V^ap persist in the presence of the Golgi disruptor BFA.

<p>A) IFA analysis of <i>T. gondii</i> grown with or without 1 µg/ml BFA for 1.5 hours. The parasites expressed the luminal apicoplast marker ^S+TRed along with FtsH1internally tagged with V5 epitopes or ATrx1-HA, which were detected with α-V5 followed by anti-mouse IgG (FITC) or anti-HA mAb directly coupled to FITC. Arrows indicate V^ap-like structures in control and BFA-treated parasites. Bar, 2 µM. B) IFA of parasites expressing the Golgi membrane protein NST1-HA (detected with anti-HA mAb coupled to FITC), treated in parallel. Bar, 2 µM. C) Quantitation. The percentage of vacuoles with parasites bearing V^ap in the presence (+) or absence (−) of BFA is depicted. Three replicates are shown for FtsH1 (circle) and one for ATrx1 (triangle). More than 125 vacuoles were analyzed for each point. At the times chosen, the proportion of parasites with apicoplasts at stage 2 (elongated oval), stage 3 (elongated bar), and stage 4 (V-shaped bar) were: FtsH1 analysis: control, 82.7%; BFA, 72.2%; ATrx1 analysis: control, 81.6%; BFA, 74.5%.</p

Effect of BFA on FtsH1 processing.

<p>Intracellular <i>T. gondii</i> expressing FtsH1 internally tagged withV5 epitopes were metabolically labeled for 30 min and then chased for various times in complete medium. BFA-treated samples included the drug throughout the pulse-chase. FtsH1 was then immunoprecipitated and subjected to SDS-PAGE followed by phosphorimaging (³⁵S panel). The blot was subsequently probed with anti-V5 mAb (Western panel). The four major forms of FtsH1 are marked according to their apparent molecular mass on SDS-PAGE: full-length (F-170), N-terminally processed (NP-154), C-terminally processed (CP-140) or dual processed (NPCP-115).</p

Protein synthesis during BFA treatment assessed by biosynthetic labeling of FtsH1, MIC5 and cytosolic GFP.

<p>Fibroblast monolayers infected with <i>T. gondii</i> expressing FtsH1 tagged internally with V5 epitopes and a cytosolic GFP (∼10⁸) were pre-incubated with or without BFA (1 µg/ml) for the indicated times prior to being labeled with ³⁵S-methionine/cysteine for 30 minutes. Samples were immunoprecipitated with anti-V5 mAb, anti-GFP, and anti-MIC5 before being separated on 7.5% (FtsH1) or 8–16% (GFP and MIC5) SDS-PAGE gels and transferred to nitrocellulose. The left panel shows phosphorimaging, the right panel shows the same lanes detected by Western blot. The four major forms of FtsH1 are marked according to their apparent molecular mass on SDS-PAGE: full-length (F-170), N-terminally processed (NP-154), C-terminally processed (CP-140) or dual processed (NPCP-115). In a 30 min labeling, the first two forms predominate <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112096#pone.0112096-Karnataki1" target="_blank">[21]</a>. The precursor (p) and mature (m) forms of MIC5 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112096#pone.0112096-Brydges1" target="_blank">[75]</a> are marked.</p

V^ap are not major vehicles for luminal protein trafficking to the apicoplast.

<p>For IFA analysis here and elsewhere unless indicated, proteins were detected by mAbs directed against epitope tags followed by fluorochrome-coupled secondary antibodies as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112096#s4" target="_blank">Methods</a>. In this case, the apicoplast membrane proteins were detected anti-HA mAb was followed by FITC-coupled secondary antibodies and ^S+TRed-V5 was detected by anti-V5 mAb followed by Texas Red-coupled antibodies to bypass the need for maturation of the HcRed chromophore. Here, as in other figures, the color coding for merged images is indicated by the text color above the merged images, while dashed lines mark the outline of the parasite. In this experiment, the parasites co-expressed ^S+TRed-V5 driven by the <i>ACP</i> promoter and epitope-tagged ApV proteins APT1-HA or ATrx1-HA. A) IFA showing the pattern seen in about 80% of parasites with ATrx1-HA in V^ap (arrows) near the apicoplast. One set of anti-V5 images is scaled normally and the second is scaled to detect fainter signals (the staining at the apicoplast is then saturated). No evidence of localization of the luminal marker ^S+TRed-V5 with V^ap was observed when scanning through the deconvolved planes. “H” marks a host cell nucleus. B) In the approximately 20% parasites with evident V^ap, occasional regions staining for membrane-associated proteins (V^ap, arrows) also showed a weak signal for the luminal marker ^S+TRed-V5. Bar, 2 µm. C) Individual parasites with V^ap as detected by the presence of ATrx1-HA were randomly chosen for quantitation of ATrx1-HA and ^S+TRed-V5 signals. The average fluorescence corresponding to each protein in a 90 pixel area covering either the apicoplast (AP), vesicles (V^ap) or adjacent regions (control) was determined and plotted for each individual parasite (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112096#s4" target="_blank">Methods</a>). The mean florescence signal seen in the parasite population is marked for each region analyzed (black lines). The raw fluorescence intensities for the adjacent regions averaged 12243 fluorescence units for ATrx1-HA and 5785 for ^S+TRed-V5, very close to the average background of 12267 (anti-HA, green line) and 6577 (anti-V5, red line) for these channels in untransfected RH parasites on the same slide.</p

Overlap between FtsH1 and GRASP55 does not reflect FtsH1 protein in the Golgi body.

<p>A) Parasites were incubated with or without 1 µg/ml BFA for 1 hour at 37°C. <i>T. gondii</i> co-expressing the Golgi matrix marker GRASP55-YFP and FtsH1 internally tagged with V5 epitopes were stained with anti-V5 mAb followed by secondary antibody coupled to DyLight 649 and Texas Red streptavidin (which detects a naturally biotinylated protein in the apicoplast lumen, AP lumen). Bar, 2 µM. B) Parasites expressing the Golgi membrane protein NST1-HA (detected with anti-HA mAb coupled to Alexa 594) served as the control, demonstrating the effectiveness of BFA. Bar, 2 µM. C) Quantitative analysis of signal overlap between internally tagged FtsH1 and GRASP55 in the presence or absence of BFA. More than 50 parasites were analyzed for each condition (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112096#s4" target="_blank">Methods</a>).</p

Vesicles Bearing <i>Toxoplasma</i> Apicoplast Membrane Proteins Persist Following Loss of the Relict Plastid or Golgi Body Disruption

<div><p><i>Toxoplasma gondii</i> and malaria parasites contain a unique and essential relict plastid called the apicoplast. Most apicoplast proteins are encoded in the nucleus and are transported to the organelle <i>via</i> the endoplasmic reticulum (ER). Three trafficking routes have been proposed for apicoplast membrane proteins: (i) vesicular trafficking from the ER to the Golgi and then to the apicoplast, (ii) contiguity between the ER membrane and the apicoplast allowing direct flow of proteins, and (iii) vesicular transport directly from the ER to the apicoplast. Previously, we identified a set of membrane proteins of the <i>T. gondii</i> apicoplast which were also detected in large vesicles near the organelle. Data presented here show that the large vesicles bearing apicoplast membrane proteins are not the major carriers of luminal proteins. The vesicles continue to appear in parasites which have lost their plastid due to mis-segregation, indicating that the vesicles are not derived from the apicoplast. To test for a role of the Golgi body in vesicle formation, parasites were treated with brefeldin A or transiently transfected with a dominant-negative mutant of Sar1, a GTPase required for ER to Golgi trafficking. The immunofluorescence patterns showed little change. These findings were confirmed using stable transfectants, which expressed the toxic dominant-negative sar1 following Cre-<i>loxP</i> mediated promoter juxtaposition. Our data support the hypothesis that the large vesicles do not mediate the trafficking of luminal proteins to the apicoplast. The results further show that the large vesicles bearing apicoplast membrane proteins continue to be observed in the absence of Golgi and plastid function. These data raise the possibility that the apicoplast proteome is generated by two novel ER to plastid trafficking pathways, plus the small set of proteins encoded by the apicoplast genome.</p></div

<i>Tg</i>ATrx1 and <i>Tg</i>ATrx2 are essential and their function requires their CXXC motifs.

<p>A. Scheme of the manipulation performed to each of the <i>Tg</i>ATrxs loci to replace their promoters with the tetracycline-regulatable promoter. The gene model in the scheme is based on <i>Tg</i>ATrx1’s gene model, however the promoter replacement occurred via the same strategy for both <i>Tg</i>ATrxs. Black boxes–exons; asterisk–stop codon. B. Western blot analysis of <i>Tg</i>ATrx1 expression using anti-<i>Tg</i>ATrx1 (i), and endogenously HA-tagged <i>Tg</i>ATrx2 using anti-HA (ii), upon ATc treatment. C. Fluorescent microscopy showing <i>Tg</i>ATrx1 (anti-<i>Tg</i>ATrx1, bottom, green) and <i>Tg</i>ATrx2 (anti-HA, top, green) depletion at 48 hours of ATc treatment (+ATc) compared to non-treated control (-ATc). D. Plaque assays performed with TATiΔKu80_PIATrx1 (top) and TATiΔKu80_PIATrx2-3HA (bottom) with (+) or without (-) ATc. E. Plaque assays performed with TATiΔKu80_PIATrx1 constitutively expressing a copy of <i>Tg</i>ATrx1^CXXC (i) or <i>Tg</i>ATrx1^CXXA (ii) and with TATiΔKu80_PIATrx2-3HA constitutively expressing a copy of <i>Tg</i>ATrx2^CXXC (iii) or <i>Tg</i>ATrx2^CXXA (iv). F. Fluorescent microscopy of the localization of <i>Tg</i>ATrx1^CXXC (i); <i>Tg</i>ATrx1^CXXA (ii); <i>Tg</i>ATrx2^CXXC (iii) and <i>Tg</i>ATrx2^CXXA (iv), all in green, co-stained with Streptavidin (SA) which labels the apicoplast acetyl CoA carboxylase (i, ii) or CPN60 (iii, iv) both in red. White broken line shows parasites’ shapes. Scale bar, 1 μm.</p

Illustrations of the two main processes studied in this work.

<p><b>(A)</b> Complex plastid evolution and the resulting architecture and protein trafficking starting from the co-translational (mRNA in blue, new polypeptide in green) translocation into the ER (light grey) through the four apicoplast compartments (grey, yellow, light-red, red) via their corresponding translocons (depicted as dark grey split ovals): Sec61, SELMA (symbiont-specific ERAD (endoplasmic reticulum-associated degradation)-like machinery), TOC/TIC (translocon of the outer/inner chloroplast membrane) <b>(B)</b> Disulphide exchange between Trx (light-grey) and its substrate (dark-grey oval), and of its dependence on the CXXC motif.</p

Inducible overexpression of <i>TgATrx2</i>^CXXC/A and substrate trap identifies gene expression components.

<p>A. Scheme of the inducible system. Triangles—LoxP sites. Arrow–promoter. B. Fluorescent microscopy analysis of <i>TgATrx2</i>^CXXA-Myc inducible expression in the presence of 50 nM Rapamycin for 48 hours, showing the switch from killer-red (red) to <i>TgATrx2</i>^CXXA-Myc (green). Scale bar, 5 μm. C/D. Western blot analyses showing the rapamycin induction of <i>TgATrx2</i>^CXXA-Myc (C) and its immunoprecipitation fractions (D) analyzed with anti-Myc antibody. E. Western blot analysis of the co-IP of HA-tagged <i>TgATrx2</i> with Myc-tagged <i>TgATrx2</i> captured with Myc-Trap beads. F. Fluorescent microscopy of the HA-endogenously tagged TGME49_292320 (green); CPN60 –red; DAPI–blue; Arrowheads highlight apicoplast staining. G. Western blot analysis of the co-IP of HA-tagged TGME49_292320 with Myc-tagged <i>TgATrx2</i> captured with Myc-Trap beads (left) and of Myc-tagged <i>TgATrx2</i> with HA-tagged TGME49_292320 captured with HA-agarose beads. In D and F, DTT elutes the disulphide bonded partners via reduction and DTT at 96°C treatment elutes all the parasite proteins bound to the beads.</p

Apicoplast protein import and gene expression are the first biogenesis pathways to reach peak reduction for <i>TgATrx1</i> and <i>TgATrx2</i> depletion respectively.

<p>Summary graphs of apicoplast numbers, LytB protein import, relative genome copy number and mRNA levels measured in both knockdown lines at 24, 48 and 72 hours with ATc. The control (no ATc) data is normalized to 100%. Error-bars are SEM. The corresponding individual graphs are found in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006836#ppat.1006836.s007" target="_blank">S5 Fig</a>.</p