Institut Curie, Centre de Recherche, Paris, France.
Nat Methods. 2012 Mar 11;9(5):493-8. doi: 10.1038/nmeth.1928.
To dissect secretory traffic, we developed the retention using selective hooks (RUSH) system. RUSH is a two-state assay based on the reversible interaction of a hook protein fused to core streptavidin and stably anchored in the donor compartment with a reporter protein of interest fused to streptavidin-binding peptide (SBP). Biotin addition causes a synchronous release of the reporter from the hook. Using the RUSH system, we analyzed different transport characteristics of various Golgi and plasma membrane reporters at physiological temperature in living cells. Using dual-color simultaneous live-cell imaging of two cargos, we observed intra- and post-Golgi segregation of cargo traffic, consistent with observation in other systems. We show preliminarily that the RUSH system is usable for automated screening. The system should help increase the understanding of the mechanisms of trafficking and enable screens for molecules that perturb pathological protein transport.
为了剖析分泌运输过程，我们开发了一种利用选择性钩子（RUSH）的保留系统。RUSH 是一种基于钩状蛋白与核心链霉亲和素可逆相互作用的双态测定法，该钩状蛋白融合到稳定锚定在供体隔室中的核心链霉亲和素上，而与链霉亲和素结合肽（SBP）融合的报告蛋白则作为受体。生物素的添加会导致报告蛋白从钩子上同步释放。利用 RUSH 系统，我们在生理温度下分析了不同的高尔基氏体和质膜报告蛋白的不同运输特性。通过对两种货物的双色同时活细胞成像，我们观察到货物运输的高尔基体内部和高尔基体后区的分隔，这与在其他系统中的观察结果一致。我们初步表明，RUSH 系统可用于自动筛选。该系统应有助于增加对运输机制的理解，并能够筛选出干扰病理性蛋白运输的分子。
