Many biological processes are executed and regulated through the molecular interactions of proteins and nucleic acids. Proximity labeling (PL) is a technology for tagging the endogenous interaction ...partners of specific protein 'baits', via genetic fusion to promiscuous enzymes that catalyze the generation of diffusible reactive species in living cells. Tagged molecules that interact with baits can then be enriched and identified by mass spectrometry or nucleic acid sequencing. Here we review the development of PL technologies and highlight studies that have applied PL to the discovery and analysis of molecular interactions. In particular, we focus on the use of PL for mapping protein-protein, protein-RNA and protein-DNA interactions in living cells and organisms.
Tobacco etch virus protease (TEV) is one of the most widely used proteases in biotechnology because of its exquisite sequence specificity. A limitation, however, is its slow catalytic rate. We ...developed a generalizable yeast-based platform for directed evolution of protease catalytic properties. Protease activity is read out via proteolytic release of a membrane-anchored transcription factor, and we temporally regulate access to TEV's cleavage substrate using a photosensory LOV domain. By gradually decreasing light exposure time, we enriched faster variants of TEV over multiple rounds of selection. Our TEV-S153N mutant (uTEV1Δ), when incorporated into the calcium integrator FLARE, improved the signal/background ratio by 27-fold, and enabled recording of neuronal activity in culture with 60-s temporal resolution. Given the widespread use of TEV in biotechnology, both our evolved TEV mutants and the directed-evolution platform used to generate them could be beneficial across a wide range of applications.
•Proximity labeling enables proteomic interrogation of the molecular components of subcellular regions as well as protein interaction networks.•Proximity labeling has been used to uncover novel ...components of the synaptic cleft and inhibitory post-synaptic density.•Different strategies have been developed to delivery small molecule probes for proximity labeling in vivo.•Proximity labeling, in combination with high-resolution imaging and genetic analysis, will advance our understanding key molecules and pathways in neurobiology.
Understanding signaling pathways in neuroscience requires high-resolution maps of the underlying protein networks. Proximity-dependent biotinylation with engineered enzymes, in combination with mass spectrometry-based quantitative proteomics, has emerged as a powerful method to dissect molecular interactions and the localizations of endogenous proteins. Recent applications to neuroscience have provided insights into the composition of sub-synaptic structures, including the synaptic cleft and inhibitory post-synaptic density. Here we compare the different enzymes and small-molecule probes for proximity labeling in the context of cultured neurons and tissue, review existing studies, and provide technical suggestions for the in vivo application of proximity labeling.
We introduce APEX-seq, a method for RNA sequencing based on direct proximity labeling of RNA using the peroxidase enzyme APEX2. APEX-seq in nine distinct subcellular locales produced a ...nanometer-resolution spatial map of the human transcriptome as a resource, revealing extensive patterns of localization for diverse RNA classes and transcript isoforms. We uncover a radial organization of the nuclear transcriptome, which is gated at the inner surface of the nuclear pore for cytoplasmic export of processed transcripts. We identify two distinct pathways of messenger RNA localization to mitochondria, each associated with specific sets of transcripts for building complementary macromolecular machines within the organelle. APEX-seq should be widely applicable to many systems, enabling comprehensive investigations of the spatial transcriptome.
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•A transcriptome-wide subcellular RNA atlas was generated by proximity labeling•Isoform-level subcellular localization patterns for over 3,200 genes identified•RNA-transcript location correlates with genome architecture and protein localization•Two modes of mRNA localization to the outer mitochondrial membrane uncovered
A newly developed technique reveals the subcellular transcriptomes at many landmarks in the nucleus and cytosol and connects mRNA localization to genome architecture, protein location, and local-translation mechanisms.
Obtaining complete protein inventories for subcellular regions is a challenge that often limits our understanding of cellular function, especially for regions that are impossible to purify and are ...therefore inaccessible to traditional proteomic analysis. We recently developed a method to map proteomes in living cells with an engineered peroxidase (APEX) that bypasses the need for organellar purification when applied to membrane-bound compartments; however, it was insufficiently specific when applied to unbounded regions that allow APEX-generated radicals to escape. Here, we combine APEX technology with a SILAC-based ratiometric tagging strategy to substantially reduce unwanted background and achieve nanometer spatial resolution. This is applied to map the proteome of the mitochondrial intermembrane space (IMS), which can freely exchange small molecules with the cytosol. Our IMS proteome of 127 proteins has >94% specificity and includes nine newly discovered mitochondrial proteins. This approach will enable scientists to map proteomes of cellular regions that were previously inaccessible.
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•Ratiometric tagging with an engineered peroxidase gives nanometer spatial resolution•Mitochondrial intermembrane space proteome mapped with >94% specificity•Nine newly discovered mitochondrial proteins confirmed by imaging and western blotting•Data support MICU1 and MICU2 localization in the mitochondrial intermembrane space
APEX is an engineered ascorbate peroxidase that can tag endogenous proteins for identification by mass spectrometry. Here, Hung et al. improve spatial specificity of APEX and analyze protein composition of the mitochondrial intermembrane space (IMS). The resulting IMS proteome of 127 proteins has >94% mitochondrial specificity and includes 9 newly discovered, validated mitochondrial proteins.
RNA–protein interactions underlie a wide range of cellular processes. Improved methods are needed to systematically map RNA–protein interactions in living cells in an unbiased manner. We used two ...approaches to target the engineered peroxidase APEX2 to specific cellular RNAs for RNA-centered proximity biotinylation of protein interaction partners. Both an MS2-MCP system and an engineered CRISPR-Cas13 system were used to deliver APEX2 to the human telomerase RNA hTR with high specificity. One-minute proximity biotinylation captured candidate binding partners for hTR, including more than a dozen proteins not previously linked to hTR. We validated the interaction between hTR and the N⁶-methyladenosine (m⁶A) demethylase ALKBH5 and showed that ALKBH5 is able to erase the m⁶A modification on endogenous hTR. ALKBH5 also modulates telomerase complex assembly and activity. MS2- and Cas13-targeted APEX2 may facilitate the discovery of novel RNA–protein interactions in living cells.
APEX is an engineered peroxidase that functions as an electron microscopy tag and a promiscuous labeling enzyme for live-cell proteomics. Because limited sensitivity precludes applications requiring ...low APEX expression, we used yeast-display evolution to improve its catalytic efficiency. APEX2 is far more active in cells, enabling the use of electron microscopy to resolve the submitochondrial localization of calcium uptake regulatory protein MICU1. APEX2 also permits superior enrichment of endogenous mitochondrial and endoplasmic reticulum membrane proteins.
Cells operate through protein interaction networks organized in space and time. Here, we describe an approach to resolve both dimensions simultaneously by using proximity labeling mediated by ...engineered ascorbic acid peroxidase (APEX). APEX has been used to capture entire organelle proteomes with high temporal resolution, but its breadth of labeling is generally thought to preclude the higher spatial resolution necessary to interrogate specific protein networks. We provide a solution to this problem by combining quantitative proteomics with a system of spatial references. As proof of principle, we apply this approach to interrogate proteins engaged by G-protein-coupled receptors as they dynamically signal and traffic in response to ligand-induced activation. The method resolves known binding partners, as well as previously unidentified network components. Validating its utility as a discovery pipeline, we establish that two of these proteins promote ubiquitin-linked receptor downregulation after prolonged activation.
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•Method to spatially identify protein interaction networks in living cells•Quantitative proteomics applied to “deconvolve” APEX proximity labeling data•Ligand-stimulated remodeling of GPCR networks captured with sub-minute resolution•Previously unknown GPCR-linked proteins identified and functionally validated
Proximity labeling coupled with quantitative proteomics captures location and timing of GPCR function in live cells.
Bring your own copper: Copper‐chelating azides undergo much faster click reactions (CuAAC) than nonchelating azides under a variety of biocompatible conditions. This kinetic enhancement allows ...site‐specific protein labeling to be performed on the surface of living cells with only 10–40 μM CuI/CuII (see scheme). Detection sensitivity was also increased for CuAAC detection of alkyne‐modified proteins and RNA.
Microscopy and mass spectrometry (MS) are complementary techniques: The former provides spatiotemporal information in living cells, but only for a handful of recombinant proteins at a time, whereas ...the latter can detect thousands of endogenous proteins simultaneously, but only in lysed samples. Here, we introduce technology that combines these strengths by offering spatially and temporally resolved proteomic maps of endogenous proteins within living cells. Our method relies on a genetically targetable peroxidase enzyme that biotinylates nearby proteins, which are subsequently purified and identified by MS. We used this approach to identify 495 proteins within the human mitochondrial matrix, including 31 not previously linked to mitochondria. The labeling was exceptionally specific and distinguished between inner membrane proteins facing the matrix versus the intermembrane space (IMS). Several proteins previously thought to reside in the IMS or outer membrane, including protoporphyrinogen oxidase, were reassigned to the matrix by our proteomic data and confirmed by electron microscopy. The specificity of peroxidase-mediated proteomic mapping in live cells, combined with its ease of use, offers biologists a powerful tool for understanding the molecular composition of living cells.
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