Protein Neighbors and Proximity Proteomics

Abstract

Within cells, proteins can co-assemble into functionally integrated and spatially restricted multicomponent complexes. Often, the affinities between individual proteins are relatively weak, and proteins within such clusters may interact only indirectly with many of their other protein neighbors. This makes proteomic characterization difficult using methods such as immunoprecipitation or cross-linking. Recently, several groups have described the use of enzyme-catalyzed proximity labeling reagents that covalently tag the neighbors of a targeted protein with a small molecule such as fluorescein or biotin. The modified proteins can then be isolated by standard pulldown methods and identified by mass spectrometry. Here we will describe the techniques as well as their similarities and differences. We discuss their applications both to study protein assemblies and to provide a new way for characterizing organelle proteomes. We stress the importance of proteomic quantitation and independent target validation in such experiments. Furthermore, we suggest that there are biophysical and cell-biological principles that dictate the appropriateness of enzyme-catalyzed proximity labeling methods to address particular biological questions of interest.

Fig. 1.

An example to illustrate the crowded nature of the cell. Here, two adjacent vascular endothelial cells are shown connected via an adherens junction (AJ) between the opposing plasma membranes (PM) of the two cells. The adherens junction contains localized clusters of cell adhesion proteins, mainly cadherins and catenins (green). Note that other plasma membrane proteins such as vascular endothelial growth factor receptors (yellow) are excluded from the clusters. Extracellular blood plasma proteins are shown in tan (upper left). Cytoplasmic proteins are shown in turquoise, and nuclear proteins are shown in purple and blue (right). The image also displays common features of the eukaryotic cell, including ribosomes (R), cytoskeletal cables (C), the nuclear pore complex (NPC), the nuclear lamina (NL), the nuclear envelope (NE), and the ER lumen/perinuclear space (ERL/PS). The macromolecules are shown at their approximate true densities and molecular dimensions. For more details about the biological context of this image, see Span et al. (73) (used with permission of David S. Goodsell, the Scripps Research Institute).

Fig. 2.

Summarized outline of the major published enzyme-catalyzed proximity labeling assays. A, in BioID (20), a bait protein fused to a promiscuous biotin protein ligase (blue) is expressed in cells. Biotin is added to initiate biotinylation of closely associated proteins (gray). B, EMARS (17). In this example, HRP-coupled cholera toxin (tan) is added to cells and binds lipid raft-associated gangliosides in the plasma membrane (blue). Fluorescein- or biotin-conjugated aryl azide with hydrogen peroxide initiates labeling of neighboring proteins (gray). C, SPPLAT (19). HRP-coupled antibody (tan) to a plasma membrane target protein (blue) is added to cells. Biotin-tyramide with hydrogen peroxide initiates labeling of neighboring proteins (gray). D, use of APEX for organelle-specific labeling. Here, APEX (tan) has been engineered to be selectively expressed in the mitochondrial matrix as described (18). Cells are briefly incubated with a biotin-tyramide derivative (biotin-phenol) with hydrogen peroxide to initiate labeling of matrix-associated proteins (gray). In all cases, cells are lysed, and labeled proteins are isolated by affinity pulldown using immobilized streptavidin (for biotinylated proteins) or anti-fluorescein antibody (for fluorescein-tagged proteins). Samples are analyzed by mass spectrometry.