Screening of Interactions with the ESCRT Machinery by a Gaussia princeps Split Luciferase-Based Complementation Assay

Rina Barouch-Bentov (1) , Yves Jacob (2) , Shirit Einav (1)

1 Stanford School of Medicine [Stanford]
2 GMV-ARN (UMR_3569 / U-Pasteur_2) - Génétique Moléculaire des Virus à ARN - Molecular Genetics of RNA Viruses

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.

Mots clés: ESCRT; Proteomics; Protein-protein interactions; Protein-fragment complementation assays

Domaines: Sciences du Vivant [q-bio]

Rina Barouch-Bentov, Yves Jacob, Shirit Einav. Screening of Interactions with the ESCRT Machinery by a Gaussia princeps Split Luciferase-Based Complementation Assay. The ESCRT Complexes, 1998, Humana, pp.291-304, 2019, Methods in Molecular Biology, 978-1-4939-9494-6. ⟨10.1007/978-1-4939-9492-2_21⟩. ⟨pasteur-04098565⟩