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      Highly effective proximate labeling in Drosophila

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          Abstract

          The protein–protein interaction (PPI) is a basic strategy for life to operate. The analysis of PPIs in multicellular organisms is very important but extremely challenging because PPIs are particularly dynamic and variable among different development stages, tissues, cells, and even organelles. Therefore, understanding PPI needs a good resolution of time and space. More importantly, understanding in vivo PPI needs to be realized in situ. Proximity-based biotinylation combined with mass spectrometry (MS) has emerged as a powerful approach to study PPI networks and protein subcellular compartmentation. TurboID, the newly engineered promiscuous ligase, has been reported to label proximate proteins effectively in various species. In Drosophila, we systematically apply TurboID-mediated biotinylation in a wide range of developmental stages and tissues, and demonstrate the feasibility of TurboID-mediated labeling system in desired cell types. For a proof-of-principle, we use the TurboID-mediated biotinylation coupled with MS to distinguish CTP synthase with or without the ability to form filamentous cytoophidia, retrieving two distinct sets of proximate proteomes. Therefore, this makes it possible to map PPIs in vivo and in situ at a defined spatiotemporal resolution, and demonstrates a referable resource for cytoophidium proteome in Drosophila.

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          Most cited references40

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          clusterProfiler: an R package for comparing biological themes among gene clusters.

          Increasing quantitative data generated from transcriptomics and proteomics require integrative strategies for analysis. Here, we present an R package, clusterProfiler that automates the process of biological-term classification and the enrichment analysis of gene clusters. The analysis module and visualization module were combined into a reusable workflow. Currently, clusterProfiler supports three species, including humans, mice, and yeast. Methods provided in this package can be easily extended to other species and ontologies. The clusterProfiler package is released under Artistic-2.0 License within Bioconductor project. The source code and vignette are freely available at http://bioconductor.org/packages/release/bioc/html/clusterProfiler.html.
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            Global quantification of mammalian gene expression control.

            Gene expression is a multistep process that involves the transcription, translation and turnover of messenger RNAs and proteins. Although it is one of the most fundamental processes of life, the entire cascade has never been quantified on a genome-wide scale. Here we simultaneously measured absolute mRNA and protein abundance and turnover by parallel metabolic pulse labelling for more than 5,000 genes in mammalian cells. Whereas mRNA and protein levels correlated better than previously thought, corresponding half-lives showed no correlation. Using a quantitative model we have obtained the first genome-scale prediction of synthesis rates of mRNAs and proteins. We find that the cellular abundance of proteins is predominantly controlled at the level of translation. Genes with similar combinations of mRNA and protein stability shared functional properties, indicating that half-lives evolved under energetic and dynamic constraints. Quantitative information about all stages of gene expression provides a rich resource and helps to provide a greater understanding of the underlying design principles.
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              Efficient proximity labeling in living cells and organisms with TurboID

              Protein interaction networks and protein compartmentalization underlie all signaling and regulatory processes in cells. Enzyme-catalyzed proximity labeling (PL) has emerged as a new approach to study the spatial and interaction characteristics of proteins in living cells. However, current PL methods require over 18 hour labeling times or utilize chemicals with limited cell permeability or high toxicity. We used yeast display-based directed evolution to engineer two promiscuous mutants of biotin ligase, TurboID and miniTurbo, which catalyze PL with much greater efficiency than BioID or BioID2, and enable 10-minute PL in cells with non-toxic and easily deliverable biotin. Furthermore, TurboID extends biotin-based PL to flies and worms.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                G3 (Bethesda)
                Genetics
                g3journal
                G3: Genes|Genomes|Genetics
                Oxford University Press
                2160-1836
                May 2021
                16 March 2021
                16 March 2021
                : 11
                : 5
                : jkab077
                Affiliations
                [1 ] School of Life Science and Technology, ShanghaiTech University , Shanghai 201210, China
                [2 ] Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences , Shanghai 200031, China
                [3 ] University of Chinese Academy of Sciences , Beijing 100049, China
                [4 ] Department of Physiology, Anatomy and Genetics, University of Oxford , Oxford OX1 3PT, UK
                Author notes
                [†]

                These authors contributed equally to this work.

                Author information
                https://orcid.org/0000-0002-7805-1786
                https://orcid.org/0000-0002-4569-2798
                https://orcid.org/0000-0002-4834-8554
                Article
                jkab077
                10.1093/g3journal/jkab077
                8104946
                33724396
                6976851d-e61b-4130-ba97-45c10e02915c
                © The Author(s) 2021. Published by Oxford University Press on behalf of Genetics Society of America.

                This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                : 17 December 2020
                : 05 February 2021
                Page count
                Pages: 11
                Funding
                Funded by: ShanghaiTech University, National Natural Science Foundation of China;
                Award ID: 31771490
                Funded by: UK Medical Research Council;
                Award ID: MC_UU_12021/3
                Award ID: MC_U137788471
                Categories
                Investigation
                AcademicSubjects/SCI01180
                AcademicSubjects/SCI01140
                AcademicSubjects/SCI00010
                AcademicSubjects/SCI00960

                Genetics
                protein–protein interaction,turboid,proximate labeling,ctp synthase,cytoophidium,mass spectrometry,drosophila

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