PerTurboID, A targeted in situ method reveals the impact of kinase deletion on its local protein environment in the cytoadhesion complex of malaria causing parasites
- The Francis Crick Institute, United Kingdom
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.
Data availability
The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE (62) partner repository with the dataset identifier PXD039125.Source data for all Western Blots and PCRs is shown in two separate Source data files.
Article and author information
Author details
Malgorzata Broncel
Funding
Francis Crick Institute (CC2132)
- Heledd Davies
- Hugo Belda
- Malgorzata Broncel
- Jill Dalimot
- Moritz Treeck
Francis Crick Institute (CC0199)
- Malgorzata Broncel
- Moritz Treeck
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
© 2023, Davies et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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PerTurboID, A targeted in situ method reveals the impact of kinase deletion on its local protein environment in the cytoadhesion complex of malaria causing parasites
eLife 12:e86367.
https://doi.org/10.7554/eLife.86367
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