Background

Most cellular processes are carried out and controlled by protein-protein interactions (PPIs) and post-translation protein modifications (PTMs). Therefore, detecting PPIs and PPI-dependent PTMs is critical for understanding normal and pathological conditions, and for potential therapeutic development. Indeed, many methodologies have been developed for identification and characterization of PPIs and PTMs. Each of these methods has its own benefits and caveats. In the 1980s, phage display and genetic yeast two-hybrid systems were developed to study PPIs (Smith, 1985; Fields and Song, 1989; Bair et al., 2008). These methods allowed, for the first time, a high-throughput screening of a large repertoire of proteins or peptides. A great benefit of these methods is the linkage to the DNA sequence encoding the identified polypeptide. However, in the phage display systems, proteins interact in the extra-cellular environment, a process that leads to many false positive interactions, due to misfolding. In contrast, complementation of the split-Gal4 transcription factor in the yeast nucleus is required to activate the reporter, and consequently limits the positive readouts of the screen. Cleverly, Stagljar and co-workers circumvented this with a split-ubiquitin system [a system previously developed by Johnsson and Varshavsky (Johnsson et al., 1994)], in which the interaction of membrane proteins induces a proteolytic cleavage that releases a fused nuclear transcription factor, activating reporter genes in the nucleus (Stagljar et al., 1998). Additionally, Michnick and co-workers developed two excellent Protein-fragment Complementation Assays (PCA) based on split dihydrofolate reductase (DHFR) and split-TEM-1 (β-lactamase) enzymes (Pelletier et al., 1998; Galarneau et al., 2002). The split-DHFR system provides growth phenotypic readouts, both in bacteria and yeast. However, seeding of fairly low cell concentrations is required to prevent non-specific growth (Levin-Kravets et al., 2021). Split-reporters that provide fluorescence or luminescence are widely used to detect PPIs. Forster resonance energy transfer by fluorescence lifetime imaging (FRET-FLIM) and bimolecular fluorescence complementation (BiFC) are useful in live cell imaging (Majoul et al., 2002; Walter et al., 2004). The advantage of this approach is the information regarding the cellular localization of the PPIs. Nevertheless, these methods require sophisticated microscopy equipment, which can frequently be a limiting factor.

Eukaryotic protein interactions are often regulated by transient PTMs that challenge the identification of these PPIs. The high sensitivity of mass-spectrometry technologies are beneficial for the detection of a large spectra of PPIs and PTMs (Blagoev et al., 2003; Peng et al., 2003). However, redundancy within the modifier enzymes challenges their linking to their respective targets. Protein microarray serves as an alternative, comprehensive approach for identification of PTMs, while linking them to their modifier enzyme (Gupta et al., 2007). Sequence and structure-based in silico approaches provide predictions of PPIs, and thus facilitate analysis using the above described methods (Marcotte et al., 1999; London et al., 2012; Keren-Kaplan et al., 2013; Jumper et al., 2021). Among over 200 different PTMs in the eukaryotic proteome, ubiquitylation is the most abundant and complex modification, with over 600 ubiquitin E3-ligases in humans. Here, we present a detailed protocol for a recently developed application to detect and quantify PPIs and ubiquitylation, using a recombinant system that is expressed in E. coli, and based on Split-chloramphenicol Acetyl-Transferase (Split-CAT) (Levin-Kravets et al., 2021). Bacterial growth efficiency is the system readout, which can be estimated as endpoint, or continuously measured to obtain kinetics readouts. Contradictory to selection systems that report growth based on synthesis of essential metabolites, such as uracil, thymidine, or histidine, which pose limits on seeding concentration, the Split-CAT system modifies the antibiotic, thus allowing high-density seeding of bacteria. The sensitivity to a wide concentration range of chloramphenicol allows the detection of a large spectrum of affinities of PPIs. The tight correlation between binding or ubiquitylation and growth efficiency allows the estimation of apparent relative affinity (Keren-Kaplan et al., 2016). The system was constructed with several compatible plasmids, which provides modularity, and facilitates its transfer to different bacteria (Figure 1).

Figure 1. Concept of the Split-CAT selection system. The DNA plasmids encoding the system are shown on the left side. The activities of the proteins encoded by the system are shown to the right. (A) Shows a scheme of the PPI study. (B) Shows a scheme of the ubiquitylation study.