Proximity Labeling of the Chlamydia trachomatis Inclusion Membrane.
Proximity Labeling of the Chlamydia trachomatis Inclusion Membrane.
复制标题
沙眼衣原体包涵体膜的邻近标记。
DOI：
10.1007/978-1-4939-9694-0_17
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发表时间：
2019
期刊：
影响因子：
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通讯作者：
Rucks,ElizabethA
中科院分区：
文献类型：
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作者：
Olson,MacyG;Jorgenson,LisaM;Widner,RayE;Rucks,ElizabethA
在研究存在于膜结合空泡内的细胞内细菌时，存在许多与原核或真核跨膜或膜相关蛋白如何在这些含病原体的空泡的膜内组织和功能有关的问题。然而，这种宿主-病原体相互作用界面已被证明难以通过实验解决。例如，开始理解蛋白质功能的一种方法是确定蛋白质结合配偶体;然而，使用标准免疫沉淀或免疫共沉淀技术检查疏水跨膜蛋白的蛋白质-蛋白质相互作用并不广泛成功。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膜蛋白不相容。在本章中，我们概述了两个邻近标记系统，以规避这些问题，研究（1）真核蛋白，本地化的膜结合包涵体形成的沙眼衣原体使用BioID，（2）衣原体蛋白插入到包涵体膜使用APEX 2。BioID是一种混杂的生物素连接酶，用于用生物素标记近端蛋白质。APEX 2是一种抗坏血酸过氧化物酶，可产生生物素-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，用于使用透射电子显微镜检查APEX 2标记亚细胞结构。我们介绍了这些方法最初是如何概念化和开发的，以便用户可以了解每个邻近标记系统的优势和局限性。我们讨论了实验设计的重要考虑因素，其中包括仔细考虑背景条件和质谱结果的统计分析。当应用在适当的背景下，有足够的控制，这些方法可以是强大的工具，了解细胞内病原体和它们的主机之间的膜界面。
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 byChlamydia trachomatisusing 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.