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. Video Abstract.

Keywords: APEX; BioID; Proximity labeling; TurboID; miniTurbo.

Fig. 1

Workflow of APEX and split-APEX. APEX targets organelles or specific protein complexes within cells. After treating living cells with biotin-phenol under H₂O₂ conditions for only 1 minute, APEX catalyses the one-electron oxidation of biotin-phenol to form biotin-phenoxy radicals. Split-APEX is divided into two parts, AP and EX, Each fragment has no activity on its own, but when recombined during molecular interactions, peroxidase activity is restored. APEX and split-APEX catalyze the single-electron oxidation of biotin-phenol to form biotin-phenoxy radicals by treating live cells with biotin-phenol for 1 min under hydrogen peroxide conditions

Fig. 2

Workflow of APEX-RIP, Proximity-CLIP and APEX-seq. APEX-RIP: Cells expressing APEX2 were treated with biotin-phenol and hydrogen peroxide to biotinylate proximal endogenous proteins. The biotinylated proteins were then cross-linked to nearby RNA using 0.1% formaldehyde. After cell lysis, the biotinylated nucleic acids were enriched by streptavidin and finally analyzed by RNA-Seq. Proximity-CLIP: RNA was first labelled with 4SU and APEX2 was fused to the localisation element (targeting to the compartment of interest), followed by treatment of cells with biotin-phenol and hydrogen peroxide to biotinylate the proximal protein. Protein-RNA cross-linking was then achieved using UV light (λ > 312 nm). Purified protein-RNA complexes after cell lysis can be used for protein mass spectrometry and RNA-Seq analysis. APEX-seq: APXE2 was first localised in the cytoplasm, cytoplasmic face of the endoplasmic reticulum membrane or nucleus, followed by initiation of APEX2 to biotinylate the proximate RNA. After affinity purification of the above RNA by streptavidin peroxidase, RNA sequencing analysis proceeded

Fig. 3

Workflow of BioID and TurboID. BioID is a humanized version of the BirA protein from E. coli with a R118G mutation, TurboID is a directed-evolution variant of BioID. Here we mark them as BirA*. When BirA* is fused to a target protein and expressed in cells, exogenously added free biotin can be converted to highly reactive biotin-5'-AMP. Within the labeling radius, proteins that either directly or indirectly interact with the fusion protein will be labeled. Subsequent selective isolation and identification of these biotinylated proximal endogenous proteins using biotin affinity purification. Finally, the resulting proteins are identified and analyzed by mass spectrometry

Fig. 4

Workflow of Split-TurboID. Split-TurboID was applied to the FRB-FKBP dimer system, and after treatment with rapamycin, the two inactive fragments of TurboID recombined to form an active enzyme that produced biotin-5’-AMP