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. 2017 Apr 27:5:e3128.
doi: 10.7717/peerj.3128. eCollection 2017.

Membrane and luminal proteins reach the apicoplast by different trafficking pathways in the malaria parasite Plasmodium falciparum

Rahul Chaudhari  1 , Vishakha Dey  2 , Aishwarya Narayan  2 , Shobhona Sharma  1 , Swati Patankar  2
Affiliations

Membrane and luminal proteins reach the apicoplast by different trafficking pathways in the malaria parasite Plasmodium falciparum

Rahul Chaudhari et al. PeerJ. .

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.

Keywords: Acyl-carrier protein (ACP-GFP); Apicoplast; Glutathione peroxidase like thioredoxin peroxidase (PfTPxGl); P. falciparum; Protein trafficking.

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Conflict of interest statement

The authors declare there are no competing interests.

Figures

Figure 1
Figure 1. Immunofluorescence images show PfTPxGl, microtubules, ACP-GFP, PfACP and PfEMP1 trafficking in AlF4−-treated D10-ACPleader-GFP parasites.
(A) PfTPxGl, ACP-GFP, PfACP and PfEMP1 localization in control parasites, (B) PfTPxGl, ACP-GFP, PfACP and PfEMP1 localization in AlF4−-treated parasites. For D10-ACPleader-GFP parasites, 98% of the 115 parasites analyzed showed disrupted PfTPxGl signal while 96% of the 147 parasites analyzed showed arrest of PfEMP1 in the parasites. Scale Bar: 10 µm, (C) Processing of apicoplast targeted protein ACP-GFP is not affected in drug-treated parasites. Western blot showing the processing of apicoplast targeted ACP-GFP and PfTPxGl in AlF4−- treated parasites. In ACP-GFP panel, upper band indicated with an arrowhead represents an unprocessed form of ACP-GFP while lower band represents processed form of ACP-GFP.
Figure 2
Figure 2. Immunofluorescence images show PfTPxGl, microtubules, ACP-GFP, PfACP and PfEMP1 trafficking in vinblastine-treated D10-ACPleader-GFP parasites.
(A) PfTPxGl, microtubules, ACP-GFP, PfACP and PfEMP1 localization in solvent control parasites, (B) PfTPxGl, microtubules, ACP-GFP, PfACP and PfEMP1 localization in vinblastine treated parasites. In these experiments, targeting to the apicoplast was inhibited in 94% of the parasites with vinblastine treatment (33 parasites counted) while an arrest of PfEMP1 was observed in 97% of the parasites (134 parasites counted). Scale Bar: 10 µm.
Figure 3
Figure 3. 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 vinblastine treatment. In these experiments, localization of PfTPxGl was reverted to the apicoplast in 47% parasites, while remaining 53% parasites showed mitochondrial localization (23 parasites counted). Scale Bar: 10 µm.
Figure 4
Figure 4. Immunofluorescence images showing the endoplasmic reticulum (ER) morphology in AlF4− and vinblastine treated parasites.
(A) PfBiP localization in control parasites for AlF4− treatment, (B) ER morphology in AlF4−- treated parasites (14 parasites were counted, none showed dispersal of ER structure), (C) PfBiP localization in solvent (PBS) control parasites for vinblastine treatment, (D) ER morphology in vinblastine-treated parasites (26 parasites were counted, none showed dispersal), (E) ER morphology in parasites reverted after vinblastine treatment (27 parasites counted). Scale Bar: 10 µm.
Figure 5
Figure 5. Immunofluorescence images showing the Golgi morphology in AlF4− and vinblastine treated parasites.
(A) PfGRASP localization in control parasites for AlF4− treatment, (B) Golgi morphology in AlF4−- treated parasites (17 parasites counted, Golgi structure was dispersed in 95% parasites), (C) PfGRASP localization in solvent (PBS) control parasites for vinblastine treatment, (D) Golgi morphology in vinblastine-treated parasites (18 parasites counted, Golgi structure was dispersed in 95% parasites), (E) Golgi morphology in parasites reverted after vinblastine treatment (11 parasites counted, Intact Golgi structure was observed in 90% parasites). Scale Bar: 10 µm.
Figure 6
Figure 6. PfTPxGl localization to the outermost membrane of the apicoplast in D10-ACPleader-GFP parasites.
(A) Western blots showing association of PfTPxGl with the organellar membranes (S-Supernatant, P-Pellet) after hypotonic lysis, (B) Staining of the isolated organellar fraction with MitoTracker Red, (C) Localization of PfTPxGl to the membranes of intact/permeabilized apicoplasts from control parasites, (D) Absence of PfTPxGl in the membranes of intact/permeabilized apicoplasts from AlF4− treated parasites. Scale Bars as indicated in the figures. (E) Thermolysin treatment of isolated organelles demonstrates outermost membrane localization of PfTPxGl.
Figure 7
Figure 7. Schematic representation of secretory protein targeting pathways in Plasmodium falciparum.
Arrows indicate the direction of the secretory protein traffic.
See this image and copyright information in PMC

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Grants and funding

This work was supported by grants from the Science and Engineering Research Board (SERB, Project File no. SB/YS/LS-354/2013) and the Board of Research in Nuclear Sciences (BRNS, Project File no. 2013/37B/18/BRNS). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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