ReLo is a simple and quick colocalization assay to identify and characterize direct protein-protein interactions

Abstract

Characterizing protein-protein interactions (PPIs) is fundamental for understanding biochemical processes. Many methods have been established to identify and study direct PPIs; however, screening and investigating PPIs involving large and poorly soluble proteins remain challenges. As a result, we developed ReLo, a simple cell culture-based method to detect and investigate interactions in a cellular context. ReLo allows both the identification of protein domains that mediate the formation of complexes and screening of interfering point mutations. ReLo is sensitive to drugs that mediate or interfere with a specific interaction. Furthermore, protein conformation- and protein arginine methylation-dependent interactions can be studied. Importantly, ReLo can be used for specifically detecting direct but not indirect PPIs and can be applied for describing the binding topology of subunits within multiprotein complexes. Because of these attributes, ReLo is a simple, quick and versatile tool for identifying and studying binary PPIs, as well as for characterizing multisubunit complexes.

Structural basis for binding of Smaug to the GPCR Smoothened and to the germline inducer Oskar

“…Therefore, to assess protein interactions to Short Oskar, it carried an N-terminal fusion to OST4, a small membrane protein that directed Short Oskar localization to the endoplasmic reticulum (ER). Using OST4-anchored Short Oskar, we have previously confirmed the known direct interaction with the DEAD-box RNA helicase Vasa (Supplemental Figure 7A) (Salgania et al, 2022;Jeske et al, 2017Jeske et al, , 2015. Here, we show that OST4-Short Oskar interacted also with Smaug (Figure 4B), which is consistent with earlier observations (Dahanukar et al, 1999;Jeske et al, 2011;Zaessinger et al, 2006).…”

“…To analyze protein interactions between proteins, we have recently developed ReLo, a cell culture-based protein-protein interaction assay that is based on a subcellular translocation readout (Salgania et al , 2022). Our previous analyses strongly suggested that interactions identified with the ReLo assay are direct (Salgania et al , 2022).…”

“…When Long Oskar and Smaug were coexpressed, we did not observe a relocalization of Smaug (Figure 4B). Short Oskar localizes to the nucleus in S2R+ cells and a fusion to the PH domain does not efficiently redirect/anchor Short Oskar to the plasma membrane (Salgania et al , 2022). Therefore, to assess protein interactions to Short Oskar, it carried an N-terminal fusion to OST4, a small membrane protein that directed Short Oskar localization to the endoplasmic reticulum (ER).…”

RNA binding proteins Smaug and Cup induce CCR4-NOT-dependent deadenylation of the nanos mRNA in a reconstituted system

“…Interactions between Smaug and individual CCR4-NOT subunits present in the MINI complex were examined by a recently described in vivo interaction assay based on intracellular protein relocalization (ReLo assay) (Salgania et al, 2022). For this assay, CCR4-NOT subunits were fused with mCherry and with the pleckstrin homology (PH) domain of rat phospholipase Cd1 and expressed as bait in Drosophila S2R+ cells; the PH domain caused their localization on the plasma membrane.…”

“…ReLo assays were performed as described (Salgania et al, 2022). In short, Drosophila S2R+ cells were seeded onto four-well chambered coverslips (Ibidi), co-transfected with the desired combination of pAc5.1 plasmids expressing the two proteins of interest, and protein localization was analyzed after two days by live confocal fluorescence microscopy.…”