Plasmodium falciparum Kelch13, Eps15 and Clathrin HC interactomes obtained using a dimerisation-induced quantitative BioID (DiQ-BioID)

In order to identify Kelch13 interaction partners in living P. falciparum parasites, we established a new type of BioID we termed dimerisation-induced quantitative BioID (DiQ-BioID) that takes advantage of the FKBP-FRB heterodimerisation system. For DiQ-BioID the target is endogenously tagged with FKBP. The promiscuous biotin ligase BirA* is fused with FRB and expressed in the cells expressing the FKBP tagged target. Upon addition of the dimerising ligand (rapalog) BirA*-FRB is recruited onto the FKBP fused target. In the presence of biotin this results in the biotinylation of the target and its interactors whereas in the control the cellular background is biotinylated. As matched parasite cultures (identical cultures grown with and without rapalog) are used, quantitative mass spectrometry was employed to identify specific interaction partners (or compartment neighbors). This was done for Kelch13 as a bait, for one of the so identified hits (Eps15) in reciprocal DiQ-BioID experiments and for the clathrin heavy chain as a negative control (a protein that like Kelch13 and Eps15 is found in foci in the cells but does not co-localise with these targets).