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Screening of Interactions with the ESCRT Machinery by a Gaussia princeps Split Luciferase-Based Complementation Assay. Methods in molecular biology (Clifton, N.J.) Barouch-Bentov, R. n., Jacob, Y. n., Einav, S. n. 2019; 1998: 291–304

Abstract

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.

View details for DOI 10.1007/978-1-4939-9492-2_21

View details for PubMedID 31250310