Lorenz Holger, Hailey Dale W, Lippincott-Schwartz Jennifer
Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bldg. 18T Library Drive, Bethesda, Maryland 20892, USA.
Nat Methods. 2006 Mar;3(3):205-10. doi: 10.1038/nmeth857.
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.
了解许多蛋白质的细胞生物学特性需要知晓其在体内的拓扑分布。在此，我们描述一种基于荧光的新技术——荧光蛋白酶保护法（FPP），用于研究蛋白质的拓扑结构以及在活细胞复杂环境中定位蛋白质亚群。在源自生化蛋白酶保护分析的FPP分析中，绿色荧光蛋白（GFP）融合蛋白被用作非侵入性工具，以快速且直接的方式获取活细胞内蛋白质拓扑结构和定位的详细信息。为证明FPP的广泛适用性，我们运用该技术确定了定位于包括内质网（ER）、高尔基体、线粒体、过氧化物酶体和自噬体在内的多种细胞器的蛋白质的拓扑结构。FPP分析成功地在适当的区室中表征了测试蛋白质的拓扑结构，这表明该技术在研究细胞内蛋白质拓扑结构和定位方面具有广泛的适用性。
