A Proximity Labeling Strategy Provides Insights into the Composition and Dynamics of Lipid Droplet Proteomes

Bersuker, Kirill; Peterson, Clark W.H.; To, Milton; Sahl, Steffen J.; Savikhin, Victoria; Grossman, Ellie; Nomura, Daniel K.; Olzmann, James A.

doi:10.1016/j.devcel.2017.11.020

Cited by 263 publications

(321 citation statements)

References 64 publications

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“…Subsequently, biotinylated proteins are enriched with streptavidin and identified by mass spectrometry. The high spatial specificity of this approach has enabled APEX to be used not only for mapping organelle proteomes (Han et al, 2017;Hung et al, 2017;Hung et al, 2016;Hung et al, 2014;Lam et al, 2014;Loh et al, 2016;Rhee et al, 2013) but also protein interaction networks in living cells (Bersuker et al, 2018;Gupta et al, 2018;Lobingier et al, 2017;Markmiller et al, 2018;Paek et al, 2017).…”

Section: Development Of Apex-seq Methodologymentioning

confidence: 99%

Atlas of Subcellular RNA Localization Revealed by APEX-seq

Fazal

Han

Kaewsapsak

et al. 2018

Preprint

112

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We introduce APEX-seq, a method for RNA sequencing based on spatial proximity to the peroxidase enzyme APEX2. APEX-seq in nine distinct subcellular locales produced a nanometer-resolution spatial map of the human transcriptome, revealing extensive and exquisite 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.(ERM), which we previously attempted but failed to map using APEX2-ERM and APEX-RIP (Kaewsapsak et al., 2017). When we performed a comparison of APEX-seq and APEX-RIP using HEK 239T cells expressing APEX on the ER membrane (facing the cytosol), we found that APEX-seq could clearly enrich secretory mRNAs on the ER membrane over cytosolic mRNAs, whereas APEX-RIP could not ( Figure 1D). Given the very close proximity between ER membrane-associated RNAs and cytosolic RNAs that are immediately adjacent to the ER, these results suggest that APEX-seq has a labeling radius of only a few nanometers. Validation of APEX-seqEncouraged by the results above, we expanded APEX-seq to nine distinct subcellular locations ( Figure 1E). Correct localization of each APEX2 fusion protein (domain structures shown in Figure S2A) in HEK-293T cells was confirmed by imaging with organelle markers ( Figure 1F). We prepared two replicates for each location ( Figure S2B and S2C, correlation coefficient r between replicates ranged from 0.97 to 1) as well as negative controls in which H2O2 was omitted from the labeling reaction. After labeling for 1 minute and cell lysis, we enriched biotinylated RNAs and prepared APEX-seq libraries with polyA+ selection. Transcripts shorter than 100 nt were excluded during purification. We used DESeq2 (Love et al., 2014) for data analysis and used a qvalue (FDR-adjusted p-value) < 0.05 and a log2fold-change > 0.75 to select for significantly enriched transcripts.Consistent with the RT-qPCR results for the mitochondrial matrix shown in Figure 1C, our sequencing-based analysis showed that all 13 mRNAs and 2 rRNAs encoded by mtDNA are strongly enriched by MITO-APEX2 labeling (Figure 2A, S2D and S2E, mean enrichment > 11fold), whereas no mRNAs encoded by the nuclear genome are enriched. Figure 2B shows good correlation (r > 0.9) between technical replicates.To assess APEX-seq in an "open" subcellular region (not enclosed by a membrane), we focused again on the ER membrane (ERM). The ERM is particularly valuable for methodology validation because there are well-established "true positives" (mRNAs encoding secreted proteins that are known to be translated at the ERM) and well-established "false positive...

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Section: Development Of Apex-seq Methodologymentioning

confidence: 99%

Atlas of Subcellular RNA Localization Revealed by APEX-seq

Fazal

Han

Kaewsapsak

et al. 2018

Preprint

112

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“…In this assay, MARCH5/MITOL (a ubiquitin E3 ligase that positively regulates membrane fission 27 ) and DNM1L served as positive controls and induced mitochondrial fragmentation as expected (Figure 3C, 3D). Expression of three other proteins, APOL2 (apolipoprotein L2), C18orf32 (a protein that traffics to lipid droplets 28 ) and CHMP7 (an ESCRT-III component) also strongly induced mitochondrial fragmentation. To evaluate the consequence of depletion of the two strongest hits on mitochondrial morphology, C18orf32 and CHMP7, we used siRNA-mediated depletion.…”

Section: Bodymentioning

confidence: 99%

A proximity-dependent biotinylation map of a human cell: an interactive web resource

Knight

Rajasekharan

et al. 2019

Preprint

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INTRODUCTIONCompartmentalization is an essential characteristic of eukaryotic cells, ensuring that cellular processes are partitioned to defined subcellular locations. High throughput microscopy 1 and biochemical fractionation coupled with mass spectrometry 2-6 have helped to define the proteomes of multiple organelles and macromolecular structures. However, many compartments have remained refractory to such methods, partly due to lysis and purification artefacts and poor subcompartment resolution. Recently developed proximity-dependent biotinylation approaches such as BioID and APEX provide an alternative avenue for defining the composition of cellular compartments in living cells (e.g. 7-10 ). Here we report an extensive BioID-based proximity map of a human cell, comprising 192 markers from 32 different compartments that identifies 35,902 unique high confidence proximity interactions and localizes 4,145 proteins expressed in HEK293 cells. The recall of our localization predictions is on par with or better than previous large-scale mass spectrometry and microscopy approaches, but with higher localization specificity. In addition to assigning compartment and subcompartment localization for many previously unlocalized proteins, our data contain finegrained localization information that, for example, allowed us to identify proteins with novel roles in mitochondrial dynamics. As a community resource, we have created humancellmap.org, a website that allows exploration of our data in detail, and aids with the analysis of BioID experiments. BODYProximity-dependent labelling approaches have rapidly grown in popularity, as they provide a robust way to label the environment in which a protein resides in living cells 7,8 . In the most widely used of these techniques, BioID, a mutant E. coli biotin ligase -BirA* (R118G) -is fused in-frame with the coding sequence of a bait polypeptide of interest, and the resulting fusion protein expressed in cultured cells. While BirA* can activate biotin to biotinoyl-AMP, the abortive mutant enzyme exhibits a reduced affinity for the activated molecule. A reactive intermediate is thus released into the local environment that can react with free epsilon amine groups on nearby lysine residues 7 . This ability for BirA* to label a local environment has led to BioID being employed by multiple laboratories to define the composition, and in some cases the overall organization, of both membrane-bound and membraneless organelles (e.g. 7-10 ).Here, we set out to map a human cell by profiling markers (consisting of full-length proteins or targeting sequences) from 32 cellular compartments. These compartments include the cytosolic face of all membrane-bound organelles, the ER lumen, subcompartments of the nucleus and mitochondria, major membraneless organelles such as the centrosome and the nucleolus, and the main cytoskeletal structures (actin, microtubules and intermediate filaments). Several proteins were also queried throughout the endomembrane system to identify components enriched at locales a...

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“…Ascorbate peroxidase (APEX), a developed proximity labelling ligase in 2013, is characterised by a rapid labelling time (1 min or less) and subsequent generation of a snapshot of proximal proteins, and thus, it enables the identification even of components that interact transiently (Lam et al, 2015;Rhee et al, 2013). APEX has been successfully utilised in the characterization of the proteomes of the mitochondrial matrix, the primary cilium, and the ER-plasma membrane junctions, as well as in the composition profiling of the lipid droplet (Bersuker et al, 2018;Hung et al, 2017;Jing et al, 2015;Mick et al, 2015). However, utilisation of toxic H2O2 during labelling and high endogenous peroxidase activity limit APEX broad application on living samples and plants (Hung et al, 2016;.…”

Section: Introductionmentioning

confidence: 99%

TurboID-mediated proximity labelling of cytoophidium proteome inDrosophila

Zhang

Liu

2019

Preprint

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Proximity-based biotinylation combined with mass spectrometry has emerged as a powerful approach to study protein interaction networks and protein subcellular compartmentation. However, low kinetics and the requirement of toxic chemicals limit the broad utilisation of current proximity labelling methods in living organisms.TurboID, the newly engineered promiscuous ligase, has been reported to label bait proteins effectively in various species. Here, we systematically demonstrated the application of TurboIDmediated biotinylation in a wide range of developmental stages and tissues, and we also verified the feasibility of TurboID-mediated labelling in desired cells via cell-type-specific GAL4 driver in Drosophila. Furthermore, using TurboID-mediated biotinylation coupled with mass spectrometry, we characterized the proximate proteome of the cytoophidium, a newly identified filamentous structure containing the metabolic enzyme CTP synthase (CTPS) in Drosophila. Our study demonstrates a referable tool and resource for research in subcellular compartments of metabolic enzymes in vivo. Running title: Cytoophidium proteome captured by TurboID

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A Proximity Labeling Strategy Provides Insights into the Composition and Dynamics of Lipid Droplet Proteomes

Cited by 263 publications

References 64 publications

Atlas of Subcellular RNA Localization Revealed by APEX-seq

Atlas of Subcellular RNA Localization Revealed by APEX-seq

A proximity-dependent biotinylation map of a human cell: an interactive web resource

TurboID-mediated proximity labelling of cytoophidium proteome inDrosophila

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