Probing the Environment of Emerin by Enhanced Ascorbate Peroxidase 2 (APEX2)-Mediated Proximity Labeling
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...
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| Format: | Article |
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MDPI AG
2020-03-01
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| Series: | Cells |
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| Online Access: | https://www.mdpi.com/2073-4409/9/3/605 |
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| author | Marret Müller Christina James Christof Lenz Henning Urlaub Ralph H. Kehlenbach |
| author_facet | Marret Müller Christina James Christof Lenz Henning Urlaub Ralph H. Kehlenbach |
| author_sort | Marret Müller |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-03-12T19:17:58Z |
| format | Article |
| id | doaj.art-7a22017d9f8b4d8d92a30a387823c1c8 |
| institution | Directory Open Access Journal |
| issn | 2073-4409 |
| language | English |
| last_indexed | 2024-03-12T19:17:58Z |
| publishDate | 2020-03-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Cells |
| spelling | doaj.art-7a22017d9f8b4d8d92a30a387823c1c82023-08-02T05:25:14ZengMDPI AGCells2073-44092020-03-019360510.3390/cells9030605cells9030605Probing the Environment of Emerin by Enhanced Ascorbate Peroxidase 2 (APEX2)-Mediated Proximity LabelingMarret Müller0Christina James1Christof Lenz2Henning Urlaub3Ralph H. Kehlenbach4Department of Molecular Biology, Faculty of Medicine, GZMB, Georg-August-University Göttingen, Humboldtallee 23, 37073 Göttingen, GermanyDepartment of Molecular Biology, Faculty of Medicine, GZMB, Georg-August-University Göttingen, Humboldtallee 23, 37073 Göttingen, GermanyBioanalytics Group, Institute of Clinical Chemistry, University Medical Center Göttingen, Robert-Koch-Straße 40, 37075 Göttingen, GermanyBioanalytics Group, Institute of Clinical Chemistry, University Medical Center Göttingen, Robert-Koch-Straße 40, 37075 Göttingen, GermanyDepartment of Molecular Biology, Faculty of Medicine, GZMB, Georg-August-University Göttingen, Humboldtallee 23, 37073 Göttingen, GermanyEmerin 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.https://www.mdpi.com/2073-4409/9/3/605proximity labelingapexrapidsemerininner nuclear membrane |
| spellingShingle | Marret Müller Christina James Christof Lenz Henning Urlaub Ralph H. Kehlenbach Probing the Environment of Emerin by Enhanced Ascorbate Peroxidase 2 (APEX2)-Mediated Proximity Labeling Cells proximity labeling apex rapids emerin inner nuclear membrane |
| title | Probing the Environment of Emerin by Enhanced Ascorbate Peroxidase 2 (APEX2)-Mediated Proximity Labeling |
| title_full | Probing the Environment of Emerin by Enhanced Ascorbate Peroxidase 2 (APEX2)-Mediated Proximity Labeling |
| title_fullStr | Probing the Environment of Emerin by Enhanced Ascorbate Peroxidase 2 (APEX2)-Mediated Proximity Labeling |
| title_full_unstemmed | Probing the Environment of Emerin by Enhanced Ascorbate Peroxidase 2 (APEX2)-Mediated Proximity Labeling |
| title_short | Probing the Environment of Emerin by Enhanced Ascorbate Peroxidase 2 (APEX2)-Mediated Proximity Labeling |
| title_sort | probing the environment of emerin by enhanced ascorbate peroxidase 2 apex2 mediated proximity labeling |
| topic | proximity labeling apex rapids emerin inner nuclear membrane |
| url | https://www.mdpi.com/2073-4409/9/3/605 |
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