Screening of Interactions with the ESCRT Machinery by a Gaussia princeps Split Luciferase-Based Complementation Assay.
ESCRT
Protein-fragment complementation assays
Protein-protein interactions
Proteomics
Journal
Methods in molecular biology (Clifton, N.J.)
ISSN: 1940-6029
Titre abrégé: Methods Mol Biol
Pays: United States
ID NLM: 9214969
Informations de publication
Date de publication:
2019
2019
Historique:
entrez:
29
6
2019
pubmed:
30
6
2019
medline:
3
3
2020
Statut:
ppublish
Résumé
The endosomal sorting complex required for transport (ESCRT) machinery comprises five complexes that act sequentially to recruit and cluster transmembrane cargo proteins (ESCRT-0), drive membrane curving (ESCRT-I and II), catalyze fission of cargo-containing vesicles (ESCRT-III and VPS/VTA1), and disassemble and recycle the ESCRT-III complex (VPS/VTA1). Since interactions between ESCRT components and cellular or microbial proteins are typically weak, transient, and involve membrane proteins, they are often difficult to study by standard technologies. Here, we describe the utility of high-throughput protein-fragment complementation assays based on the reconstitution of a split luciferase reporter to screen for interactions between any protein and a library of ESCRT proteins in mammalian cells and provide a detailed protocol for these assays.
Identifiants
pubmed: 31250310
doi: 10.1007/978-1-4939-9492-2_21
doi:
Substances chimiques
Endosomal Sorting Complexes Required for Transport
0
Recombinant Proteins
0
Green Fluorescent Proteins
147336-22-9
Luciferases
EC 1.13.12.-
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
291-304Références
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