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Titel: Lights, Camera, Interaction: Studying Protein–Protein Interactions of the ER Protein Translocase in Living Cells
VerfasserIn: Sicking, Mark
Jung, Martin
Lang, Sven
Sprache: Englisch
Titel: International Journal of Molecular Sciences
Bandnummer: 22
Heft: 19
Verlag/Plattform: MDPI
Erscheinungsjahr: 2021
Freie Schlagwörter: bimolecular luminescence complementation
competition
split luciferase
membrane proteins
protein–protein interactions
Sec61 complex
Sec63
synthetic peptide complementation
TRAP complex
ER protein translocase
DDC-Sachgruppe: 610 Medizin, Gesundheit
Dokumenttyp: Journalartikel / Zeitschriftenartikel
Abstract: 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.
DOI der Erstveröffentlichung: 10.3390/ijms221910358
Link zu diesem Datensatz: urn:nbn:de:bsz:291--ds-348314
hdl:20.500.11880/31846
http://dx.doi.org/10.22028/D291-34831
ISSN: 1422-0067
Datum des Eintrags: 13-Okt-2021
Bezeichnung des in Beziehung stehenden Objekts: Supplementary Materials
In Beziehung stehendes Objekt: https://www.mdpi.com/article/10.3390/ijms221910358/s1
Fakultät: M - Medizinische Fakultät
Fachrichtung: M - Medizinische Biochemie und Molekularbiologie
Professur: M - Keiner Professur zugeordnet
Sammlung:SciDok - Der Wissenschaftsserver der Universität des Saarlandes

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