, University Muenster, Badestr 9, D-48149 Muenster, Germany.
出版信息
Biochem Biophys Res Commun. 2010 Jun 4;396(3):679-83. doi: 10.1016/j.bbrc.2010.04.161. Epub 2010 May 4.
In extension to previously applied techniques like yeast two-hybrid and GST pull-down assays, we successfully established a FACS-based FRET analysis to investigate the interaction of the Mint3 adaptor protein and the small Rab GTPase Rab6A in living mammalian cells. A Mint3 mutant containing only the PTB domain (Mint3Delta6) is able to interact with the constitutively active form of Rab6A. Mint3Delta4, a mutant lacking part of the PTB domain was unable to interact with Rab6A in GST pull-down analysis and did not produce FRET signals, when co-expressed with active Rab6A. We demonstrate that this FACS-based FRET analysis is a suitable method for interaction studies between two proteins in living cells.
除了先前应用的技术，如酵母双杂交和 GST 下拉分析，我们还成功建立了基于 FACS 的 FRET 分析，以研究 Mint3 衔接蛋白与小 Rab GTPase Rab6A 在活哺乳动物细胞中的相互作用。仅包含 PTB 结构域的 Mint3 突变体（Mint3Delta6）能够与 Rab6A 的组成性激活形式相互作用。在 GST 下拉分析中，缺乏部分 PTB 结构域的 Mint3Delta4 突变体不能与 Rab6A 相互作用，并且当与活性 Rab6A 共表达时，也不会产生 FRET 信号。我们证明这种基于 FACS 的 FRET 分析是研究活细胞中两种蛋白质相互作用的合适方法。
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