VivosX, a disulfide crosslinking method to capture site-specific, protein-protein interactions in yeast and human cells
- Stony Brook University, United States
- University of Minnesota Medical School, United States
Abstract
VivosX is an in vivo disulfide crosslinking approach that utilizes a pair of strategically positioned cysteines on two proteins to probe physical interactions within cells. Histone H2A.Z, which often replaces one or both copies of H2A in nucleosomes downstream of promoters, was used to validate VivosX. Disulfide crosslinks between cysteine-modified H2A.Z and/or H2A histones within nucleosomes were induced using a membrane-permeable oxidant. VivosX detected different combinations of H2A.Z and H2A within nucleosomes in yeast cells. This assay correctly reported the change in global H2A.Z occupancy previously observed when the deposition and eviction pathways of H2A.Z were perturbed. Homotypic H2A.Z/H2A.Z (ZZ) nucleosomes accumulated when assembly of the transcription preinitiation complex was blocked, revealing that the transcription machinery preferentially disassembles ZZ nucleosomes. VivosX works in human cells and distinguishes ZZ nucleosomes with one or two ubiquitin moieties, demonstrating that it can be used to detect protein-protein interactions inside cells from different species.
Data availability
Source data files of western blotting quantification are included as Figure Supplements
Article and author information
Author details
Funding
National Institute of General Medical Sciences (RO1 GM104111)
- Ed Luk
National Cancer Institute (RO1 CA200652)
- Cheuk T Leung
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
© 2018, Mohan et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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VivosX, a disulfide crosslinking method to capture site-specific, protein-protein interactions in yeast and human cells
eLife 7:e36654.
https://doi.org/10.7554/eLife.36654
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