Immunofluorescence microscopy of PfCRT-GFP over-expressing parasites treated with either Brefeldin A or Dynasore.

<p>PfCRT was over-expressed as a GFP-fusion protein using an ATet-inducible expression system and treated with either BFA (5 µg/mL) for 3 h or Dynasore (40 µM/mL) for 2 h. As a control a second parasite population was treated with an equivalent volume of carrier alone (ethanol and DMSO, respectively). Following the BFA treatment, immunofluorescence microscopy was performed on fixed cells with mouse anti-GFP, Alexa-594 goat anti-mouse IgG and DAPI. Representative parasites in trophozoite and schizont stage are shown for control and treated parasites. (<b>A</b>) The control parasites show the restricted FV localisation of PfCRT-GFP (false-coloured in green). (<b>B</b>) BFA treated parasites show an accumulation of fluorescence around the DAPI-stained nuclei, consistent with ER localisation in addition to the FV localisation (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038781#pone-0038781-g001" target="_blank">Figure 1</a>). (<b>C</b>) Treatment of parasites with Dynasore resulted in an accumulation of fluorescence around the DAPI-stained nuclei in addition to the FV membrane localisation, similar to the observed effect of BFA treatment (<b>B</b>).</p

IC₅₀ of different <i>P. falciparum</i> transgenic lines and treatments after 48 hours chloroquine treatment.

<p>IC₅₀ of different <i>P. falciparum</i> transgenic lines and treatments after 48 hours chloroquine treatment.</p

An FtsH Protease Is Recruited to the Mitochondrion of <i>Plasmodium falciparum</i>

<div><p>The two organelles, apicoplast and mitochondrion, of the malaria parasite <i>Plasmodium falciparum</i> have unique morphology in liver and blood stages; they undergo complex branching and looping prior to division and segregation into daughter merozoites. Little is known about the molecular processes and proteins involved in organelle biogenesis in the parasite. We report the identification of an AAA+/FtsH protease homolog (<i>Pf</i>FtsH1) that exhibits ATP- and Zn²⁺-dependent protease activity. <i>Pf</i>FtsH1 undergoes processing, forms oligomeric assemblies, and is associated with the membrane fraction of the parasite cell. Generation of a transfectant parasite line with hemagglutinin-tagged <i>Pf</i>FtsH1, and immunofluorescence assay with anti-<i>Pf</i>FtsH1 Ab demonstrated that the protein localises to <i>P. falciparum</i> mitochondria. Phylogenetic analysis and the single transmembrane region identifiable in <i>Pf</i>FtsH1 suggest that it is an i-AAA like inner mitochondrial membrane protein. Expression of <i>Pf</i>FtsH1 in <i>Escherichia coli</i> converted a fraction of bacterial cells into division-defective filamentous forms implying a sequestering effect of the <i>Plasmodium</i> factor on the bacterial homolog, indicative of functional conservation with <i>Ec</i>FtsH. These results identify a membrane-associated mitochondrial AAA+/FtsH protease as a candidate regulatory protein for organelle biogenesis in <i>P. falciparum</i>.</p> </div

Analysis of the <i>de novo</i> biogenesis and development of the FV in live cells using PfCRT-GFP as a marker of the FV membrane.

<p>PfCRT was over-expressed as a GFP-fusion protein using an ATc-inducible expression system. (<b>A</b>) Live DAPI-stained cells were imaged. The earliest detectable and distinct localisation of PfCRT-GFP was observed in mid ring stage parasites, co-localising with a round spherical shape in proximity to the DAPI-stained nucleus. The characteristic dark haemozoin crystal was not yet visible in these parasites. In addition to FV labelling, some fluorescence was detectable at the ER. PfCRT-GFP labelling of the FV membrane was observed throughout the whole intra-erythrocytic life cycle. In accordance with an increase of the haemozoin crystal, the ring-like labelling of the FV membrane expanded as the parasite grew. An unexpected second PfCRT-GFP-labelled sphere was observed in schizont stage parasites. In some parasites this additional structure was associated/attached to the FV membrane (lower schizont panel). (<b>B</b>) An overlay with the corresponding brightfield (BF) image shows that in some parasites the additional PfCRT-GFP enclosed compartment (white arrow) is separate from the FV and surrounds a dark structure, possibly haemozoin.</p

Over-expression of sensitive and resistant form of PfCRT as GFP-fusion protein.

<p>(<b>A</b>) Schematic representation of the inducible expression plasmid pT150KPfCRTGFP. Expression of the transcription activator TATi3 is under the control of the 5' UTR of PTEX150. In the presence of the transcription inhibitor anhydrotetracycline (ATc), TATi3-binding to the Tet operon (TetO) is inhibited and no expression of GFP-tagged PfCRT occurs. In the absence of ATc, TATi3 binds to TetO, initiating the over-expression of PfCRT-GFP. Cam5': 5' UTR of Calmodulin, hDHFR: human dihydrofolate reductase. (<b>B</b>) Western Blot analysis of induced over-expression of sensitive (PfCRT^S) and resistant (PfCRT^R) form of PfCRT as GFP-fusion. Presence of PfCRT^S and PfCRT^R as GFP-fusions, labelled with mouse anti-GFP, is confirmed by the presence of a band at 62 kDa only in the parasites cultured in the absence of ATc. <i>P. falciparum</i> wild-type strain 3D7 represents the negative control. Labelling with rabbit anti-GAPDH shows equal loading in these lanes. (<b>C</b>) Fluorescence microscopy of GFP-fusions of sensitive (PfCRT^S, top) and resistant (PfCRT^R, bottom) form of PfCRT. PfCRT^S and PfCRT^R were over-expressed as GFP-fusion proteins using an ATc-inducible expression system. Live cell images of DAPI-stained infected red blood cells show that both forms of PfCRT localise to the FV membrane. Later stages of the asexual life cycle show ring/dot-like structures (white arrow) within the FV, possibly degraded GFP-fusion proteins.</p

Defective cytokinesis observed in a fraction of <i>E. coli</i> cells expressing <i>Pf</i>FtsH_int.

<p><i>E. coli</i> cells transformed with pGEX + RIG or pGEX-<i>Pf</i>FtsH_int +RIG were grown for 3 h at 20°C after induction, fixed and stained with DAPI.</p

Localization of <i>Pf</i>FtsH1 to the mitochondrion is confirmed by immunofluorescence with anti-FtsH1 Ab.

<p>Confocal immunofluorescence microscopy of <i>P. falciparum</i> 3D7 infected erythrocytes using Mitotracker Red and anti-FtsH1 Ab shows localization of <i>Pf</i>FtsH1 in the parasite mitochondrion.</p

ATP- and Zn²⁺-dependent protease activity of <i>Pf</i>FtsH1.

<p>(A) <i>Pf</i>FtsH1 cleaves α-casein in a time-dependent manner in the presence of zinc and ATP. Addition of EDTA inhibits protease activity of <i>Pf</i>FtsH1 indicating the requirement of Zn²⁺ for <i>Pf</i>FtsH1-catalysed proteolysis. (B) <i>Pf</i>FtsH1 binds ATP as indicated by quenching of intrinsic fluorescence from the single tryptophan residue of the recombinant protein upon incubation with ATP. (C) ATP hydrolysis, and not just binding of the nucleotide to <i>Pf</i>FtsH1, is required for proteolytic activity. Proteolysis of α-casein was measured in the presence of ATP or its non-hydrolysable analog AMPPNP, and control sets lacking nucleotide or the enzyme. Proteolysis was observed only in the presence of ATP.</p

Recombinant expression of <i>Pf</i>FtsH1 in <i>E. coli</i> and its detection in the parasite lysate.

<p>(A) Line-drawing showing <i>Pf</i>FtsH1 stretches expressed in <i>E. coli</i> and the probable protein cleavage site. (B) Purified GST-<i>Pf</i>FtsH_int visualised in a coomassie-stained SDS-PA gel (left panel) and western blot analysis of purified protein using anti-GST Ab (right panel). (C) Purified His-<i>Pf</i>FtsH1 ATPase + protease domain on a coomassie-stained SDS-PA gel (left panel) and western blot of the protein with anti-His Ab (right panel). (D) <i>P. falciparum</i> lysate probed with anti-<i>Pf</i>FtsH1 Ab (I) detects a ~101 kDa band and a major ~66 kDa band. A minor band is also seen at ~72 kDa. No signal is detected with pre-immune serum (Pre-I).</p

Localisation of <i>Pf</i>FtsH1 in a <i>P. falciparum</i> 3D7 transfectant line carrying C-terminal 3xHA-tagged <i>Pf</i>FtsH1.

<p>(A) Western with anti-HA mAb recognises an intact ~105 kDa (FtsH + HA tag) product and a ~38 kDa band likely to represent the cleaved ~35 kDa C-terminal region fused with HA. (B) Immunofluorescence localization of <i>Pf</i>FtsH1-HA using the anti-HA mAb and antibody against the apicoplast marker ACP. No overlap of <i>Pf</i>FtsH1-HA signal was observed with the apicoplast marker. (C) <i>Pf</i>FtsH1 co-localizes with the mitochondrial signal in trophozoites (upper panel) and appears as punctuate signals lining the organelle defined by the mitochondrial stain Mitotracker Red in schizonts (lower panel). (D) Confocal microscopy <i>Pf</i>FtsH1-HA expressing parasites showing co-localisation of <i>Pf</i>FtsH1 with the mitochondrion which is stained with Mitotracker Red.</p