Department of Molecular Biology, Faculty of Medicine, GZMB, Georg-August-University Göttingen, Humboldtallee 23, 37073 Göttingen, Germany.
Bioanalytics Group, Institute of Clinical Chemistry, University Medical Center Göttingen, Robert-Koch-Straße 40, 37075 Göttingen, Germany.
Cells. 2020 Mar 3;9(3):605. doi: 10.3390/cells9030605.
Emerin is one of the best characterized proteins of the inner nuclear membrane, but can also occur at the level of the endoplasmic reticulum. We now use enhanced ascorbate peroxidase 2 (APEX2) to probe the environment of emerin. APEX2 can be used as a genetic tag that produces short-lived yet highly reactive biotin species, allowing the modification of proteins that interact with or are in very close proximity to the tagged protein. Biotinylated proteins can be isolated using immobilized streptavidin and analyzed by mass spectrometry. As an alternative to the standard approach with a genetic fusion of APEX2 to emerin, we also used RAPIDS (rapamycin- and APEX-dependent identification of proteins by SILAC), a method with improved specificity, where the peroxidase interacts with the protein of interest (i.e., emerin) only upon addition of rapamycin to the cells. We compare these different approaches, which, together, identify well-known interaction partners of emerin like lamin A and the lamina associated polypeptide 1 (LAP1), as well as novel proximity partners.
emerin 是核内膜中研究得最为透彻的蛋白之一，但也可以出现在内质网水平。我们现在使用增强型过氧化物酶 2 (APEX2) 来探测 emerin 的环境。APEX2 可用作遗传标记，产生短暂但高度反应性的生物素种类，从而修饰与标记蛋白相互作用或非常接近的蛋白。通过固定化链霉亲和素可以分离生物素化蛋白，并通过质谱分析进行分析。作为 APEX2 与 emerin 基因融合的标准方法的替代方法，我们还使用了 RAPIDS（雷帕霉素和 APEX 依赖性 SILAC 鉴定蛋白），这是一种特异性得到改善的方法，其中过氧化物酶仅在向细胞中添加雷帕霉素时才与感兴趣的蛋白（即 emerin）相互作用。我们比较了这些不同的方法，这些方法共同鉴定出了 emerin 的一些已知相互作用伙伴，如 lamin A 和 lamina associated polypeptide 1 (LAP1)，以及一些新的接近伙伴。
