Lights, Camera, Interaction: Studying Protein–Protein Interactions of the ER Protein Translocase in Living Cells
Various landmark studies have revealed structures and functions of the Sec61/SecY complex in all domains of live demonstrating the conserved nature of this ancestral protein translocase. While the bacterial homolog of the Sec61 complex resides in the plasma membrane, the eukaryotic counterpart manag...
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| Format: | Article |
| Language: | English |
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MDPI AG
2021-09-01
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| Series: | International Journal of Molecular Sciences |
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| Online Access: | https://www.mdpi.com/1422-0067/22/19/10358 |
| _version_ | 1797516315404009472 |
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| author | Mark Sicking Martin Jung Sven Lang |
| author_facet | Mark Sicking Martin Jung Sven Lang |
| author_sort | Mark Sicking |
| collection | DOAJ |
| description | Various landmark studies have revealed structures and functions of the Sec61/SecY complex in all domains of live demonstrating the conserved nature of this ancestral protein translocase. While the bacterial homolog of the Sec61 complex resides in the plasma membrane, the eukaryotic counterpart manages the transfer of precursor proteins into or across the membrane of the endoplasmic reticulum (ER). Sec61 complexes are accompanied by a set of dynamically recruited auxiliary proteins assisting the transport of certain precursor polypeptides. TRAP and Sec62/Sec63 are two auxiliary protein complexes in mammalian cells that have been characterized by structural and biochemical methods. Using these ER membrane protein complexes for our proof-of-concept study, we aimed to detect interactions of membrane proteins in living mammalian cells under physiological conditions. Bimolecular luminescence complementation and competition was used to demonstrate multiple protein–protein interactions of different topological layouts. In addition to the interaction of the soluble catalytic and regulatory subunits of the cytosolic protein kinase A, we detected interactions of ER membrane proteins that either belong to the same multimeric protein complex (intra-complex interactions: Sec61α–Sec61β, TRAPα–TRAPβ) or protein complexes in juxtaposition (inter-complex interactions: Sec61α–TRAPα, Sec61α–Sec63, and Sec61β–Sec63). In the process, we established further control elements like synthetic peptide complementation for expression profiling of fusion constructs and protease-mediated reporter degradation demonstrating the cytosolic localization of a reporter complementation. Ease of use and flexibility of the approach presented here will spur further research regarding the dynamics of protein–protein interactions in response to changing cellular conditions in living cells. |
| first_indexed | 2024-03-10T06:59:30Z |
| format | Article |
| id | doaj.art-09bca699379f4a3d9bc6ed9b2853e494 |
| institution | Directory Open Access Journal |
| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-03-10T06:59:30Z |
| publishDate | 2021-09-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | International Journal of Molecular Sciences |
| spelling | doaj.art-09bca699379f4a3d9bc6ed9b2853e4942023-11-22T16:08:23ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672021-09-0122191035810.3390/ijms221910358Lights, Camera, Interaction: Studying Protein–Protein Interactions of the ER Protein Translocase in Living CellsMark Sicking0Martin Jung1Sven Lang2Department of Medical Biochemistry and Molecular Biology, Saarland University, 66421 Homburg, GermanyDepartment of Medical Biochemistry and Molecular Biology, Saarland University, 66421 Homburg, GermanyDepartment of Medical Biochemistry and Molecular Biology, Saarland University, 66421 Homburg, GermanyVarious landmark studies have revealed structures and functions of the Sec61/SecY complex in all domains of live demonstrating the conserved nature of this ancestral protein translocase. While the bacterial homolog of the Sec61 complex resides in the plasma membrane, the eukaryotic counterpart manages the transfer of precursor proteins into or across the membrane of the endoplasmic reticulum (ER). Sec61 complexes are accompanied by a set of dynamically recruited auxiliary proteins assisting the transport of certain precursor polypeptides. TRAP and Sec62/Sec63 are two auxiliary protein complexes in mammalian cells that have been characterized by structural and biochemical methods. Using these ER membrane protein complexes for our proof-of-concept study, we aimed to detect interactions of membrane proteins in living mammalian cells under physiological conditions. Bimolecular luminescence complementation and competition was used to demonstrate multiple protein–protein interactions of different topological layouts. In addition to the interaction of the soluble catalytic and regulatory subunits of the cytosolic protein kinase A, we detected interactions of ER membrane proteins that either belong to the same multimeric protein complex (intra-complex interactions: Sec61α–Sec61β, TRAPα–TRAPβ) or protein complexes in juxtaposition (inter-complex interactions: Sec61α–TRAPα, Sec61α–Sec63, and Sec61β–Sec63). In the process, we established further control elements like synthetic peptide complementation for expression profiling of fusion constructs and protease-mediated reporter degradation demonstrating the cytosolic localization of a reporter complementation. Ease of use and flexibility of the approach presented here will spur further research regarding the dynamics of protein–protein interactions in response to changing cellular conditions in living cells.https://www.mdpi.com/1422-0067/22/19/10358bimolecular luminescence complementationcompetitionsplit luciferasemembrane proteinsprotein–protein interactionsSec61 complex |
| spellingShingle | Mark Sicking Martin Jung Sven Lang Lights, Camera, Interaction: Studying Protein–Protein Interactions of the ER Protein Translocase in Living Cells International Journal of Molecular Sciences bimolecular luminescence complementation competition split luciferase membrane proteins protein–protein interactions Sec61 complex |
| title | Lights, Camera, Interaction: Studying Protein–Protein Interactions of the ER Protein Translocase in Living Cells |
| title_full | Lights, Camera, Interaction: Studying Protein–Protein Interactions of the ER Protein Translocase in Living Cells |
| title_fullStr | Lights, Camera, Interaction: Studying Protein–Protein Interactions of the ER Protein Translocase in Living Cells |
| title_full_unstemmed | Lights, Camera, Interaction: Studying Protein–Protein Interactions of the ER Protein Translocase in Living Cells |
| title_short | Lights, Camera, Interaction: Studying Protein–Protein Interactions of the ER Protein Translocase in Living Cells |
| title_sort | lights camera interaction studying protein protein interactions of the er protein translocase in living cells |
| topic | bimolecular luminescence complementation competition split luciferase membrane proteins protein–protein interactions Sec61 complex |
| url | https://www.mdpi.com/1422-0067/22/19/10358 |
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