Fluorescence protease protection of GFP chimeras to reveal protein topology and subcellular localization
Fluorescence protease protection of GFP chimeras to reveal protein topology and subcellular localization
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DOI：
10.1038/nmeth857
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发表时间：
2006-03-01
期刊：
影响因子：
48
通讯作者：
Lippincott-Schwartz, J
中科院分区：
文献类型：
--
作者：
Lorenz, H;Hailey, DW;Lippincott-Schwartz, J
了解许多蛋白质的细胞生物学需要了解它们在体内的拓扑分布。在这里，我们描述了一种新的基于荧光的技术，荧光蛋白酶保护(FPP)，用于研究蛋白质的拓扑结构，并在活细胞的复杂环境中定位蛋白质亚群。在FPP分析中，GFP融合蛋白被用作非侵入性工具，以快速和直接的方式获得活细胞内的蛋白质拓扑和定位细节。为了证明FPP的广泛适用性，我们使用该技术定义了定位于多种细胞器的蛋白质的拓扑结构，包括内质网(ER)、高尔基体、线粒体、过氧体和自噬小体。FPP分析成功地表征了被测试蛋白质在其适当间隔内的拓扑结构，这表明该技术在研究蛋白质拓扑结构和细胞内的定位方面具有广泛的适用性。
Understanding the cell biology of many proteins requires knowledge of their in vivo topological distribution. Here we describe a new fluorescence-based technique, fluorescence protease protection (FPP), for investigating the topology of proteins and for localizing protein subpopulations within the complex environment of the Living cell. In the FPP assay, adapted from biochemical protease protection assays, GFP fusion proteins are used as noninvasive tools to obtain details of protein topology and localization within Living cells in a rapid and straightforward manner. To demonstrate the broad applicability of FPP, we used the technique to define the topology of proteins localized to a wide range of organelles including the endoplasmic reticulum (ER), Golgi apparatus, mitochondria, peroxisomes and autophagosomes. The success of the FPP assay in characterizing the topology of the tested proteins within their appropriate compartments suggests this technique has wide applicability in studying protein topology and localization within the cell.