Proximity Labeling of the Chlamydia trachomatis Inclusion Membrane

Macy G. Olson, Lisa M. Jorgenson, Ray E. Widner, Elizabeth A. Rucks

Research output: Chapter in Book/Report/Conference proceedingChapter

1 Scopus citations

Abstract

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.

Original languageEnglish (US)
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages245-278
Number of pages34
DOIs
StatePublished - 2019

Publication series

NameMethods in Molecular Biology
Volume2042
ISSN (Print)1064-3745
ISSN (Electronic)1940-6029

Keywords

  • APEX2
  • BioID
  • Chlamydia trachomatis
  • Host–pathogen interactions
  • Proximity labeling

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

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    Olson, M. G., Jorgenson, L. M., Widner, R. E., & Rucks, E. A. (2019). Proximity Labeling of the Chlamydia trachomatis Inclusion Membrane. In Methods in Molecular Biology (pp. 245-278). (Methods in Molecular Biology; Vol. 2042). Humana Press Inc.. https://doi.org/10.1007/978-1-4939-9694-0_17