Inner membrane complex proteomics reveals a palmitoylation regulation critical for intraerythrocytic development of malaria parasite

  1. Pengge Qian
  2. Xu Wang
  3. Chuan-Qi Zhong
  4. Jiaxu Wang
  5. Mengya Cai
  6. Wang Nguitragool
  7. Jian Li
  8. Huiting Cui
  9. Jing Yuan

  • Curated by eLife

    eLife logo

    Evaluation Summary:

    This paper is of interest to scientists within the field of apicomplexans cytoskeleton and malaria parasite proliferation. A series of compelling experimental manipulations identify potential new pellicle proteins and dissect the role of a protein acyl-transferase for the development of the intraerythrocytic stages of Plasmodiun yoelii and the palmitoylation status of two potential substrates.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

Reviewed by

Read the full article See related articles

Listed in

Log in to save this article

Abstract

Malaria is caused by infection of the erythrocytes by the parasites Plasmodium . Inside the erythrocytes, the parasites multiply via schizogony, an unconventional cell division mode. The inner membrane complex (IMC), an organelle located beneath the parasite plasma membrane, serving as the platform for protein anchorage, is essential for schizogony. So far, the complete repertoire of IMC proteins and their localization determinants remain unclear. Here we used biotin ligase (TurboID)-based proximity labeling to compile the proteome of the schizont IMC of the rodent malaria parasite Plasmodium yoelii . In total, 300 TurboID-interacting proteins were identified. 18 of 21 selected candidates were confirmed to localize in the IMC, indicating good reliability. In light of the existing palmitome of Plasmodium falciparum , 83 proteins of the P. yoelii IMC proteome are potentially palmitoylated. We further identified DHHC2 as the major resident palmitoyl-acyl-transferase of the IMC. Depletion of DHHC2 led to defective schizont segmentation and growth arrest both in vitro and in vivo. DHHC2 was found to palmitoylate two critical IMC proteins CDPK1 and GAP45 for their IMC localization. In summary, this study reports an inventory of new IMC proteins and demonstrates a central role of DHHC2 in governing the IMC localization of proteins during the schizont development.

Article activity feed

  1. Version published to 10.7554/elife.77447 on eLife
  2. eLife

    Author Response

    Reviewer #1 (Public Review):

    Apicomplexan parasites, including the malaria parasite Plasmodium, possess a characteristic inner membrane complex (IMC), which plays an essential role in maintaining the parasite shape and regulating motility. In this interesting study, Qian and colleagues determine the IMC proteome of erythrocytic stages in the rodent malaria parasite Plasmodium yoelii, and identify the palmitoyl-acyl-transferase DHHC2 as a key enzyme that regulates the localization of IMC proteins through palmitoylation. The authors used a proximity biotinylation strategy based on TurboID to identify by mass spectrometry 300 proteins associated with the IMC. Using genetic tagging they could confirm IMC localization of 19 out of 22 selected candidate proteins. This analysis revealed that many of the IMC proteins are predicted to undergo palmitoylation, a modification that is known to play a role in protein localization to the IMC. The authors identified 3 candidate IMC palmitoyl-acyl-transferase, including DHHC2, which was most highly expressed and predicted to interact with many of the identified IMC proteins. Using conditional protein depletion based on the auxin degron system, the authors demonstrate that DHHC2 is essential for blood stage growth, schizont segmentation and merozoite invasion, and show that DHHC2 palmitoylates GAP45 and CDPK1, two essential IMC proteins. Altogether this study provides a comprehensive view of the IMC proteome and the role of palmitoylation in Plasmodium erythrocytic stages.

    The study identifies a large number of putative IMC proteins, with only partial overlap with other studies performed in P. falciparum. This suggests a potentially high rate of false positives, although 11 out of 14 new proteins were confirmed to be localized in the IMC. The authors should explain how they selected the 14 candidate proteins. This is important to ensure the absence of bias. Do these proteins correspond to the 22 proteins with assigned GO term IMC (line 219)? It is likely that many of the identified proteins correspond to trafficking-related proteins, as discussed in the text, or plasma membrane proteins (such as PMP1) or cytosolic proteins (since the ligase is exposed on the cytosolic face of the IMC).

    In the initial manuscript of this study, we chosen 22 hits for their protein subcellular localization test. There are 8 protein hits previously validated localizing at IMC in other Plasmodium species and 14 hit candidates whose localization at IMC have not been validated in the Plasmodium. These 22 hits were chosen basically in a random manner, but they showed various (high, middle, and low) levels in the enrichment ratio detected by the TurboID-mediated proximity labeling (Figure 2A).

    Reviewer #3 (Public Review):

    In this manuscript, the authors use TurboID proximity labeling to identify novel components of the Plasmodium inner membrane complex. They then verify many of the identified candidate proteins, demonstrating the utility of the approach. They build on this by identifying the major palmitoylacyltransferase involved in tethering IMC proteins to the membranes of the organelles and directly test the importance of palmitoylation in the trafficking and function of several key IMC proteins. The experiments are supported by extensive controls throughout the paper. Overall, this is a robust paper that provides an important addition to the field. Specific comments are below.

    1. line 149 and S2A. The authors use the term "signal peptide" for the N-terminal 20 amino acids of ISP1 that target TurboID to the IMC

    a. "signal peptide" is likely to be confused with a secretory signal peptide for entrance into the lumen of the ER/golgi. There is no predicted signal peptide on ISP1 (Signal P Prediction), instead ISP1 is likely both myristoylated and palmitoylated as previously shown in T. gondii. This tethers the protein to the cytoplasmic face of the IMC (and potentially to vesicles targeting to the IMC).

    b. Line 565-573 This becomes more confusing in the discussion when the authors claim that the "signal peptide" directs the fusion through the ER/Golgi secretory pathway. Together, this would be better stated as an "IMC targeting peptide" to avoid confusion with this well-established nomenclature (and modify the discussion accordingly)

    Thanks for reviewer’s good suggestion. We changed the “IMC signal peptide” to the “IMC targeting peptide” through the text.

    1. Regarding predicted acylation of TurboID identified IMC protein candidates

    a) It would be useful to state how many of the proteins are predicted to be palmitoylated and add this to table S1.

    We have added a new column containing related information in Table S1.

    b) Since myristoylation also plays a role in IMC trafficking, it would also be useful to state how many of the proteins are predicted to be myristoylated (also add to S1).

    We have added a new column containing related information in Table S1

  3. eLife

    Evaluation Summary:

    This paper is of interest to scientists within the field of apicomplexans cytoskeleton and malaria parasite proliferation. A series of compelling experimental manipulations identify potential new pellicle proteins and dissect the role of a protein acyl-transferase for the development of the intraerythrocytic stages of Plasmodiun yoelii and the palmitoylation status of two potential substrates.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

  4. eLife

    Reviewer #1 (Public Review):

    Apicomplexan parasites, including the malaria parasite Plasmodium, possess a characteristic inner membrane complex (IMC), which plays an essential role in maintaining the parasite shape and regulating motility. In this interesting study, Qian and colleagues determine the IMC proteome of erythrocytic stages in the rodent malaria parasite Plasmodium yoelii, and identify the palmitoyl-acyl-transferase DHHC2 as a key enzyme that regulates the localization of IMC proteins through palmitoylation. The authors used a proximity biotinylation strategy based on TurboID to identify by mass spectrometry 300 proteins associated with the IMC. Using genetic tagging they could confirm IMC localization of 19 out of 22 selected candidate proteins. This analysis revealed that many of the IMC proteins are predicted to undergo palmitoylation, a modification that is known to play a role in protein localization to the IMC. The authors identified 3 candidate IMC palmitoyl-acyl-transferase, including DHHC2, which was most highly expressed and predicted to interact with many of the identified IMC proteins. Using conditional protein depletion based on the auxin degron system, the authors demonstrate that DHHC2 is essential for blood stage growth, schizont segmentation and merozoite invasion, and show that DHHC2 palmitoylates GAP45 and CDPK1, two essential IMC proteins. Altogether this study provides a comprehensive view of the IMC proteome and the role of palmitoylation in Plasmodium erythrocytic stages.

    Strengths:

    -This study provides a comprehensive view of the IMC proteome and identifies new IMC proteins
    -The work is robust and involves cutting-edge approaches such as TurboID (used for the first time in Plasmodium), auxin degron system, CRISPR-Cas9 targeted mutagenesis of DHHC2 substrates.
    -This study convincingly demonstrates the role of DHHC2 in the palmitoylation of IMC proteins in Plasmodium blood stages, providing potential targets for the development of novel anti-malarial strategies.

    Limitations:

    -The screen identified a large number of candidate IMC proteins, raising the possibility that some are false positives.
    -There could be alternative explanations for the observed depletion of GAP45 and CDPK1 from the IMC upon mutagenesis, DHHC2 depletion or 2BP treatment, including lower expression or disruption of the IMC.

  5. eLife

    Reviewer #2 (Public Review):

    In this research article, Quian et al. investigate the composition of the inner membrane complex (IMC) of Plasmodiun yoelii schizonts by proximity labelling and identify 11 new proteins in the pellicle of the parasite attesting for the robustness of the enrichment. They also dissect the role of DHHC2, an IMC-resident protein acyl-transferase by generating an inducible knock-down of this enzyme. By doing so, they convincingly demonstrate the essential role of DHHC2 for the survival of the parasite in the mouse model and show that it is required for the intraerythrocytic development of the parasites and more specifically for the segmentation of the schizonts. In addition, the authors demonstrate that GAP45 and CDPK1 are palmitoylated proteins that are not found associated to the membrane in DHHC2 depleted parasites.

    Finally, point mutations have been generated in these two proteins to determine the cysteine(s) that might be palmitoylated by DHHC2.

    Overall the article is well written, the experiment are well conducted, well controlled and analyzed and the role of DHHC2 in the parasite development is nicely illustrated. However, the dissection of GAP45 and CDPK1 needs further analysis and discussion, respectively. Indeed, on one hand, the GAP45 construct used to assess the IMC targeting palmitoylation sites might be already altered by the C-terminal tagging while on the other hand, CDPK1 is described in the literature as a plasma membrane protein like its orthologue TgCDPK3.

    The data presented in this study are of high quality. Some immunofluorescence needs to be improved in their brightness to better determine where are localized the proteins of interest and some inconsistencies of MSP1 staining between the figures need to be discussed (likely resulting from different timing of auxin and 2-bromopalmitate exposure).

  6. eLife

    Reviewer #3 (Public Review):

    In this manuscript, the authors use TurboID proximity labeling to identify novel components of the Plasmodium inner membrane complex. They then verify many of the identified candidate proteins, demonstrating the utility of the approach. They build on this by identifying the major palmitoylacyltransferase involved in tethering IMC proteins to the membranes of the organelles and directly test the importance of palmitoylation in the trafficking and function of several key IMC proteins. The experiments are supported by extensive controls throughout the paper. Overall, this is a robust paper that provides an important addition to the field. Specific comments are below.

    1. line 149 and S2A. The authors use the term "signal peptide" for the N-terminal 20 amino acids of ISP1 that target TurboID to the IMC
    a. "signal peptide" is likely to be confused with a secretory signal peptide for entrance into the lumen of the ER/golgi. There is no predicted signal peptide on ISP1 (Signal P Prediction), instead ISP1 is likely both myristoylated and palmitoylated as previously shown in T. gondii. This tethers the protein to the cytoplasmic face of the IMC (and potentially to vesicles targeting to the IMC).

    b. Line 565-573 This becomes more confusing in the discussion when the authors claim that the "signal peptide" directs the fusion through the ER/Golgi secretory pathway.

    Together, this would be better stated as an "IMC targeting peptide" to avoid confusion with this well-established nomenclature (and modify the discussion accordingly)

    2. Regarding predicted acylation of TurboID identified IMC protein candidates
    a) It would be useful to state how many of the proteins are predicted to be palmitoylated and add this to table S1.
    b) Since myristoylation also plays a role in IMC trafficking, it would also be useful to state how many of the proteins are predicted to be myristoylated (also add to S1).

  7. Version published to 10.1101/2022.01.28.478263v1 on bioRxiv