Olson Macy G, Jorgenson Lisa M, Widner Ray E, Rucks Elizabeth A
Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, NE, USA.
Methods Mol Biol. 2019;2042:245-278. doi: 10.1007/978-1-4939-9694-0_17.
In the study of intracellular bacteria that reside within a membrane-bound vacuole, there are many questions related to how prokaryotic or eukaryotic transmembrane or membrane-associated proteins are organized and function within the membranes of these pathogen-containing vacuoles. Yet this host-pathogen interaction interface has proven difficult to experimentally resolve. For example, one method to begin to understand protein function is to determine the protein-binding partners; however, examining protein-protein interactions of hydrophobic transmembrane proteins is not widely successful using standard immunoprecipitation or coimmunoprecipitation techniques. In these scenarios, the lysis conditions that maintain protein-protein interactions are not compatible with solubilizing hydrophobic membrane proteins. In this chapter, we outline two proximity labeling systems to circumvent these issues to study (1) eukaryotic proteins that localize to the membrane-bound inclusion formed by Chlamydia trachomatis using BioID, and (2) chlamydial proteins that are inserted into the inclusion membrane using APEX2. BioID is a promiscuous biotin ligase to tag proximal proteins with biotin. APEX2 is an ascorbate peroxidase that creates biotin-phenoxyl radicals to label proximal proteins with biotin or 3,3'-diaminobenzidine intermediates for examination of APEX2 labeling of subcellular structures using transmission electron microscopy. We present how these methods were originally conceptualized and developed, so that the user can understand the strengths and limitations of each proximity labeling system. We discuss important considerations regarding experimental design, which include careful consideration of background conditions and statistical analysis of mass spectrometry results. When applied in the appropriate context with adequate controls, these methods can be powerful tools toward understanding membrane interfaces between intracellular pathogens and their hosts.
在对存在于膜结合液泡内的细胞内细菌的研究中，有许多问题涉及原核或真核跨膜蛋白或膜相关蛋白如何在这些含病原体液泡的膜内组织和发挥功能。然而，事实证明，这个宿主 - 病原体相互作用界面很难通过实验来解析。例如，一种开始了解蛋白质功能的方法是确定蛋白质结合伙伴；然而，使用标准免疫沉淀或共免疫沉淀技术来检测疏水性跨膜蛋白的蛋白质 - 蛋白质相互作用并不十分成功。在这些情况下，维持蛋白质 - 蛋白质相互作用的裂解条件与溶解疏水性膜蛋白不兼容。在本章中，我们概述了两种邻近标记系统，以规避这些问题来进行研究：（1）使用BioID研究定位于沙眼衣原体形成的膜结合包涵体的真核蛋白质，以及（2）使用APEX2研究插入包涵体膜的衣原体蛋白质。BioID是一种滥交生物素连接酶，用于用生物素标记近端蛋白质。APEX2是一种抗坏血酸过氧化物酶，它产生生物素 - 苯氧基自由基，用生物素或3,3'-二氨基联苯胺中间体标记近端蛋白质，以便使用透射电子显微镜检查APEX2对亚细胞结构的标记。我们介绍了这些方法最初是如何构思和开发的，以便用户能够理解每个邻近标记系统的优点和局限性。我们讨论了关于实验设计的重要考虑因素，包括仔细考虑背景条件和对质谱结果的统计分析。当在适当的背景下并进行充分的对照应用时，这些方法可以成为理解细胞内病原体与其宿主之间膜界面的有力工具。
