Probing the Environment of Emerin by Enhanced Ascorbate Peroxidase 2 (APEX2)-Mediated Proximity Labeling.

Cells
Marret MüllerRalph H Kehlenbach

Abstract

Emerin is one of the best characterized proteins of the inner nuclear membrane, but can also occur at the level of the endoplasmic reticulum. We now use enhanced ascorbate peroxidase 2 (APEX2) to probe the environment of emerin. APEX2 can be used as a genetic tag that produces short-lived yet highly reactive biotin species, allowing the modification of proteins that interact with or are in very close proximity to the tagged protein. Biotinylated proteins can be isolated using immobilized streptavidin and analyzed by mass spectrometry. As an alternative to the standard approach with a genetic fusion of APEX2 to emerin, we also used RAPIDS (rapamycin- and APEX-dependent identification of proteins by SILAC), a method with improved specificity, where the peroxidase interacts with the protein of interest (i.e., emerin) only upon addition of rapamycin to the cells. We compare these different approaches, which, together, identify well-known interaction partners of emerin like lamin A and the lamina associated polypeptide 1 (LAP1), as well as novel proximity partners.

References

Aug 1, 1987·Molecular and Cellular Biology·C Chen, H Okayama
Sep 2, 1994·Journal of Molecular Biology·P CharneauF Clavel
Aug 7, 2002·FEBS Letters·John M K MislowElizabeth M McNally
Sep 6, 2007·The Journal of Cell Biology·Georgia SalpingidouChris J Hutchison
May 13, 2008·Trends in Cell Biology·Sima LevNili Dahan
Sep 30, 2010·Molecular & Cellular Proteomics : MCP·Gavin S WilkieEric C Schirmer
Mar 30, 2011·The Journal of Cell Biology·Nikolaj ZulegerEric C Schirmer
May 10, 2012·The Journal of Biological Chemistry·Justin DemmerleJames M Holaska
Sep 20, 2012·Nucleus·Nadia KorfaliEric C Schirmer
Jul 23, 2013·Nucleus·Jason M BerkKatherine L Wilson
Apr 15, 2014·Nature Biotechnology·Juan A VizcaínoHenning Hermjakob
Jul 24, 2014·Journal of Cell Science·Jason M BerkKatherine L Wilson
Nov 25, 2014·Nature Methods·Stephanie S LamAlice Y Ting
Jun 10, 2015·The Journal of Cell Biology·Rosemarie UngrichtUlrike Kutay
Nov 21, 2015·Developmental Cell·David U MickMaxence V Nachury
Oct 25, 2016·Trends in Cell Biology·Dae In Kim, Kyle J Roux
Sep 15, 2019·The Journal of Biological Chemistry·Christina JamesRalph H Kehlenbach

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Citations

Jun 2, 2020·Frontiers in Genetics·Henning Ummethum, Stephan Hamperl
Aug 8, 2021·Cells·Christina James, Ralph H Kehlenbach

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Datasets Mentioned

BETA
PXD009783

Methods Mentioned

BETA
co-immunoprecipitation
affinity capture
PCR
fluorescence microscopy
Protein Assay
Coimmunoprecipitation
immunoprecipitation
transfection

Software Mentioned

Perseus
WebGestalt ( WEB Gene SeT AnaLysis Toolkit
BioID
RAPIDS
MaxQuant

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