PerTurboID: A targeted in situ method to measure changes in a local protein environment reveals the impact of kinase deletion on cytoadhesion in malaria causing parasites

Abstract

Reverse genetics is key to understanding protein function, but the mechanistic connection between a gene of interest and the observed phenotype is not always clear. Here we describe the use of proximity labeling using TurboID and site-specific quantification of biotinylated peptides to measure changes to the local protein environment of selected targets upon perturbation. We apply this technique, which we call PerTurboID, to understand how the P. falciparum exported kinase, FIKK4.1, regulates the function of the major virulence factor of the malaria causing parasite, PfEMP1. We generated independent TurboID fusions of 2 proteins that are predicted substrates of FIKK4.1 in a FIKK4.1 conditional KO parasite line. Comparing the abundance of site-specific biotinylated peptides between wildtype and kinase deletion lines reveals the differential accessibility of proteins to biotinylation, indicating changes to localization, protein-protein interactions, or protein structure which are mediated by FIKK4.1 activity. We further show that FIKK4.1 is likely the only FIKK kinase that controls surface levels of PfEMP1, but not other surface antigens, on the infected red blood cell under standard culture conditions. We believe PerTurboID is broadly applicable to study the impact of genetic or environmental perturbation on a selected cellular niche.

Novel secretory organelles of parasite origin ‐ at the center of host‐parasite interaction

“…One elegant application of proximity labeling has recently been shown in a preprint published by Davies et al [212] The authors used TurboID-based proximity labeling (PerTurboID) and observed that several MC proteins with N-terminal transmembrane domains were inserted into the membrane of the organelle in the opposite direction. [212] The study further assessed the role the kinase FIKK4.1 plays in the function of PfEMP1, which shows the potential of proximity labeling in deciphering PfEMP1. [212] Further, non-genetic approaches are an appropriate way of examining the exportome of P. falciparum and proteins destined for the MC and the RBC surface.…”

“…[212] The study further assessed the role the kinase FIKK4.1 plays in the function of PfEMP1, which shows the potential of proximity labeling in deciphering PfEMP1. [212] Further, non-genetic approaches are an appropriate way of examining the exportome of P. falciparum and proteins destined for the MC and the RBC surface. Ready-to-use small molecular probes (e.g., for click chemistry) from the field of chemical biology permit a fast assessment of proteins at every developmental stage of P. falciparum using different experimental approaches.…”

“…[ 212 ] The study further assessed the role the kinase FIKK4.1 plays in the function of PfEMP1, which shows the potential of proximity labeling in deciphering PfEMP1. [ 212 ]…”