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

Chaudhari, Rahul; Dey, Vishakha; Narayan, Aishwarya; Sharma, Shobhona; Patankar, Swati

doi:10.7287/peerj.preprints.2813v2

Cited by 3 publications

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“…Mutational analysis of the N terminus of PfTPx Gl defined a signal anchor sequence between residues 1-21-a signal sequence that targets the protein to the membrane of the endoplasmic reticulum. Membrane anchorage is consistent with results seen in P. falciparum organelles [13], and here, use of the heterologous system was able to define the N terminus of the protein as responsible for membrane anchorage and for targeting.…”

Section: The Use Of Toxoplasma Gondii Identifies the Nterminal Signalsupporting

confidence: 88%

“…Small molecule inhibitors have previously been used for the characterization of trafficking pathways used by PfTPx Gl to reach the apicoplast and mitochondrion. Inhibition of vesicle fusion by blocking heterotrimeric G proteins showed a complete disruption of PfTPx Gl targeting to the apicoplast and only a partial disruption of targeting to the mitochondrion [13].…”

Section: Resultsmentioning

confidence: 99%

“…Taken together, these observations led us to test whether the protein was anchored to the membrane by an N‐terminal transmembrane domain. Indeed, PfTPx Gl has been demonstrated to be membrane bound in P. falciparum .…”

Section: Resultsmentioning

confidence: 99%

“…Incidentally, the bipartite targeting signal of ACP is cleaved during this process, while the size of PfTPx Gl remains unaltered . Further investigation revealed that PfTPx Gl is anchored to the apicoplast membrane . This led us to speculate that membrane anchorage itself might play a role in the altered targeting pathway of this protein when compared with that of luminal ACP.…”

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confidence: 96%

“…Another apicoplast protein, glutathione peroxidase‐like thioredoxin peroxidase (PfTPx Gl , PlasmoDB Gene ID PF3D7_1212000) targets to the apicoplast via the Golgi, unlike the apicoplast luminal acyl carrier protein (ACP), that in the same paper was found to route independently of the Golgi . Additionally, in another report, PfTPx Gl trafficking is inhibited by treatment with tetrafluoroaluminate, which blocks the fusion of heterotrimeric G protein‐dependent vesicles to their target membranes, while ACP trafficking is completely unaffected . Incidentally, the bipartite targeting signal of ACP is cleaved during this process, while the size of PfTPx Gl remains unaltered .…”

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confidence: 99%

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Heterologous expression in Toxoplasma gondii reveals a topogenic signal anchor in a Plasmodium apicoplast protein

et al. 2018

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Glutathione peroxidase‐like thioredoxin peroxidase (Pf TP x Gl ) is an antioxidant enzyme trafficked to the apicoplast, a secondary endosymbiotic organelle, in Plasmodium falciparum . Apicoplast trafficking signals usually consist of N‐terminal signal and transit peptides, but the trafficking signal of Pf TP x Gl appears to exhibit important differences. As transfection is a protracted process in P. falciparum , we expressed the N terminus of Pf TP x Gl as a GFP fusion protein in a related apicomplexan, Toxoplasma gondii , in order to dissect its trafficking signals. We show that Pf TP x Gl possesses an N‐terminal signal anchor that takes the protein to the endoplasmic reticulum in Toxoplasma —this is the first step in the apicoplast targeting pathway. We dissected the residues important for endomembrane system uptake, membrane anchorage, orientation, spacing, and cleavage. Protease protection assays and fluorescence complementation revealed that the C terminus of the protein lies in the ER lumen, a topology that is proposed to be retained in the apicoplast. Additionally, we examined one mutant, responsible for altered Pf TP x Gl targeting in Toxoplasma , in Plasmodium . This study has demonstrated that Pf TP x Gl belongs to an emergent class of proteins that possess signal anchors, unlike the canonical bipartite targeting signals employed for the trafficking of luminal apicoplast proteins. This work adds to the mounting evidence that the signals involved in the targeting of apicoplast membrane proteins may not be as straightforward as those of luminal proteins, and also highlights the usefulness of T. gondii as a heterologous system in certain aspects of this study, such as reducing screening time and facilitating the verification of membrane topology.

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Section: The Use Of Toxoplasma Gondii Identifies the Nterminal Signalsupporting

confidence: 88%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 96%

mentioning

confidence: 99%

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Heterologous expression in Toxoplasma gondii reveals a topogenic signal anchor in a Plasmodium apicoplast protein

et al. 2018

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Live and Let Dye: Visualizing the Cellular Compartments of the Malaria Parasite Plasmodium falciparum

et al. 2019

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Dually localised proteins found in both the apicoplast and mitochondrion utilize the Golgi‐dependent pathway for apicoplast targeting in Toxoplasma gondii

Prasad

Mastud

Patankar

2020

Biology of the Cell

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Background InformationLike other apicomplexan parasites, Toxoplasma gondii harbours a four‐membraned endosymbiotic organelle – the apicoplast. Apicoplast proteins are nuclear encoded and trafficked to the organelle through the endoplasmic reticulum (ER). From the ER to the apicoplast, two distinct protein trafficking pathways can be used. One such pathway is the cell's secretory pathway involving the Golgi, whereas the other is a unique Golgi‐independent pathway. Using different experimental approaches, many apicoplast proteins have been shown to utilize the Golgi‐independent pathway, whereas a handful of reports show that a few proteins use the Golgi‐dependent pathway. This has led to an emphasis towards the unique Golgi‐independent pathway when apicoplast protein trafficking is discussed in the literature. Additionally, the molecular features that drive proteins to each pathway are not known.ResultsIn this report, we systematically test eight apicoplast proteins, using a C‐terminal HDEL sequence to assess the role of the Golgi in their transport. We demonstrate that dually localised proteins of the apicoplast and mitochondrion (TgSOD2, TgTPx1/2 and TgACN/IRP) are trafficked through the Golgi, whereas proteins localised exclusively to the apicoplast are trafficked independent of the Golgi. Mutants of the dually localised proteins that localised exclusively to the apicoplast also showed trafficking through the Golgi. Phylogenetic analysis of TgSOD2, TgTPx1/2 and TgACN/IRP suggested that the evolutionary origins of TgSOD2 and TgTPx1/2 lie in the mitochondrion, whereas TgACN/IRP appears to have originated from the apicoplast.Conclusions and SignificanceCollectively, with these results, for the first time, we establish that the driver of the Golgi‐dependent trafficking route to the apicoplast is the dual localisation of the protein to the apicoplast and the mitochondrion.

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

Abstract: 25The secretory pathway in Plasmodium falciparum has evolved to transport proteins to 26 the host cell membrane and to an endosymbiotic organelle, the apicoplast. The latter can 27 occur via the ER or the ER-Golgi route. Here, we study these three routes using proteins

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Heterologous expression in Toxoplasma gondii reveals a topogenic signal anchor in a Plasmodium apicoplast protein

Heterologous expression in Toxoplasma gondii reveals a topogenic signal anchor in a Plasmodium apicoplast protein

Live and Let Dye: Visualizing the Cellular Compartments of the Malaria Parasite Plasmodium falciparum

Dually localised proteins found in both the apicoplast and mitochondrion utilize the Golgi‐dependent pathway for apicoplast targeting in Toxoplasma gondii

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