Directed evolution of TurboID for efficient proximity labeling in living cells and organisms

Abstract

Protein interaction networks and protein compartmentation underlie every signaling process and regulatory mechanism in cells. Recently, proximity labeling (PL) has emerged as a new approach to study the spatial and interaction characteristics of proteins in living cells. However, the two enzymes commonly used for PL come with tradeoffs -BioID is slow, requiring tagging times of 18-24 hours, while APEX peroxidase uses substrates that have limited cell permeability and high toxicity. To address these problems, we used yeast display-based directed evolution to engineer two mutants of biotin ligase, TurboID and miniTurbo, with much greater catalytic efficiency than BioID, and the ability to carry out PL in cells in much shorter time windows (as little as 10 minutes) with non-toxic and easily deliverable biotin. In addition to shortening PL time by 100-fold and increasing PL yield in cell culture, TurboID enabled biotin-based PL in new settings, including yeast, Drosophila, and C. elegans. Main textProximity labeling (PL) has emerged as an alternative to immunoprecipitation and biochemical fractionation for the proteomic analysis of macromolecular complexes, organelles, and protein interaction networks 1 . In PL, a promiscuous labeling enzyme is targeted by genetic fusion to a specific protein or subcellular region. Addition of a small molecule substrate, such as biotin, initiates covalent tagging of endogenous proteins within a few nanometers of the promiscuous enzyme (Figure 1a). Subsequently, the biotinylated proteins are harvested using streptavidin-coated beads and identified by mass spectrometry (MS).Two enzymes are commonly used for PL: APEX2, an engineered variant of soybean ascorbate peroxidase 2,3 , and BirA-R118G (here, referred to as "BioID"), a point mutant of E. coli biotin ligase 4,5 . The main advantage of APEX2 is its speed: proximal proteins can be tagged in 1 minute or less, enabling dynamic analysis of protein interaction networks 6,7 . However, APEX labeling requires the use of H2O2, which is toxic to cells and difficult to deliver into live organisms without causing severe tissue damage. By contrast, BioID is attractive because of the simplicity of its labeling protocol and non-toxic labeling conditions -only biotin needs to be added to initiate tagging. These attributes have resulted in over 100 applications of BioID over the past 5 years, in cultured mammalian cells 5,8,9 , plant protoplasts 10 , parasites [11][12][13][14][15][16][17][18][19] , slime mold 20,21 , and mouse 22 . BioID has been used, for example, to map the protein composition of the centrosome-cilium interface 8 and the inhibitory postsynaptic region 22 , each with nanometer spatial specificity. The major disadvantage of BioID, however, is its slow kinetics, which necessitates labeling with biotin for 18-24 hours, and sometimes much longer 22 , to accumulate sufficient quantities of biotinylated material for proteomic analysis. This precludes the use of BioID for studying dynamic processes that occur on the timescale of...

Proximity RNA labeling by APEX-Seq Reveals the Organization of Translation Initiation Complexes and Repressive RNA Granules

“…The most dramatic advances in proximity labeling involve protein biotinylation, through the use of enzymes that produce diffusible reactive intermediates --either short-lived radicals [1][2][3] , or longer lived adenylate esters [4][5][6][7][8] . These protein labeling tools can only indirectly address the organization of DNA in chromatin and RNA in various nuclear and cytosolic granules 9,10 .…”

Split-BioID — Proteomic Analysis of Context-specific Protein Complexes in Their Native Cellular Environment

“…However, although a homodimerizing protein was successfully biotinylated, whether the assay can also be used to label and identify proteins that assemble around a pair of interacting proteins remains to be demonstrated. Very recently, directed evolution was used to create TurboID and miniTurbo, two variants of BirA* with enhanced activity that allow much shorter labeling time windows, down to 10 min 26 . Adapting split-BioID to these new variants will further extend the use of this technique to a broader field of applications.…”

Emerging proteomic approaches to identify the underlying pathophysiology of neurodevelopmental and neurodegenerative disorders

“…For these reasons, BioID is generally used to identify a history of interacting proteins, while APEX can capture interacting proteins during a short period of time. A newer version of BioID, called TurboID, allows biotin labeling within a 10-min time frame, creating the opportunity to study dynamic changes in PINs [94].…”