Filling the Void: Proximity-Based Labeling of Proteins in Living Cells

Abstract

There are inherent limitations with traditional methods to study protein behavior or to determine the constituency of proteins in discrete subcellular compartments. In response to these limitations, several methods have recently been developed that use proximity-dependent labeling. By fusing proteins to enzymes that generate reactive molecules, most commonly biotin, proximate proteins are covalently labeled to enable their isolation and identification. In this review we describe current methods for proximity-dependent labeling in living cells and discuss their applications and future use in the study of protein behavior.

Keywords: APEX; BioID; protein–protein interactions; proteomics; proximity-dependent labeling; subcellular proteome.

Figure 1. Key Figure: Proximity-dependent protein biotinylation to study protein behavior and subcellular proteomics in live cells

There are a multiple methods that use proximity-dependent protein labeling to study protein behavior and constituency in living cells. (A) Binary candidate methods utilize fusion of a labeling enzyme or an acceptor motif to bait and prey proteins to assess protein interactions. (B) HRP-based methods are used at the cell surface to label proteins associated with a bait protein. (C) With APEX-methods a peroxidase is fused to a targeting motif (TM) or protein and used to label proximate to map the proteome of discrete subcellular compartments. (D) Using a promiscuous biotin ligase fused to a bait protein BioID-methods generate a history of protein association over time to screen for candidate protein-protein interactions or the constituency of subcellular structures.

Figure 2. Proximity-dependent protein labeling can be used to detect candidate binary interactions

The biotin ligase BirA is fused to a bait protein and a biotin acceptor peptide (BAP) is fused to a candidate prey protein. Upon cellular expression of both proteins, biotinylation (denoted by orange circles with B) of the BAP-fused prey protein indicates their proximity and suggests that an interaction between bait and prey has occurred. This approach can be used (A) within a cell or (B) between two different cells.

Figure 3. Using BioID to generate a history of PPIs or to identify constituents of subcellular domains

Cellular expression of a protein of interest (bait) fused to a promiscuous biotin ligase called BioID enables biotinylation (denoted by orange circles with B) of proximate proteins within approximately 10nm (light orange disc). This protein labeling is initiated by the addition of a supraphysiological concentration of biotin and requires many hours to generate substantial labeling. Following cell lysis and protein denaturation, biotinylated proteins are captured by affinity purification for identification by mass spectrometry to reveal a history of protein associations that occurred during the labeling period.

Figure 4. Proximity dependent labeling on the cell surface with HRP

Horseradish peroxidase is not active with cells but when it is either (A) expressed on the cell surface as a fusion protein or (B) targeted to the cell surface by fusion with an antibody it can be used to label proximate proteins. By providing reactive molecules, such as arylazides or tyramines and H₂O₂, conjugated to biotin or fluorescein, the HRP generates a reactive molecule that covalently labels with proximate proteins within 300 nm. The biotin or fluorescein label enables protein identification. Methods that use this approach, including EMARS and SPPLAT, can be used to identify proximate proteins on the cell surface.

Figure 5. Mapping the proteome of subcellular compartments with APEX

An enzyme that promiscuously labels proteins, such as the mutant peroxidase used for APEX, is specifically targeted to a distinct subcellular compartment by fusion of a strong targeting motif (TM). The motif could be a minimal sequence needed for targeting or a full-length protein that is mobile within the compartment. Following incubation with biotin-phenol and a pulse of H₂O₂, the APEX enzyme rapidly produces a reactive biotin that covalently biotinylates (denoted by orange circles with B) proteins constituents within the compartment enabling their selective isolation and identification by mass spectrometry.