Spatiotemporal profiling of cytosolic signaling complexes in living cells by selective proximity proteomics

Mi Ke, Xiao Yuan, An He, Peiyuan Yu, Wendong Chen, Yu Shi, Tony Hunter, Peng Zou and Ruijun Tian ()
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Mi Ke: Southern University of Science and Technology
Xiao Yuan: Southern University of Science and Technology
An He: Southern University of Science and Technology
Peiyuan Yu: Southern University of Science and Technology
Wendong Chen: Southern University of Science and Technology
Yu Shi: Molecular and Cell Biology Laboratory, Salk Institute for Biological Studies
Tony Hunter: Molecular and Cell Biology Laboratory, Salk Institute for Biological Studies
Peng Zou: Peking University
Ruijun Tian: Southern University of Science and Technology

Nature Communications, 2021, vol. 12, issue 1, 1-13

Abstract: Abstract Signaling complexes are often organized in a spatiotemporal manner and on a minute timescale. Proximity labeling based on engineered ascorbate peroxidase APEX2 pioneered in situ capture of spatiotemporal membrane protein complexes in living cells, but its application to cytosolic proteins remains limited due to the high labeling background. Here, we develop proximity labeling probes with increased labeling selectivity. These probes, in combination with label-free quantitative proteomics, allow exploring cytosolic protein assemblies such as phosphotyrosine-mediated protein complexes formed in response to minute-scale EGF stimulation. As proof-of-concept, we systematically profile the spatiotemporal interactome of the EGFR signaling component STS1. For STS1 core complexes, our proximity proteomics approach shows comparable performance to affinity purification-mass spectrometry-based temporal interactome profiling, while also capturing additional—especially endosomally-located—protein complexes. In summary, we provide a generic approach for exploring the interactome of mobile cytosolic proteins in living cells at a temporal resolution of minutes.

Date: 2021
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DOI: 10.1038/s41467-020-20367-x

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