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      Proteomic mapping of cytosol-facing outer mitochondrial and ER membranes in living human cells by proximity biotinylation

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          Abstract

          The cytosol-facing membranes of cellular organelles contain proteins that enable signal transduction, regulation of morphology and trafficking, protein import and export, and other specialized processes. Discovery of these proteins by traditional biochemical fractionation can be plagued with contaminants and loss of key components. Using peroxidase-mediated proximity biotinylation, we captured and identified endogenous proteins on the outer mitochondrial membrane (OMM) and endoplasmic reticulum membrane (ERM) of living human fibroblasts. The proteomes of 137 and 634 proteins, respectively, are highly specific and highlight 94 potentially novel mitochondrial or ER proteins. Dataset intersection identified protein candidates potentially localized to mitochondria-ER contact sites. We found that one candidate, the tail-anchored, PDZ-domain-containing OMM protein SYNJ2BP, dramatically increases mitochondrial contacts with rough ER when overexpressed. Immunoprecipitation-mass spectrometry identified ribosome-binding protein 1 (RRBP1) as SYNJ2BP’s ERM binding partner. Our results highlight the power of proximity biotinylation to yield insights into the molecular composition and function of intracellular membranes.

          DOI: http://dx.doi.org/10.7554/eLife.24463.001

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          Gene Ontology: tool for the unification of biology

          Genomic sequencing has made it clear that a large fraction of the genes specifying the core biological functions are shared by all eukaryotes. Knowledge of the biological role of such shared proteins in one organism can often be transferred to other organisms. The goal of the Gene Ontology Consortium is to produce a dynamic, controlled vocabulary that can be applied to all eukaryotes even as knowledge of gene and protein roles in cells is accumulating and changing. To this end, three independent ontologies accessible on the World-Wide Web (http://www.geneontology.org) are being constructed: biological process, molecular function and cellular component.
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            Directed evolution of APEX2 for electron microscopy and proteomics

            APEX is an engineered peroxidase that functions both as an electron microscopy tag, and as a promiscuous labeling enzyme for live-cell proteomics. Because the limited sensitivity of APEX precludes applications requiring low APEX expression, we used yeast display evolution to improve its catalytic efficiency. Our evolved APEX2 is far more active in cells, enabling the superior enrichment of endogenous mitochondrial and endoplasmic reticulum membrane proteins and the use of electron microscopy to resolve the sub-mitochondrial localization of calcium uptake regulatory protein MICU1.
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              The endoplasmic reticulum: structure, function and response to cellular signaling

              The endoplasmic reticulum (ER) is a large, dynamic structure that serves many roles in the cell including calcium storage, protein synthesis and lipid metabolism. The diverse functions of the ER are performed by distinct domains; consisting of tubules, sheets and the nuclear envelope. Several proteins that contribute to the overall architecture and dynamics of the ER have been identified, but many questions remain as to how the ER changes shape in response to cellular cues, cell type, cell cycle state and during development of the organism. Here we discuss what is known about the dynamics of the ER, what questions remain, and how coordinated responses add to the layers of regulation in this dynamic organelle.
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                Author and article information

                Contributors
                Role: Reviewing editor
                Journal
                eLife
                Elife
                eLife
                eLife
                eLife
                eLife Sciences Publications, Ltd
                2050-084X
                25 April 2017
                2017
                : 6
                : e24463
                Affiliations
                [1 ]deptDepartment of Chemistry , Massachusetts Institute of Technology , Cambridge, United States
                [2 ]Broad Institute of MIT and Harvard , Cambridge, United States
                [3 ]deptDepartment of Molecular Biology , Howard Hughes Medical Institute, Massachusetts General Hospital, Harvard Medical School , Boston, United States
                Morgridge Institute for Research , United States
                Morgridge Institute for Research , United States
                Author notes
                [‡]

                Departments of Developmental Biology and Genetics, Stanford University, Stanford, United States.

                [§]

                Department of Molecular Biology, Howard Hughes Medical Institute, Massachusetts General Hospital, Harvard Medical School, Boston, United States.

                [¶]

                Departments of Biology, Genetics, and Chemistry, Stanford University, Stanford, United States.

                [†]

                These authors contributed equally to this work.

                Author information
                http://orcid.org/0000-0003-3972-2820
                http://orcid.org/0000-0002-2687-3470
                http://orcid.org/0000-0001-9924-642X
                http://orcid.org/0000-0002-8277-5226
                Article
                24463
                10.7554/eLife.24463
                5404927
                28441135
                e73e77d8-8f63-4ce4-9050-819f1b1e01c0
                © 2017, Hung et al

                This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

                History
                : 20 December 2016
                : 14 March 2017
                Funding
                Funded by: FundRef http://dx.doi.org/10.13039/100000002, National Institutes of Health;
                Award ID: R01 CA186568
                Award Recipient :
                Funded by: FundRef http://dx.doi.org/10.13039/100000011, Howard Hughes Medical Institute;
                Award ID: HHMI Collaborative Innovation Award
                Award Recipient :
                Funded by: FundRef http://dx.doi.org/10.13039/100000002, National Institutes of Health;
                Award ID: R01 GM077465
                Award Recipient :
                Funded by: FundRef http://dx.doi.org/10.13039/100000001, National Science Foundation;
                Award ID: Graduate Research Fellowship
                Award Recipient :
                Funded by: FundRef http://dx.doi.org/10.13039/100000005, U.S. Department of Defense;
                Award ID: National Defense Science and Engineering Graduate (NDSEG) Fellowship
                Award Recipient :
                Funded by: FundRef http://dx.doi.org/10.13039/100000011, Howard Hughes Medical Institute;
                Award ID: Investigator
                Award Recipient :
                The funders had no role in study design, data collection, and interpretation, or the decision to submit the work for publication.
                Categories
                Tools and Resources
                Biochemistry
                Cell Biology
                Custom metadata
                2.5
                One minute biotinylation with APEX2 peroxidase in living cells identifies established and new components of mitochondrial and ER membranes; dataset intersection and overexpression screen identifies SYNJ2BP overexpression as inducing mitochondria-rough ER contacts.

                Life sciences
                microscopy,promiscuous enzymatic labeling,subcellular regions,apex2,mitochondria-er junctions,mitochondria-associated membrane,human

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