An apicoplast localized ubiquitylation system is required for the import of nuclear-encoded plastid proteins

Abstract

Apicomplexan parasites are responsible for numerous important human diseases including toxoplasmosis, cryptosporidiosis, and most importantly malaria. There is a constant need for new antimalarials, and one of most keenly pursued drug targets is an ancient algal endosymbiont, the apicoplast. The apicoplast is essential for parasite survival, and several aspects of its metabolism and maintenance have been validated as targets of anti-parasitic drug treatment. Most apicoplast proteins are nuclear encoded and have to be imported into the organelle. Recently, a protein translocon typically required for endoplasmic reticulum associated protein degradation (ERAD) has been proposed to act in apicoplast protein import. Here, we show ubiquitylation to be a conserved and essential component of this process. We identify apicoplast localized ubiquitin activating, conjugating and ligating enzymes in Toxoplasma gondii and Plasmodium falciparum and observe biochemical activity by in vitro reconstitution. Using conditional gene ablation and complementation analysis we link this activity to apicoplast protein import and parasite survival. Our studies suggest ubiquitylation to be a mechanistic requirement of apicoplast protein import independent to the proteasomal degradation pathway.

Figure 1. Ubiquitylation proteins localize to the apicoplast in Plasmodium falciparum and Toxoplasma gondii.

(A–E) Immunofluorescence assays detecting the respective ubiquitylation factor indicated (white lettering) in the left-most panel. Proteins were detected by tagging with an HA or GFP epitope in the genomic locus (A, D), by ectopic fusion with full (B) or partial coding sequence (E), or by using an antibody raised again recombinant protein (C). Staining for the apicoplast markers Cpn60 and ACP is shown in the second lane. Merged images also show DAPI staining for P. falciparum. Insert in (B) shows a 200% enlargement. Western blots of T. gondii (F, G, transgene as indicated) or P. falciparum (H) protein samples reacted with anti-HA and anti-PfE2_Ap antibodies. Predicted sizes of initial translation product of TgE2_Ap, PfE2_Ap and TgE1_Ap are 70.4 kDa, 32.8 kDa and 315.9 kDa respectively.

Figure 2. TgE2_Ap is localized to the periphery of the apicoplast.

(A, B) Transmission electron micrograph of cryo-sections prepared from the TgE2_Ap -HA parasite line and labeled with anti-HA antibody and antiglobulin conjugated to colloidal gold. Gold beads are observed at the periphery of an organelle that is surrounded by four membranes.

Figure 3. Recombinant Plasmodium falciparum apicoplast proteins show ubiquitin activation, conjugation and ligation activity.

(A) Schematic maps of recombinant proteins indicating protein domains and purification tags used and the predicted molecular weight of the fusion protein (not to scale). (B) Western blot of recombinant proteins detected with antibodies to the indicated tag. (C) Schematic outline of the biochemical assay, ubiquitin is incubated with recombinant E1, E2 and E3 enzymes in the presence of ATP, human enzymes are shown in red, parasite enzymes in green (this color scheme is used as reference in panels D–H). Ubiquitylation is measured by Western blot detected polyubiquitin chains (PolyUb) either free, or linked to the E3 RING domain. The antibody symbol indicates the specific protein detected in each panel. PfE3w_Ap (D) or PfE3c_Ap (E) were incubated with human UBA1 and different UBCs as indicated. Note ubiquitylation using UBC5a (E3 associated higher molecular weight) and UBC13 (free Ub chains, only selected panels are shown in E). (F) Only the use of UBC5a results in ubiquitylation attached to PFE3_Ap visible as higher molecular weight species recognized by the antibody to the GST tag of E3. (G) Reconstitution of ubiquitylation using recombinant Plasmodium E1_Ap and E2_Ap and PfE3w_Ap or PfE3c_Ap respectively. (H) The presumptive E2/E3 complex immunoprecipitated from P. falciparum parasites using an antibody to PfE2_Ap shows ubiquitylation activity when incubated with biotinylated human ubiquitin and UBA1 (detected with Streptavidin, SA).

Figure 4. Genetic ablation of E2_Ap in T. gondii results in a block of apicoplast protein import and parasite growth.

(A) The TgE2_Ap locus was modified homologous recombination and insertion of a DHFR selectable marker and a regulatable promoter (arrow, black circles indicate tet operator elements). Positions of diagnostic primers (half arrows) and probe (black line) are indicated. (B) PCR test for insertion, note that only the recombined locus is suitable to template the 2.8 kbp product. (C) Southern analysis hybridizing genomic DNA form parental (ΔKu80/TATI) or mutant ((i)ΔTgE2_Ap) parasites with a probe derived from the first exon of TgE2_Ap. (D) Real time PCR analysis of TgE2_Ap expression in (i)ΔTgE2_Ap parasites upon ATc treatment (normalized to the mRNA of the major surface protein SAG1, untreated control set to 100%, error bars show SD, n = 3) detects the expected DNA fragments. PCR analysis of clones shows integration of the promoter. (E) Growth of (i)ΔTgE2_Ap was measured by fluorescence (circles no drug, squares ATc, triangles 3 days of pretreatment with ATc prior to plate inoculation) and (F) plaque assay. (G) Apicoplast protein import in (i)ΔTgE2_Ap was measured by following PDH(E2) lipoylation ( highlighted by box). Note loss of band upon ATc treatment and persistence of mitochondrial lipoylation (Mito (E2)-LA); Please note that there are two lipoylated proteins in the T. gondii mitochondrion , , * human PDH-E2. (H) Quantification of protein import (as measured in G, squares, data shown is representative of four experiments) and number of apicoplasts per parasite (circles, n = 3, SD shown) in (i)ΔTgE2_Ap over the course of ATc treatment. (I) Western blots probing FNR-RFP in (i)ΔTgE2Ap upon ATc treatment (p, precursor; m, mature protein). Note reduction in mature band upon treatment. Immunofluorescence assays showing (i)ΔTgE2Ap in the absence (J) or presence (K) of ATc. 38% of parasite vacuoles showed labeling outside the apicoplast, likely due to back up of cargo into the ER at 48 h of treatment (<3% in untreated parasites).

Figure 5. Active site residues are required for functional complementation of the TgE2_Ap null mutant.

(A) MacPyMOL model of the secondary structure of TgE2_Ap (alignable residues A489-T608) using S. cerevesiae Ubc-4 enzyme as template and. Note three conserved α-helices (blue) and four β-strands (magenta). The active site cysteine residue is shown in red the HVH triad in green. (B) Multiple sequence alignment of the active site (cysteine, red: triad, green) from P. falciparum, T. gondii, the diatom Thalassiosira pseudonana and yeast (complete alignment shown in Fig. S3 in Text S1). (C) Wild type and point mutants of the TgE2_Ap coding sequence were introduced into the UPRT locus of TgE2_Ap (C) under 5FUrd selection, correct insertion was established by PCR (D) and phenotypic complementation was assessed by plaque assay in the presence or absence of ATc (E).