The development of proximity labeling technology and its applications in mammals, plants, and microorganisms

Abstract

Protein‒protein, protein‒RNA, and protein‒DNA interaction networks form the basis of cellular regulation and signal transduction, making it crucial to explore these interaction networks to understand complex biological processes. Traditional methods such as affinity purification and yeast two-hybrid assays have been shown to have limitations, as they can only isolate high-affinity molecular interactions under nonphysiological conditions or in vitro. Moreover, these methods have shortcomings for organelle isolation and protein subcellular localization. To address these issues, proximity labeling techniques have been developed. This technology not only overcomes the limitations of traditional methods but also offers unique advantages in studying protein spatial characteristics and molecular interactions within living cells. Currently, this technique not only is indispensable in research on mammalian nucleoprotein interactions but also provides a reliable approach for studying nonmammalian cells, such as plants, parasites and viruses. Given these advantages, this article provides a detailed introduction to the principles of proximity labeling techniques and the development of labeling enzymes. The focus is on summarizing the recent applications of TurboID and miniTurbo in mammals, plants, and microorganisms.

ACKR3 Proximity Labeling Identifies Novel G protein- and β-arrestin-independent GPCR Interacting Proteins

“…An inherent limitation in proximity labeling is the identification of biologically inconsequential proteins due to non-specific binding of proteins during purification, bystander effects, and endogenous biotinylation of proteins, among others ( 56–58 ). To more confidently identify biologically meaningful GIPs, we selected three other GPCR-based proximity labeling datasets performed in HEK293 cells for further analyses ( Figure 2D) .…”

Signaling mechanisms underlying activity‐dependent integration of adult‐born neurons in the mouse olfactory bulb

“…Coupling terminal‐specific stimulation, MERFISH (Chen, Boettiger, et al, 2015), and immunohistochemistry with super‐resolution imaging techniques such as expansion microscopy (Chen, Tillberg, & Boyden, 2015) and/or STORM (Rust et al, 2006) also promises to provide a deeper insight into the input‐specific plasticity contributed by such mechanisms. Finally, in vivo proximity‐based proteomics through methods such as BioID and TurboID can reveal dynamic protein interactomes with spatiotemporal specificity (Guo et al, 2023; Xu et al, 2021), which may also provide needed clues to novel protein interactions within spines or synapses.…”

Dynamic changes in the proximitome of neutral sphingomyelinase-2 (nSMase2) in TNFα stimulated Jurkat cells

“…However, these approaches have limitations in capturing rapid changes of weak and transient interactions, e.g., during the first few minutes of TNFα exposure. To overcome these challenges, proximity-dependent labeling techniques in living cells, coupled with affinity purification, have emerged as an excellent tool to capture and resolve protein networks ( 29 – 32 ). If genetically tagged with the engineered peroxidase APEX2, proteins of interest and their proximitome can be rapidly biotinylated in living cells pre-loaded with biotin-phenol (BP) upon adding hydrogen peroxide (H 2 O 2 ).…”