BioID screening of biotinylation sites using the avidin-like protein Tamavidin 2-REV identifies global interactors of stimulator of interferon genes (STING)

Kou Motani,Hidetaka Kosako

doi:10.1074/jbc.ra120.014323

Abstract

Stimulator of interferon genes (STING) mediates cytosolic DNA-induced innate immune signaling via membrane trafficking. The global identification of proteins that spatiotemporally interact with STING will provide a better understanding of its trafficking mechanisms and of STING signaling pathways. Proximity-dependent biotin identification (BioID) is a powerful technology to identify physiologically relevant protein-protein interactions in living cells. However, biotinylated peptides are rarely detected in the conventional BioID method, which uses streptavidin beads to pull down biotinylated proteins, because the biotin-streptavidin interaction is too strong. As a result, only nonbiotinylated peptides are identified, which cannot be distinguished from peptides of nonspecifically pull-downed proteins. Here, we developed a simple method to efficiently and specifically enrich biotinylated peptides using Tamavidin 2-REV, an engineered avidin-like protein with reversible biotin-binding capability. Using RAW264.7 macrophages stably expressing TurboID-fused STING, we identified and quantified >4,000 biotinylated peptides of STING-proximal proteins. Various endoplasmic reticulum-associated proteins were biotinylated in unstimulated cells, and STING activation caused biotinylation of many proteins located in the Golgi and endosomes. These proteins included those known to interact with activated STING, such as TANK-binding kinase 1 (TBK1), several palmitoyl transferases, and p62/sequestosome 1 (SQSTM1). Furthermore, interferon-induced transmembrane protein 3 (IFITM3), an endolysosome-localized antiviral protein, bound to STING at the late activation stage. These dynamic interaction profiles will provide detailed insights into STING signaling; we propose that our approach using Tamavidin 2-REV would be useful for BioID-based and other biotinylation-based peptide identification methods.

Highlights

The presence of pathogenic or self-DNA in the cytosol is a danger signal that triggers the host innate immune system and leads to the production of cyclic GMP-AMP [1,2,3,4]. cGAMP functions as a second messenger that binds to and activates the adaptor protein Stimulator of interferon genes (STING), which consists of a four-pass transmembrane domain at the N terminus and a cytosolic cGAMP-binding domain at the C terminus [5,6,7,8]
The STING interactome obtained in this study will provide new insights into the STING signaling network, and our approach using Tamavidin 2-REV would enable researchers to utilize biotin identification (BioID) and other identification methods based on protein biotinylation more and effectively
To observe TurboID-STING-mediated protein biotinylation in situ, TurboID-STING-expressing cells were treated with or without DMXAA for 1 h in the presence of biotin, fixed, and stained with fluorescent streptavidin. Both V5-tagged TurboID-STING and biotinylated proteins exhibited a mesh-like distribution throughout the cytoplasm, which is consistent with the endoplasmic reticulum (ER) localization of STING at the steady state (Fig. 1E, left)

Summary

Introduction

The presence of pathogenic or self-DNA in the cytosol is a danger signal that triggers the host innate immune system and leads to the production of cyclic GMP-AMP (cGAMP) [1,2,3,4]. cGAMP functions as a second messenger that binds to and activates the adaptor protein STING, which consists of a four-pass transmembrane domain at the N terminus and a cytosolic cGAMP-binding domain at the C terminus [5,6,7,8]. We have improved the BioID method to efficiently and enrich biotinylated peptides by using Tamavidin 2-REV, an engineered avidin-like protein with reversible biotin-binding capability. By using immobilized Tamavidin 2-REV beads, over 4,000 biotinylated peptides were identified and quantified from cells stably expressing STING fused to TurboID [30].

Results

Conclusion