Membrane and luminal proteins reach the apicoplast by different trafficking pathways in the malaria parasite Plasmodium falciparum
- Published
- Accepted
- Subject Areas
- Cell Biology, Parasitology
- Keywords
- Apicoplast, Acyl-carrier protein (ACP-GFP), P. falciparum, Glutathione peroxidase like thioredoxin peroxidase (PfTPxGl), Protein trafficking
- Copyright
- © 2017 Chaudhari et al.
- Licence
- This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, reproduction and adaptation in any medium and for any purpose provided that it is properly attributed. For attribution, the original author(s), title, publication source (PeerJ Preprints) and either DOI or URL of the article must be cited.
- Cite this article
- 2017. Membrane and luminal proteins reach the apicoplast by different trafficking pathways in the malaria parasite Plasmodium falciparum. PeerJ Preprints 5:e2813v2 https://doi.org/10.7287/peerj.preprints.2813v2
Abstract
The secretory pathway in Plasmodium falciparum has evolved to transport proteins to the host cell membrane and to an endosymbiotic organelle, the apicoplast. The latter can occur via the ER or the ER-Golgi route. Here, we study these three routes using proteins Erythrocyte Membrane Protein-1 (PfEMP1), Acyl Carrier Protein (ACP) and glutathione peroxidase-like thioredoxin peroxidase (PfTPxGl) and inhibitors of vesicular transport. As expected, the G protein dependent vesicular fusion inhibitor AlF4- and microtubule destabilizing drug vinblastine block the trafficking of PfEMP-1, a protein secreted to the host cell membrane. However, while both PfTPxGl and ACP are targeted to the apicoplast, only ACP trafficking remains unaffected by these treatments. This implies that G-protein dependent vesicles do not play a role in classical apicoplast protein targeting. Unlike the soluble protein ACP, we show that PfTPxGl is localized to the outermost membrane of the apicoplast. Thus, the parasite apicoplast acquires proteins via two different pathways: first, the vesicular trafficking pathway appears to handle not only secretory proteins, but an apicoplast membrane protein, PfTPxGl. Second, trafficking of apicoplast luminal proteins appear to be independent of G-protein coupled vesicles.
Author Comment
This is a version with updated figures. Subheadings (like anti-GFP, PfTPxGl) have been added for different immunofluorescence panels in Figure 6 which were missing earlier. We would like to note that these subheadings are crucial to understand the Figure 6 correctly. The content of the Figure 6 has not been changed.
Supplemental Information
Supplementary Images showing raw data for the Western blots
Combinations and dilutions of the antibodies and their method of generation and checking the specificity
Quantification data for various drug treatments
S2 (A): Percentage distribution of PfTPxGl in control, drug treated and drug washed out parasites. (B): Percentage of parasites showing intact/collapsed ER-Golgi morphology in drug treated and drug washed out cells. (C): Percentage of parasites showing PfEMP1 and KAHRP distribution in drug treated and drug washed out parasites.
Effect of Aluminum tetrafluoride (AlF4-) treatment on P. falciparum cultures.
(A) Dose response curve fit for P. falciparum treated at different concentrations of Aluminum tetrafluoride (AlF4-). IC50 value calculated non-linear regression of the sigmoidal dose response equation from OriginPro was found to be 1.23±0.16 µM. 95% confidence interval was found to be ±0.16. Note that no parasite survival was observed at higher AlF4- concentrations of 7.5 and 10 µM at 24 hours. (B) Images showing a normal morphology of P. falciparum treated with Aluminum tetrafluoride (AlF4-) and vineblastine at IC50 concentration of 1.2 µM and 100 nM respectively for 18±2 hours.
Immunofluorescence images showing PfKAHRP trafficking in control, AlF4--treated and vinblastine treated D10-ACPleader-GFP parasites
In AlF4- treatment, KAHRP trafficking to the RBC cytosol and surface was inhibited in 97% of the parasites 87 parasites analyzed. In vinblastine treated cultures, KAHRP trafficking to the RBC cytosol and surface was inhibited in 98% of the 56 parasites analyzed (See Supplementary Table 2 for quantification). Scale Bar: 10 µm.
Immunofluorescence images showing PfTPxGl and PfFC (mitochondrial marker protein) trafficking in AlF4-, nocodazole and vineblsatine treated 3D7 parasites
(A) PfTPxGl and PfFC co-localization in AlF4--treated parasites, (B) PfTPxGl and PfFC co-localization in vinblastine-treated parasites, (C) PfTPxGl and PfFC co-localization in nocodazole-treated parasites. In this experiment, PfTPxGl was found to be co-localized with the mitochondrial marker protein PfFC in 40% of the treated parasites suggesting that trafficking of PfTPxGl to the mitochondrion may be partially disrupted by the treatments. Scale Bar: 10 µm.
Immunofluorescence images showing PfTPxGl trafficking in AlF4-, nocodazole and vineblsatine treated 3D7 parasites
(A) PfTPxGl localization in solvent control parasites, (B) PfTPxGl localization in AlF4--treated parasites, (C) PfTPxGl localization in nocodazole-treated parasites, (D) PfTPxGl localization in vinblastine-treated parasites, (E) PfTPxGl localization in parasites reverted after nocodazole treatment, (F) PfTPxGl localization in parasites reverted after vinblastine treatment. PfTPxGl targeting was disrupted in 97% (35 parasites counted) of nocodazole-treated parasites and in 96% (33 parasites counted) of vinblastine-treated parasites. For AlF4--treatment, 92% of the 72 parasites analyzed showed disrupted PfTPxGl signal. In reversion experiments, localization of PfTPxGl in vinblastine washed out parasites was reverted to the apicoplast in 52% parasites (48 parasites counted), while in nocodazole washed out parasites 49% showed apicoplast localization (59 parasites counted) (See Supplementary Table 2 for quantification). Scale Bar: 10 µm.
Immunofluorescence images showing PfTPxGl and microtubules in nocodazole-treated D10-ACPleader-GFP parasites
(A) In these experiments, targeting to the apicoplast was inhibited in 97% of the parasites with nocodazole treatment (35 parasites counted), (B) Immunofluorescence images showing PfTPxGl and microtubules in D10-ACPleader-GFP parasites with drug washed out. Reversion of PfTPxGl localization to the organelles and intact microtubular structures observed in parasites in drug washed out medium after nocodazole treatment. In reversion experiment, localization of PfTPxGl in nocodazole washed out parasites was reverted to the apicoplast in 45% parasites (22 parasites counted). Scale Bar: 10 µm.
Immunofluorescence images showing the endoplasmic reticulum (ER) morphology in AlF4- and vinblastine treated parasites
(A) PfBiP localization in control parasites, (B) ER morphology in AlF4-- treated parasites (C) ER morphology in vinblastine-treated parasites, (D) ER morphology in parasites reverted after vinblastine treatment. Scale Bar: 10 µm.