RPM-1 in vivo affinity-purification proteomics using C. elegans

To uncover putative RPM-1 protein-protein interactions, we performed a comprehensive series of in vivo affinity-purification proteomics experiments using C. elegans. We utilized integrated transgenes that express RPM-1 fused with a Protein G::Streptavidin binding peptide (GS) affinity tag expressed using the native rpm-1 promoter on an rpm-1 protein null background. Negative control animals expressed an integrated transgene where the GS tag was fused to GFP (GS::GFP) and expressed by the rpm-1 promoter. We also designed a biochemical “trap” using an RPM-1 ligase-dead (RPM-1 LD) site-directed mutant to identify ubiquitination substrates. RPM-1 LD would enrich substrates by trapping them in stalled ubiquitination complexes, as well as preventing proteasome-mediated degradation and elevating substrate levels. RPM-1-binding proteins that are not ubiquitination substrates would bind to both RPM-1 and RPM-1 LD.