Tellier G , Lenne A , Cailliau-Maggio K , Cabezas-Cruz A , Valdés JJ , Martoriati A , Aliouat el M , Gosset P , Delaire B , Fréville A , Pierrot C , Khalife J .

	Figure 1. Molecular cloning, sequences analysis of PfeIF2β and its expression by P. falciparum. (A) Analysis of P. falciparum (PF3D7_1010600) and human eIF2β (GenBank AAA52383.1). Sequences were aligned using ClustalW Multiple Alignment (BioEdit). The identical and semiconserved amino acids are highlighted in black and gray respectively. Lysine blocks and GTP binding domains are underlined with orange and blue lines respectively. Green line corresponds to the domain of the superfamily eIF2/eIF5. The blue boxes contain the ≪zinc finger≫ motif (C2-C2motif). The potential PP1-binding motifs “RVxF” and “FxxR/KxR/K” are in red and green boxes respectively. Phosphorylable amino acids reported in PlasmoDB are in red. (B) Detection of endogenous PfeIF2β in asynchronous cultures of P. falciparum. Blots were probed with pre-immune serum (lane 1) or with anti-PfeIF2β serum (lane 2). The blots were revealed as described in Section Materials and Methods.
	Figure 2. Phylogenetic position and relevant domains of eIF2β from Apicomplexan parasites. (A) The figure displays the maximum likelihood phylogenetic tree of eIF2β amino acid sequences from 23 apicomplexan, 5 mammals, 1 amphibian, 8 fish, 9 plants, 8 arthropods, 12 archaea, 1 yeast (fungi), 2 amoebozoa, 1 cercozoa, 1 foraminifera, and 5 excavata. All sequences selected for the analysis belonged to eIF5/eIF2B family (pfam01873), and contained the characteristic eIF2B domain (Accession number: PRK03988). The position eIF2β from P. falciparum is shown (red branch and circle). Numerals at internal branches represent bootstrap values. Only bootstrap values higher than 50 are shown. The accession number of each sequence is provided in the figure. The name of species was abbreviated as follow: Apicomplexan: Pfal, Plasmodium falciparum; Pber, P. berghei; Pkno, P. knowlesi; Pviv, P. vivax; Pyyo, P. yoelii yoelii; Prei, P. reichenowi; Pcyn, P. cynomolgi; Pinu, P. inui; Pvvi, P. vinckei vinckei; Bequ, Babesia equi; Bbig, B. bigemina; Bmic, B. microtis; Bbov, B. bovis; Tann, Theileria annulata; Tori, T. orientalis; Tpar, T. parva; Hham, Hammondia hammondi; Eace, Eimeria acervulina; Ncan, Neospora caninum; Cpar, Cryptosporidium parvum; Cmur, C. muris; Tgon, Toxoplasma gondii; and Gnip, Gregarina niphandrodes; Mammals: Hsap, Homo sapiens; Mmus, Mus musculus; Btau, Bos taurus; Ggor, Gorilla gorilla; Cfer, Camelus ferus; Amphibian: Xlae, Xenopus laevis; Fish: Drer, Danio rerio; Ssal, Salmo salar; Eluc, Esox Lucius; Omyk, Oncorhynchus mykiss; Locu, Lepisosteus oculatus; Spar, Stegastes partitus; Csem, Cynoglossus semilaevis; Nbri, Neolamprologus brichardi; Plant: Atha, Arabidopsis thaliana; Pvul, Phaseolus vulgaris; Csat, Cucumis sativus; Sind, Sesamum indicum; Csat, Camelina sativa; Vvin, Vitis vinifera; Ptri, Populus trichocarpa; Peu, Populus euphratica; Brap, Brassica rapa; Insects: Drosophila melanogaster; Ccap, Ceratitis capitata; Bdor, Bactrocera dorsalis; Adar, Anopheles darling; Asin, A. sinensis; Agam, A. gambiae; Mdom, Musca domestica; Nvit, Nasonia vitripennis; Archeae: Nequ, Nanoarchaeum equitans; Ssol, Sulfolobus solfataricus; PNA2, Pyrococcus sp. NA2; TES1, Thermococcus sp. ES1; Tbar, T. barophilus; Tgam, T. gammatolerans EJ3; Paby, Pyrococcus abyssi GE5; Pyay, P. yayanosii CH1; Pfur, P. furiosus; Mvan, Methanococcus vannielii SB; Mkan, Methanopyrus kandleri AV19; Smar, Staphylothermus marinus F1; and the Fungi: Spom, Schizosaccharomyces pombe; Amoebozoa: Edis, Entamoeba dispar; Ehis, Entomoeba histolytica; Cercozoa: Pbra, Plasmodiophora brassicae; Foraminifera: Rfil, Reticulomyxa filose and Excavata: Tviv, Trypanosoma vivax; Tgra, T. grayi; Tbb, T. brucei brucei; Tbg, T. brucei gambiense; Tcru, T. cruzi. The accession number of each sequence is provided in Table S2. (B) Simplified representation of eIF2β sequence for each group in the tree. Data regarding functional domains was collected from Asano et al. (1999) and Fréville et al. (2014).
	Figure 3. Modeling of the tertiary structure of PfeIF2β. (A) The figure shows the tertiary structure for the initiation factor of P. falciparum (predicted model) based on the initiation factor of Pyrococcus furiosus (PDB: 2DCU, chain B) in 180 turns. The structure is color-coded from the N-terminus (blue) to the C-terminus (red). The helix-turn-helix (HTH) and the zinc-binding domain (ZBD) are indicated (colored according to the respective position). The zinc ion is represented as a gray sphere. (B) The panel is a superposition of the alpha-carbon backbone of both structures (RMSD = 2.3Å).
	Figure 4. Interaction between PfeIF2β and its partners PfeIF2γ and PfeIF5. (A) GST pull-down assays. Glutathione beads alone (lane 1) or coupled with GST alone (lane 2), or GST-PfeIF2γ (lane 3) were incubated with 6His-tagged PfeIF2β wild-type. After washings, proteins bound to the beads were separated by SDS-PAGE and blotted to nitrocellulose. Immunoblot (IB) analysis was performed with mAb anti-His antibodies (upper blot) and mAb anti-GST (lower blot). As control, 20% of the input of PfeIF2β protein detected was used and immunoblotted with anti-His antibody (lane 4). (B) Interaction of PfeIF2β with PfeIF2γ in Xenopus oocytes. His-tagged PfeIF2γ recombinant protein and cRNA of PfeIF2β producing HA-tagged protein were micro-injected in fresh oocytes. Co-immunoprecipitations were carried out with anti-His (recognizing recombinant PfeIF2γ tagged with 6-His) (upper blot) or with anti-HA (recognizing PfeIF2β tagged HA) (lower blot) antibodies from micro-injected Xenopus extracts. Immunoprecipitates from Xenopus oocytes micro-injected with cRNA PfeIF2β alone (lane 1), PfeIF2γ protein alone (lane 2), or PfeIF2β and PfeIF2γ (lane 3) were eluted, separated by SDS-PAGE and transferred to nitrocellulose membrane. Immunoblot (IB) analysis was performed with anti-His or anti-HA antibodies. (C) Interaction of PfeIF2β with PfeIF5. His-tagged PfeIF2β recombinant protein and cRNA of PfeIF5 producing HA-tagged protein were micro-injected in fresh oocytes. Co-immunoprecipitations were performed as described in (B). Immunoprecipitates (IP) from Xenopus oocytes micro-injected with PfeIF5 cRNA alone (lane 1), PfeIF2β protein alone (lane 2), or PfeIF2β and PfeIF5 cRNA (lane 3) were eluted, separated by SDS-PAGE and transferred to nitrocellulose membrane. Immunoblots were performed as described in (B).
	Figure 5. Interaction studies of PfeIF2β with PfPP1. (A) Binding of His-tagged PfPP1 recombinant protein to endogenous PfeIF2β expressed by P. falciparum. Total soluble proteins extracted from asynchronous cultures (2 mg) were pre-cleared on Ni-NTA-agarose beads before and incubated overnight with PfPP1-6His affinity Ni-NTA column. After washings, eluted proteins were separated by SDS-PAGE and blotted onto nitrocellulose. Using anti-His mAb, lane 1 confirmed the presence of His-PP1. Pre-immune serum and anti-PfeIF2β antisera were used in lanes 2 and 3 respectively. Lane 3 showed the presence of PfeIF2β. As positive control, the presence of PfeIF2β in the total parasite extracts using the PfeIF2β antisera is shown in lane 4. (B) Direct binding of PfeIF2β to PfPP1 by GST pull-down assays. Glutathione beads alone (lane 1) or coupled with GST alone (lane 2), or PfPP1-GST (lane 3) were incubated with 6His-tagged PfeIF2β wild-type. After washings, proteins bound to the beads were separated by SDS-PAGE and blotted onto nitrocellulose. Immunoblot analysis was performed with anti-His mAb (upper blot) and anti-GST mAb (lower blot). (C) Scheme representing the different versions of PfeIF2β recombinant proteins (wild-type or mutated) used in this study. (D) Mapping of PfeIF2β binding motifs to PfPP1. Glutathione agarose beads coupled with GST alone (lanes 1, 3, 5, 7, and 9), or GST-PfPP1 (lanes 2, 4, 6, 8, 10) were incubated with 6His-tagged PfeIF2β wild-type (lanes 3 and 4), or PfeIF2β 103KAAA106 (lanes 5 and 6), or PfeIF2β 29AGEAKA34 (lanes 7 and 8), or PfeIF2β 103KAAA106/29AGEAKA34 (lanes 9 and 10). After washings, proteins bound to the beads were separated by SDS-PAGE and blotted to nitrocellulose. Immunoblot analysis was performed with anti-GST mAb (upper blot) and with anti-His mAb (lower blot). The inputs represent 0.5 μg of each 6His-tagged protein (lanes 11, 12, 13, and 14).
	Figure 6. Effect of PfeIF2β on GVBD induction in Xenopus oocytes. (A) Induction of Germinal Vesicle Break Down (GVBD) in Xenopus oocytes by PfeIF2β. Each oocyte was micro-injected with 60 ng of PfeIF2β wild-type, or PfeIF2β 103KAAA106, or PfeIF2β 29AGEAKA34, or PfeIF2β 103KAAA106/29AGEAKA34 recombinant protein. Appearance of GVBD was monitored 15 h after injection. Each experiment was performed using a set of 20 oocytes. Results are presented as percentage ± SEM of four independent experiments (20 oocytes for each protein). (B) Binding of PfeIF2β to Xenopus oocytes PP1. Co-immunoprecipitation experiments with anti-His (upper blot) or anti-XePP1 (lower blot) antibodies were carried out on extracts obtained from oocytes micro-injected with wild-type, single mutated, or double mutated proteins. The anti-mouse IgG antibody was used as a control. Immunoprecipitates from oocytes were eluted, separated by SDS-PAGE and transferred to a nitrocellulose membrane. Immunoblot analysis was performed with anti-His antibodies (recognizing PfeIF2β) or anti-XePP1 antibodies.
	Figure 7. Targeted gene disruption of the PfeIF2β locus. (A) Gene-targeting construct for gene disruption by single homologous recombination using pCAM-BSD, and the locus resulting from integration of the Knock-Out construct. (B) Analysis of pCAM-BSD-PfeIF2β-transfected 3D7 cultures by PCR; lanes 1–3 correspond to DNA extracted from wild-type parasites; lanes 4–6 correspond to DNA extracted from transfected parasites. Lanes 1 and 4 represent the detection of the full-length wild-type locus (PCR with p25 and p28); lanes 2 and 5 represent the detection of episomal DNA (PCR with p29 and p30); lanes 3 and 6 represent the detection of integration of the insert (PCR with p24 and p30). The absence of a PCR product in lane 6 indicates the lack of homologous integration.
	Figure 8. HA-tagging of the PfeIF2β locus. (A) Epitope tagging of PfeIF2β by Knock-In strategy. Insertion of an HA epitope tag at the C-terminus of PfeIF2β single homologous recombination. (B) Analysis of pCAM-BSD-HA-PfeIF2β-transfected 3D7 cultures by PCR; lanes 1–2 correspond to DNA extracted from wild type parasites; lanes 3–4 correspond to DNA extracted from transfected parasites. Lanes 1 and 3 represent the detection of the wild-type locus (PCR with p25 and p28); lanes 2 and 4 represent the detection of integration at the 5′ end of the insert (PCR with p24 and p31). The presence of a PCR product (arrow) and its sequencing confirmed the integration of a tagged HA-PfeiF2b gene in the locus. (C) Expression of HA-PfeIF2β was checked by Western-blot with anti-HA-biotin antibody after separation on SDS-PAGE. Lane 1 represents the culture of wild-type parasites and lane 2 represents the culture of transfected parasites.
	Figure 9. Localization of PfeIF2β. (A) Immunolocalization assays. Asynchronous cultures of PfeIF2β-HA tag recombinant strain of P. falciparum 3D7 were fixed with formalin and paraffin embedded. Sections were incubated with an anti-HA tag (biotin) antibody recognized by a streptavidine-Alexa fluor 488-labeled conjugated added with DAPI to label nuclei. Fluorescence staining was analyzed using a Zeiss LSM880 confocal microscope. The merged image of the double stained (PfeIF2β-HA tag, DAPI) and differential interference contrast (DIC) images are also presented. Immunofluorescence assays revealed a cytoplasmic localization of PfeIF2β in the trophozoite (panel 1), young schizont (panel 2), or mature schizont (panel 3) stages. Note that no staining was observed in ring-stage. No staining was observed when the primary antibody (anti-HA) was omitted (not shown). Bar = 5 μm. (B) Immunoblot analysis on nuclear and cytoplasm fractions from asynchronous parasites cultures. The quality control of nuclear (N) and cytoplasm (C) fractions were checked using Anti-SOD1 (upper panel) and anti-H3 antibodies (middle panel) respectively. The (lower) panel showed the presence of PfeIF2β in both fractions. (C) A representative Western blot assay showing the detection of PfeIF2β in cytoplasm and nuclear extracts (upper panel) from ring-stage (R), trophozoit-stage (T), and schizont-stage (S). Equal amount of nuclear and cytoplasmic proteins (10 μg) extracted from each stage were loaded. The (lower) panel showed the detection of Pf-actin 1 in the different nuclear and cytoplasmic fractions used.

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