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
Curation statements for this article:-
Curated by eLife
eLife assessment
This important study combines conditional mutagenesis with proximity labeling to evaluate alterations in a sub-cellular proteome upon a perturbing event. The approach is applied to the deletion of a kinase involved in trafficking of adhesins to the malaria parasite-infected erythrocyte surface and the evidence supporting the conclusions is compelling. The work will be of broad interest to cell biologists and biochemists.
This article has been Reviewed by the following groups
Listed in
- Evaluated articles (eLife)
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 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.
Article activity feed
-
-
Author Response
Reviewer #1 (Public Review):
Davies et al. examined the role of the malaria parasite's FIKK4.1 protein kinase in trafficking and host membrane insertion of key proteins that are exported by the intracellular P. falciparum parasite. FIKK4.1 is one of 18 FIKK serine/threonine kinases exported into the host erythrocyte; these kinases phosphorylate both host proteins and exported parasite proteins. FIKK4.1 has previously been implicated in rigidification of the erythrocyte cytoskeleton. It is also known to affect trafficking and insertion of PfEMP1, the parasite's primary cytoadherence ligand, on the host cell surface. In the present studies, the authors perform sophisticated gene-editing experiments that combine conditional knockout of FIKK4.1 with tagging of two kinase targets with the TurboID proximity biotin-labeling …
Author Response
Reviewer #1 (Public Review):
Davies et al. examined the role of the malaria parasite's FIKK4.1 protein kinase in trafficking and host membrane insertion of key proteins that are exported by the intracellular P. falciparum parasite. FIKK4.1 is one of 18 FIKK serine/threonine kinases exported into the host erythrocyte; these kinases phosphorylate both host proteins and exported parasite proteins. FIKK4.1 has previously been implicated in rigidification of the erythrocyte cytoskeleton. It is also known to affect trafficking and insertion of PfEMP1, the parasite's primary cytoadherence ligand, on the host cell surface. In the present studies, the authors perform sophisticated gene-editing experiments that combine conditional knockout of FIKK4.1 with tagging of two kinase targets with the TurboID proximity biotin-labeling enzyme to explore phosphorylation-dependent changes in target protein localization, structure, or protein-protein interactions. Using conditional knockout of each exported FIKK kinase, they determine that FIKK4.1 is the only kinase that regulates PfEMP1 surface exposure and that it does not appear to modulate surface translocation of RIFINs, a family of parasite antigens involved in immune evasion. The combination of gene-editing, proximity labeling and mass spectrometry, and biochemical studies in the paper is to be lauded. These findings identify key targets of exported kinases and will guide future studies of host cell remodeling.
Key limitations of the study:
- TurboID tagging of FIKK4.1 followed by proximity labeling and mass spectrometry of biotinylated proteins revealed parasite-stage dependent labeling of 101 parasite proteins and 39 human proteins that come in contact with FIKK4.1. Although TurboID is a more efficient biotin ligase produced through directed evolution, nonspecific biotinylation of proteins that do not form biologically relevant interactions remains an issue. Biotin addition for 4 hours, as used here and in most studies using this ligase, allows for labeling of proteins that undergo random collisions with the TurboID-tagged protein. While there was clear enrichment of exported proteins in the FIKK4.1-tagged parasite at mature schizont stages when FIKK4.1 is in the host cytosol, only 66% of the proteins labeled were exported, consistent with labeling and recovery of irrelevant proteins. As the authors performed appropriate controls and interpreted their findings cautiously, this limitation results primarily from finite efficiency of TurboID, trace levels of endogenous biotin within cells, and other complexities associated with the technology.
We agree with the reviewer that there are limitations to TurboID and the mere presence of a protein in a dataset does not imply functional relevance (which is also true for IP data). However, it is highly complementary to data obtained through other methods (in our case previous cytoadhesion data and phosphoproteome data) and as we show here, can give high resolution information on the local protein environment of a protein. This is illustrated by highly significant protein-specific interaction datasets for PTP4 and KAHRP obtained from biological triplicate experiments. The site-specific protocol we use later in the paper allows us to eliminate unbiotinylated proteins non-specifically binding to beads which is a major advantage, evidenced by the much higher ratio of exported proteins observed in the PTP4 and KAHRP-turboID datasets.
- The production of dual-edited parasites carrying conditional knockout of FIKK4.1 and TurboID tagging of either KAHRP or PTP4 permitted examination of changes in localization of exported proteins upon their phosphorylation by FIKK4.1. KAHRP and PTP4 are excellent choices for these experiments because they are established targets of the kinase and good candidates for effectors involved in PfEMP1 membrane insertion. Some 30-40 proteins exhibited significant changes in biotinylation by these TurboID-tagged proteins, suggesting altered localization or structure upon loss of FIKK4.1 kinase activity. PfEMP1 trafficking proteins (PTPs), Maurer's cleft proteins, exported heat shock proteins, and components of PSAC, a parasite-associated nutrient uptake channel, all exhibited changes. Although FIKK4.1 is not essential for in vitro parasite propagation, altered localization could result either directly from changes in phosphorylation status of the protein itself or could reflect indirect effects on the cell from loss of FIKK4.1.
The reviewer is correct in that we cannot exclude that it is not only loss of FIKK4.1 mediated phosphorylation sites that leads to the observed changes, but that the loss of the FIKK4.1 kinase domain affects the localisation of other proteins. Conditional inactivation of the FIKK4.1 kinase domain while retaining the overall protein would have been a more elegant approach. However, we do not predict the kinase domain of FIKK4.1 to be a strong structural component given that kinase domains often evolved to have low affinity interactions with their multiple targets and are less likely to act as scaffolding parts. As the reviewer points out, because we observed no growth defect upon deletion of FIKK4.1. Therefore we can be quite certain that the observed changes are not due to indirect effects caused by differences in growth but are a direct effect by the loss of the kinase domain and FIKK4.1’s enzymatic activity.
- As a consequence of these two limitations, these experiments could not conclusively implicate either KAHRP or a specific PTP in PfEMP1 surface translocation. Whether specific Maurer's cleft proteins or the nutrient channel components contribute to PfEMP1 surface translocation could also not be addressed. The authors' Discussion section is appropriately cautious in interpreting changes in biotinylation upon FIKK4.1 disruption. Although a large amount of data has been generated in this sophisticated study, the precise mechanism of PfEPM1 trafficking and membrane insertion remains elusive.
We agree with the reviewer that we do not definitively explain the mechanism of FIKK4.1 in PfEMP1 surface translocation. But we identify several promising candidates for modulating its effect, some of which (for example PTP4) have previously shown to be relevant for PfEMP1 surface translocation. We also identify unexpected proteins which can now be investigated further. New methods in high resolution Cryo-EM imaging may allow us to image individual protein density in knobs and visualize the observed changes in the future. Further PerTurboID experiments with individual components will likely draw an ever finer picture. Here we focus on emphasising the potential of PerTurboID for identifying connections between proteins, and to observe changes to protein characteristics which would be missed by other techniques.
Reviewer #2 (Public Review):
Davies et al combine TurboID with conditional mutagenesis to reveal how a perturbing event alters the accessibility of a sub-cellular proteome to proximity biotinylation. The approach builds on established techniques for antibody-mediated enrichment of biotinylated peptides (rather than purification of whole biotinylated proteins by avidin) to enable mapping of the specific lysines that are biotinylated by TurboID and how access to these sites changes between conditions. The insights gained have a range of potential implications touching on protein trafficking/localization, complex dynamics and membrane topology. The authors apply this strategy to study trafficking of the key P. falciparum adhesin PfEMP1 to the infected erythrocyte surface. This group has previously shown that the exported parasite kinase FIKK4.1 is important for this process but the specific mechanism is unknown. In the first part of the present study, the authors develop PerTurboID and analyze the altered biotinylation patterns upon FIKK4.1 deletion in parasite lines bearing TurboID tags on PTP4 or KAHRP, two proteins required for this pathway and likely direct substrates of FIKK4.1. Numerous changes in site-specific biotinylation are quantitatively assessed on hundreds of proteins and possible implications for these changes are discussed, including topology of parasite integral membrane proteins exported into the RBC compartment as well as how the conformation of the RhopH complex might be altered upon RBC membrane integration. In a final set of experiments, the authors show that among 18 exported FIKK kinases, FIKK4.1 is uniquely important to PfEMP1 surface display but not to the distinct RIFIN class of parasite proteins that are also trafficked to the RBC surface. On the whole, the data are compelling and provide an important new approach that advances the proximity labeling toolkit.
While the resolution of PerTurboID captures the site-specific changes in biotinylation abundance and position that occur upon loss of FIKK4.1, a limitation of the study is that these observations do not necessarily clarify the model for how FIKK4.1 is controlling the PfEMP1 trafficking pathway. The authors convincingly show that FIKK4.1 uniquely supports PfEMP1 surface presentation and cytoadhesion. However, this is not connected to the PerTurboID data in a way that provides a mechanism for how this is achieved by FIKK4.1 activity and in my opinion doesn't deliver on the title claim to "reveal the impact of kinase deletion on cytoadhesion". Certainly the changes in biotinylation suggest a range of interesting possibilities related to the accessibility and topology of proteins within and beyond the PfEMP1 trafficking pathway; however, it is hard to interpret the relationship of these changes to the process in view. For instance, deletion of FIKK4.1 increases biotinylation of several Maurer's clefts proteins in both the PTP4- and KAHRP-TurboID experiments but why this is or whether it is significant for PfEMP1 transport is unclear.
We agree with the reviewer that we do not definitively confirm the relationship between the changes observed in protein accessibility and the role of FIKK4.1 in PfEMP1 transport. We discuss a number of likely options based on what is known of the candidate genes, but validation would require extensive further work beyond the scope of this paper. We have focussed on demonstrating the value of PerTurboID as a technique for measuring molecular-level changes which would be missed by other methods, providing a list of proteins which are likely involved in modulating FIKK4.1 activity and PfEMP1 trafficking through an interconnected network. We believe the technique will be very useful for understanding gene function in other scenarios. However, we changed the title to be more specific to proteins in the cytoadhesion complex and associated proteins, and not cytoadhesion per se.
-
eLife assessment
This important study combines conditional mutagenesis with proximity labeling to evaluate alterations in a sub-cellular proteome upon a perturbing event. The approach is applied to the deletion of a kinase involved in trafficking of adhesins to the malaria parasite-infected erythrocyte surface and the evidence supporting the conclusions is compelling. The work will be of broad interest to cell biologists and biochemists.
-
Reviewer #1 (Public Review):
Davies et al. examined the role of the malaria parasite's FIKK4.1 protein kinase in trafficking and host membrane insertion of key proteins that are exported by the intracellular P. falciparum parasite. FIKK4.1 is one of 18 FIKK serine/threonine kinases exported into the host erythrocyte; these kinases phosphorylate both host proteins and exported parasite proteins. FIKK4.1 has previously been implicated in rigidification of the erythrocyte cytoskeleton. It is also known to affect trafficking and insertion of PfEMP1, the parasite's primary cytoadherence ligand, on the host cell surface. In the present studies, the authors perform sophisticated gene-editing experiments that combine conditional knockout of FIKK4.1 with tagging of two kinase targets with the TurboID proximity biotin-labeling enzyme to explore …
Reviewer #1 (Public Review):
Davies et al. examined the role of the malaria parasite's FIKK4.1 protein kinase in trafficking and host membrane insertion of key proteins that are exported by the intracellular P. falciparum parasite. FIKK4.1 is one of 18 FIKK serine/threonine kinases exported into the host erythrocyte; these kinases phosphorylate both host proteins and exported parasite proteins. FIKK4.1 has previously been implicated in rigidification of the erythrocyte cytoskeleton. It is also known to affect trafficking and insertion of PfEMP1, the parasite's primary cytoadherence ligand, on the host cell surface. In the present studies, the authors perform sophisticated gene-editing experiments that combine conditional knockout of FIKK4.1 with tagging of two kinase targets with the TurboID proximity biotin-labeling enzyme to explore phosphorylation-dependent changes in target protein localization, structure, or protein-protein interactions. Using conditional knockout of each exported FIKK kinase, they determine that FIKK4.1 is the only kinase that regulates PfEMP1 surface exposure and that it does not appear to modulate surface translocation of RIFINs, a family of parasite antigens involved in immune evasion. The combination of gene-editing, proximity labeling and mass spectrometry, and biochemical studies in the paper is to be lauded. These findings identify key targets of exported kinases and will guide future studies of host cell remodeling.
Key limitations of the study:
1. TurboID tagging of FIKK4.1 followed by proximity labeling and mass spectrometry of biotinylated proteins revealed parasite-stage dependent labeling of 101 parasite proteins and 39 human proteins that come in contact with FIKK4.1. Although TurboID is a more efficient biotin ligase produced through directed evolution, nonspecific biotinylation of proteins that do not form biologically relevant interactions remains an issue. Biotin addition for 4 hours, as used here and in most studies using this ligase, allows for labeling of proteins that undergo random collisions with the TurboID-tagged protein. While there was clear enrichment of exported proteins in the FIKK4.1-tagged parasite at mature schizont stages when FIKK4.1 is in the host cytosol, only 66% of the proteins labeled were exported, consistent with labeling and recovery of irrelevant proteins. As the authors performed appropriate controls and interpreted their findings cautiously, this limitation results primarily from finite efficiency of TurboID, trace levels of endogenous biotin within cells, and other complexities associated with the technology.
2. The production of dual-edited parasites carrying conditional knockout of FIKK4.1 and TurboID tagging of either KAHRP or PTP4 permitted examination of changes in localization of exported proteins upon their phosphorylation by FIKK4.1. KAHRP and PTP4 are excellent choices for these experiments because they are established targets of the kinase and good candidates for effectors involved in PfEMP1 membrane insertion. Some 30-40 proteins exhibited significant changes in biotinylation by these TurboID-tagged proteins, suggesting altered localization or structure upon loss of FIKK4.1 kinase activity. PfEMP1 trafficking proteins (PTPs), Maurer's cleft proteins, exported heat shock proteins, and components of PSAC, a parasite-associated nutrient uptake channel, all exhibited changes. Although FIKK4.1 is not essential for in vitro parasite propagation, altered localization could result either directly from changes in phosphorylation status of the protein itself or could reflect indirect effects on the cell from loss of FIKK4.1.
3. As a consequence of these two limitations, these experiments could not conclusively implicate either KAHRP or a specific PTP in PfEMP1 surface translocation. Whether specific Maurer's cleft proteins or the nutrient channel components contribute to PfEMP1 surface translocation could also not be addressed. The authors' Discussion section is appropriately cautious in interpreting changes in biotinylation upon FIKK4.1 disruption. Although a large amount of data has been generated in this sophisticated study, the precise mechanism of PfEPM1 trafficking and membrane insertion remains elusive.
-
Reviewer #2 (Public Review):
Davies et al combine TurboID with conditional mutagenesis to reveal how a perturbing event alters the accessibility of a sub-cellular proteome to proximity biotinylation. The approach builds on established techniques for antibody-mediated enrichment of biotinylated peptides (rather than purification of whole biotinylated proteins by avidin) to enable mapping of the specific lysines that are biotinylated by TurboID and how access to these sites changes between conditions. The insights gained have a range of potential implications touching on protein trafficking/localization, complex dynamics and membrane topology. The authors apply this strategy to study trafficking of the key P. falciparum adhesin PfEMP1 to the infected erythrocyte surface. This group has previously shown that the exported parasite kinase …
Reviewer #2 (Public Review):
Davies et al combine TurboID with conditional mutagenesis to reveal how a perturbing event alters the accessibility of a sub-cellular proteome to proximity biotinylation. The approach builds on established techniques for antibody-mediated enrichment of biotinylated peptides (rather than purification of whole biotinylated proteins by avidin) to enable mapping of the specific lysines that are biotinylated by TurboID and how access to these sites changes between conditions. The insights gained have a range of potential implications touching on protein trafficking/localization, complex dynamics and membrane topology. The authors apply this strategy to study trafficking of the key P. falciparum adhesin PfEMP1 to the infected erythrocyte surface. This group has previously shown that the exported parasite kinase FIKK4.1 is important for this process but the specific mechanism is unknown. In the first part of the present study, the authors develop PerTurboID and analyze the altered biotinylation patterns upon FIKK4.1 deletion in parasite lines bearing TurboID tags on PTP4 or KAHRP, two proteins required for this pathway and likely direct substrates of FIKK4.1. Numerous changes in site-specific biotinylation are quantitatively assessed on hundreds of proteins and possible implications for these changes are discussed, including topology of parasite integral membrane proteins exported into the RBC compartment as well as how the conformation of the RhopH complex might be altered upon RBC membrane integration. In a final set of experiments, the authors show that among 18 exported FIKK kinases, FIKK4.1 is uniquely important to PfEMP1 surface display but not to the distinct RIFIN class of parasite proteins that are also trafficked to the RBC surface. On the whole, the data are compelling and provide an important new approach that advances the proximity labeling toolkit.
While the resolution of PerTurboID captures the site-specific changes in biotinylation abundance and position that occur upon loss of FIKK4.1, a limitation of the study is that these observations do not necessarily clarify the model for how FIKK4.1 is controlling the PfEMP1 trafficking pathway. The authors convincingly show that FIKK4.1 uniquely supports PfEMP1 surface presentation and cytoadhesion. However, this is not connected to the PerTurboID data in a way that provides a mechanism for how this is achieved by FIKK4.1 activity and in my opinion doesn't deliver on the title claim to "reveal the impact of kinase deletion on cytoadhesion". Certainly the changes in biotinylation suggest a range of interesting possibilities related to the accessibility and topology of proteins within and beyond the PfEMP1 trafficking pathway; however, it is hard to interpret the relationship of these changes to the process in view. For instance, deletion of FIKK4.1 increases biotinylation of several Maurer's clefts proteins in both the PTP4- and KAHRP-TurboID experiments but why this is or whether it is significant for PfEMP1 transport is unclear.
-
Reviewer #3 (Public Review):
The authors aim to gain a more comprehensive understanding of the role of FIKK4.1 in parasite biology. To achieve this, they used a novel approach termed PerTurboID that allows them to map changes in the conformational and interaction environment of proteins that are in close proximity of the tagged gene of interest. Here the authors focus on two proteins KHARP and PTP4 who are known targets of FIKK4.1 and assessed the impact of the genetic disruption of the kinase on the interaction environment of these proteins. The experimental strategy identifies a range of changes that indicate that changes go beyond the direct targets of FIKK4.1 and therefore creates new insights of interaction networks that are regulated by this specific kinase.
The strength of this approach is not only that it can identify new …
Reviewer #3 (Public Review):
The authors aim to gain a more comprehensive understanding of the role of FIKK4.1 in parasite biology. To achieve this, they used a novel approach termed PerTurboID that allows them to map changes in the conformational and interaction environment of proteins that are in close proximity of the tagged gene of interest. Here the authors focus on two proteins KHARP and PTP4 who are known targets of FIKK4.1 and assessed the impact of the genetic disruption of the kinase on the interaction environment of these proteins. The experimental strategy identifies a range of changes that indicate that changes go beyond the direct targets of FIKK4.1 and therefore creates new insights of interaction networks that are regulated by this specific kinase.
The strength of this approach is not only that it can identify new interaction networks relating to FIKK4.1 but that serves as a proof of concept that can be used for a wide range of applications in parasite biology. At the same time as the authors have noted themselves the extent of the biotin pulse is important and most likely needs to be calibrated for every specific application. In addition, this approach is only suitable for proteins that can be tagged without impacting their function.
The authors present very convincing evidence that the PerTurboID is suitable to study FIKK kinases in parasites and have used this to shed new light on how FIKK4.1 is involved directly or indirectly in a wider range of biological activities in the parasite.
The main impact of this work is that it provides a wider understanding of the relationship between a specific kinase and structural as well as biological consequences. The methodology is also very powerful and will have a wide range of applications.
-
