Proximity labeling in mammalian cells with TurboID and split-TurboID

Cho, Kelvin F.; Branon, Tess C; Udeshi, Namrata D.; Myers, Samuel A.; Carr, Steven A.; Ting, Alice Y.

doi:10.1038/s41596-020-0399-0

Cited by 196 publications

(197 citation statements)

References 112 publications

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“…Cells were transiently transfected using Lipofectamine 3000 (Thermo Fisher Scientific) according to manufacturer’s instructions. Biotinylation experiments using TurboID-PopTag condensates were performed as described in detail 64 . Cells grown on coverslips were fixed for 24 h after transfection in 4 % formaldehyde in PBS.…”

Section: Methodsmentioning

confidence: 99%

A modular platform for engineering function of natural and synthetic biomolecular condensates

Lasker

Boeynaems

et al. 2021

Preprint

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Phase separation is emerging as a universal principle for how cells use dynamic subcompartmentalization to organize biochemical reactions in time and space. Yet, whether the emergent physical properties of these biomolecular condensates are important for their biological function remains unclear. The intrinsically disordered protein PopZ forms membraneless condensates at the poles of the bacterium Caulobacter crescentus and selectively sequesters kinase-signaling cascades to regulate asymmetric cell division. By dissecting the molecular grammar underlying PopZ phase separation, we find that unlike many eukaryotic examples, where unstructured regions drive condensation, a structured domain of PopZ drives condensation, while conserved repulsive features of the disordered region modulate material properties. By generating rationally designed PopZ mutants, we find that the exact material properties of PopZ condensates directly determine cellular fitness, providing direct evidence for the physiological importance of the emergent properties of biomolecular condensates. Our work codifies a clear set of design principles illuminating how sequence variation in a disordered domain alters the function of a widely conserved bacterial condensate. We used these insights to repurpose PopZ as a modular platform for generating synthetic condensates of tunable function in human cells.

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Section: Methodsmentioning

confidence: 99%

A modular platform for engineering function of natural and synthetic biomolecular condensates

Lasker

Boeynaems

et al. 2021

Preprint

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“…Additionally, using an original TurboID-based time course labeling approach [34], we showed the accumulation of CPMPs overtime suggesting the involvement of CLUH in a dynamic biological process. The analysis of the two time points TurboID data allowed us to discriminate between stable and transient CLUH interactants by taking advantage of the variation of enrichment overtime that is inversely correlated to the residency time near CLUH.…”

Section: Discussionmentioning

confidence: 90%

“…TurboID proximity labeling is based on previously described protocol [34]. Briefly, HCT116 stable cell line expressing TurboID-CLUH and TurboID-GFP were grown for 30 minutes and 16 hours in presence of 50 µM of biotin (Sigma Aldrich).…”

Section: Turboid Proximity Labelingmentioning

confidence: 99%

CLUH interactome reveals an association to SPAG5 and a proximity to the translation of mitochondrial protein

Hemono

Haller

Chicher

et al. 2021

Preprint

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Mitochondria require thousands of proteins to fulfil their essential function in energy production and other fundamental biological processes. These proteins are mostly encoded by the nuclear genome, translated in the cytoplasm before being imported into the organelle. RNA binding proteins (RBPs) are central players in the regulation of this process by affecting mRNA translation, stability or localization. CLUH is an RBP recognizing specifically mRNAs coding for mitochondrial proteins, but its precise molecular function and interacting partners remain undiscovered in mammals. Here we reveal for the first time CLUH interactome in mammalian cells. Using both co-IP and BioID proximity-labeling approaches, we identify novel molecular partners interacting stably or transiently with CLUH in HCT116 cells and mouse embryonic stem cells. We reveal a stable RNA-independent interaction of CLUH with itself and with SPAG5 in cytosolic granular structures. More importantly, we uncover an unexpected proximity of CLUH to mitochondrial proteins and their cognate mRNAs in the cytosol. Additionally, our data highlight the importance of CLUH TPR domain for its interactions with both proteins and mRNAs. Overall, through the analysis of CLUH interactome, our study sheds a new light on CLUH molecular function by highlighting its association to the translation and subcellular localization of some mRNAs coding for mitochondrial proteins.

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“…After confirmation of biotinylation of proteins and V5 expression by Western blot, biotin-tagged proteins were enriched using a previously published protocol 19 with slight modifications. Briefly, for each sample, 83 μ L of streptavidin magnetic beads (Thermo, 88817) in a 1.5 mL Eppendorf LoBind tube were washed twice with 1 mL RIPA lysis buffer (50 mM Tris, 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% Triton X-100) on rotation for 2 min.…”

Section: Biotinylated Protein Enrichmentmentioning

confidence: 99%

“…This allows for the investigation of dynamic biological processes with greater proteomic breadth. In vivo applications of TurboID have been limited to investigating protein-protein interactions by fusing TurboID to the protein of interest or obtaining peripheral cell type-specific secretome profiles 19 . In the context of the brain, the proteomic profiles of astrocyte-synapse interactions were characterized in the mouse brain by introducing TurboID via adeno-associated viruses (AAV) 20 , but brain cell type-specific proteomics using TurboID has been limited by lack of a suitable mouse model.…”

Section: Introductionmentioning

confidence: 99%

Cell type-specific biotin labeling in vivo resolves regional neuronal proteomic differences in mouse brain

Rayaprolu

Bitarafan

Betarbet

et al. 2021

Preprint

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Isolation and proteomic profiling of brain cell types, particularly neurons, pose several technical challenges which limit our ability to resolve distinct cellular phenotypes in neurological diseases. Therefore, we generated a novel mouse line that enables cell type-specific expression of a biotin ligase, TurboID, via Cre-lox strategy for in vivo proximity-dependent biotinylation of proteins. Using adenoviral-based and transgenic approaches, we show striking protein biotinylation in neuronal cell bodies and axons throughout the mouse brain. We quantified more than 2,000 neuron-derived proteins following enrichment that mapped to numerous subcellular compartments. Synaptic, transmembrane transporters, ion channel subunits, and disease-relevant druggable targets were among the most significantly enriched proteins. Remarkably, we resolved brain region-specific proteomic profiles of Camk2a neurons with distinct functional molecular signatures and disease associations that may underlie regional neuronal vulnerability. Leveraging the neuronal specificity of this in vivo biotinylation strategy, we used an antibody-based approach to uncover regionally unique patterns of neuron-derived signaling phospho-proteins and cytokines, particularly in the cortex and cerebellum. Our work provides a proteomic framework to investigate cell type-specific mechanisms driving physiological and pathological states of the brain as well as complex tissues beyond the brain.

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Proximity labeling in mammalian cells with TurboID and split-TurboID

Cited by 196 publications

References 112 publications

A modular platform for engineering function of natural and synthetic biomolecular condensates

A modular platform for engineering function of natural and synthetic biomolecular condensates

CLUH interactome reveals an association to SPAG5 and a proximity to the translation of mitochondrial protein

Cell type-specific biotin labeling in vivo resolves regional neuronal proteomic differences in mouse brain

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