Context-Specific and Proximity-Dependent Labeling for the Proteomic Analysis of Spatiotemporally Defined Protein Complex
Proximity-dependent labeling techniques such as BioID and APEX2 allow the biotinylation of proteins proximal to a protein of interest in living cells. Following streptavidin pulldown and mass spectrometry analysis, this enables the identification of nativ
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Introduction In all living organisms, thousands of proteins mediate most cellular functions. To do so, proteins usually do not act alone. Rather, they assemble with other proteins to build dynamic macromolecular complexes that can remodel according to the exact functions that need to be exerted. The protein–protein interactions (PPI) involved in such complexes are often deregulated in disease and are thus promising targets for therapeutics [1]. Identifying and characterizing PPI networks are hence of prime importance when trying to understand how a protein of interest (POI) works. To complement classical pulldown approaches in which the POI is isolated by affinity purification (AP) and co-purifying proteins identified by mass spectrometry (MS) analysis, proximitydependent biotinylation techniques were recently introduced that allow the labeling of proteins vicinal to the POI in living cells. Two such techniques are currently available: APEX2- and BioID-mediated labeling [2, 3]. The former relies on an engineered peroxidase that activates a biotin-phenol substrate that is
Arnaud Poterszman (ed.), Multiprotein Complexes: Methods and Protocols, Methods in Molecular Biology, vol. 2247, https://doi.org/10.1007/978-1-0716-1126-5_17, © Springer Science+Business Media, LLC, part of Springer Nature 2021
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fed to the cells, whereas the latter uses an abortive variant of a protein biotin ligase (BPL) that activates native biotin. In both the cases, the activated substrate diffuses around the enzyme and leads to the biotinylation of proximal proteins in an estimated range of approximately 10 nm for BioID [4]. When fused to a POI, both enzymes thus mediate the biotinylation of nearby factors including interacting proteins. These biotinylated proteins can then be efficiently isolated by streptavidin pulldown and identified by MS. As opposed to AP-MS, APEX2- and BioIDMS do not aim at purifying assembled protein complexes by pulling one of its components but rather identify proteins that were marked within cells because they were in close proximity to the POI. In proximity-dependent labeling approaches, it thus does not matter whether interacting proteins are still associated during the pulldown procedure, making these techniques more powerful than AP-MS for detecting transient interactions [5] or PPI that depend on intact or poorly soluble cellular structures [6]. Yet, a limitation to both approaches is that they cannot easily resolve the remodeling and/or maturation of protein complexes. In the common situation in which a POI is part of several distinct complexes depending on the cellular context, both AP- and APEX2/BioID-MS will identify all possible PPI but will not assign individual interactions to specific context-dependent complexes. We and others have recently introduced split-BioID assays [7, 8] in which BirA*, the BPL of BioID, is split into two inactive and poorly interacting fragments that can reassemble into an active enzyme when fused with two interacting proteins. Provided a PO
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