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 Plasmodium 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 two 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.
Keywords: P. falciparum; cell biology; host–pathogen interaction; infectious disease; malaria; mass-spectrometry; microbiology; parasitic diseases; phosphoregulation; protein kinases.
Enzymes known as protein kinases regulate a huge variety of biological processes inside cells by attaching small tags known as phosphate groups onto specific locations on certain proteins. For example, the parasite that causes malaria infections in humans and great apes, injects a protein kinase called FIKK4.1 into certain cells in its host. This enzyme then adds phosphate groups to various parasite and host proteins that, in turn, causes them to form a large group of proteins (known as the cytoadhesion complex) to protect the parasite from being cleared by the hosts’ immune defences. However, it remains unclear how and where the complex forms, and how the parasite regulates it. Proximity labelling is a well-established method that allows researchers to label and identify proteins that are near to a protein of interest. To investigate how the FIKK4.1 enzyme alters host cells to make the cytoadhesion complex, Davies et al. combined proximity labelling with methods that disturb the normal state of cells at a specific timepoint during development. The team used this new approach – named PerTurboID – to identify the proteins surrounding three components in the cytoadhesion complex. This made it possible to create a map of proteins that FIKK4.1 is likely to modify to build and control the cytoadhesion complex. Further experiments examined what happened to these surrounding proteins when FIKK4.1 was inactivated. This revealed that some protein targets of FIKK4.1 become either more or less accessible to other enzymes that attach a molecule known as biotin to proteins. This could be a result of structural changes in the cytoadhesion complex that are normally regulated by the FIKK4.1 kinase. In the future, PerTurboID may be useful to study how genetics or environmental changes affect other groups of proteins within specific environments inside cells, such as protein complexes required for DNA replication or cell division, or assembly of temporal structures required for cell movement.
© 2023, Davies et al.