Figure 1.
Discovering new apicoplast proteins weighing multiple data sources.
(A) Schematic outline of the acquisition and evolution of the apicoplast. An algal cell (blue) carrying a chloroplast (green) enters an endosymbiotic relationship with a protist host. The establishment of a protein import system allows for massive gene transfer and the development of a stable union. The initial compartments of the endosymbionts are highly conserved over long periods of evolutionary time. PPC, the periplastid compartment is highlighted in blue. (B) Overview of the pipeline used for the identification, prioritization, confirmation and disruption of apicoplast proteins. The graphs show mRNA abundance profiles over two consecutive tachyzoite cell-cycles as described in [36]. Profiles are shown for those proteins used as seeds (left), the resulting candidate pool (middle), and for the newly identified apicoplast proteins (right).
Figure 2.
Localization of 30 previously uncharacterized proteins by endogenous epitope tagging.
Fluorescence microscopy analysis of parasites expressing endogenously HA-tagged proteins. (A) shows examples of a luminal apicoplast protein (top panel), a protein localizing to the periphery of the apicoplast (middle panel), and an example for the second patchy peripheral pattern observed (bottom panel). Numbers on the left represent the numbers of proteins showing the described pattern. (B) shows representative image of ATrx2 (top panel) and of PPP1 (bottom panel) localizations. (C) Representative images of tags that resulted in the identification of non-apicoplast proteins. Likely target organelles are indicated. Scale bar = 5 µm. HA in green, CPN60 in red, numbers reflect the ToxoDB gene ID as detailed in the Results section.
Table 1.
Summary of the bioinformatics and experimental data for each of the eleven new apicoplast proteins found in this work.
Figure 3.
PPP1 is a resident of the apicoplast periphery and most likely the periplastid compartment.
(A) Fluorescence microscopy analysis of parasites expressing endogenously HA-tagged PPP1 (in green in both panels), co-stained with the luminal marker CPN60 (red in upper panel) and peripheral marker ATrx1 (red in lower panel). Note that in both cases the signals do not overlap completely. Scale bar = 5 µM, and scale bar for magnified insert = 1 µM. (B) Electronmicrographs of parasites expressing endogenously HA-tagged PPP1 using immuno-gold staining after cryo-sectioning. The images on the right show only the apicoplast. Scale bar - 1 µm. (C) T. pseudonana cells were transfected with a 267353–GFP encoding plasmid and stable transgenics were established by drug selection. Expression of the transgene was induced by nitrate. Note GFP fluorescence (green) at the center of the chloroplast (imaging chlorophyll autofluorescence in red). Scale bar 2 µm.
Figure 4.
PPP1 is conserved specifically among members of the red algal lineage.
(A) Multiple protein alignment of the putative orthologues of PPP1. Color gradient corresponds to sequence similarity, deep blue represents 100% identity. Size of black bars indicates the level of conservation across the consensus. (B) Neighbor joining tree of PPP1. Bootstrap values are indicated for each node.
Figure 5.
Deletion of the TgKu80 gene in TATi transactivator parasite line.
(A) PCR analysis using primers specific for the TgKu80 coding region (left), targeting construct integration (middle), or an unrelated control region (right) using genomic DNA from RHΔHX, TATIΔTgKu80 and RHΔTgKu80 [38] as template. Position of primers is indicated in panel B. (B) Schematic representations of homologous recombination between the BLE modified TOXOW30 cosmid and the TgKu80 locus. The probe used for the southern blot presented in panel C is indicated and sizes expected after BglII digest are shown. (C) Southern blot analysis of the TATiΔTgKu80 clone and its parental TATi line demonstrating TgKu80 disruption.
Figure 6.
Isolation of a conditional PPP1 mutant by promoter replacement.
(A) Schematic representation of the two-step manipulation of the PPP1 locus: First the gene is endogenously tagged by single cross-over (cassette in green), followed by promoter replacement by double-homologous recombination (cassette in blue). A legend for the tag and promoters used is show in the upper right corner. (B) Southern blot analysis before and after tagging and promoter replacement. Strains, restriction enzymes and expected band-sizes are indicated to the right. Asterisks indicate bands resulting from additional non-homologous integration of the pLIC_3HA plasmid.
Figure 7.
PPP1 is essential for parasites growth.
(A) Western blot analysis was of proteins extracts from PPP1(HA) and (Tet)PPP1(HA) using anti HA antibody. Note comparable levels of PPP1 when expressed from the native promoter (N) or the inducible T7S4 promoter (I). Protein levels decline swiftly upon ATc treatment, numbers indicate days of culture in 0.5 µg/ml ATc. Tubulin (lower panel) serves as a loading control. (B) Fluorescence microscopy analysis of (Tet)PPP1(HA parasites using anti-HA antibody grown in the presence and absence of ATc. (C) Plaque assays performed in the absence (-) or presence (+) of ATc. Parasite line name is indicated to the left. (D) Fluorescence microscopy of parasites expressing a second copy of PPP1, which is labeled using an anti-Ty antibody (red). The apicoplast marker used is the endogenously tagged ACP-YFP (AY, green). Scale bar = 5 µm. (E) Fluorescence growth assays show growth deficiency in ATc treated (Tet)PPP1(HA). Circles, -ATc; squares, +ATc. Each data point represents the mean of six wells, and the error bar gives the standard deviation. (F) Fluorescence microscopic analysis of endogenously tagged 039680 using anti-HA (green) and the luminal marker CPN60 (red) show that 039680 is likely a luminal apicoplast protein. (G) PCR analysis of the promoter replacement clone of 039680 with diagnostic primer sets is consistent with locus modification. The size of amplicons matches the prediction for integration at both the 5' and the 3' insertion site (900 and 1800 bp respectively). (H) Plaque assays performed with the promoter replaced 039680 cell line in the presence or absence ATc show that this gene is required for growth.
Figure 8.
Loss of PPP1 results in apicoplast protein import defect.
(A) To study apicoplast biogenesis in the PPP1 mutant we engineered a reporter by tagging the ACP locus with YFP. No overt apicoplast division defects were observed upon 48 h ATc treatment. However, note accumulation of fluorescence outside of the apicoplast in treated parasites after 48 h (arrowheads). (B) Western blot analysis following the maturation of ACP-YFP in the (Tet)PPP1(HA) mutant. Parasites were grown in ATc for the times indicated. Anti-GFP antibody was used to detect ACP-YFP. Note the pronounced loss of the mature form of the protein. (C) Bands in B were quantified by densitometry and the ratio of mature to premature protein is shown for each time point. (D) Pulse-chase analysis of protein synthesis and post-translational lipoylation of apicoplast (single band labeled PDH-E2) and mitochondrial proteins (two bands marked mito-E2). Parasites were metabolically labeled as detailed in [13] and lipoylated proteins were isolated by immunoprecipitation using a specific antibody. One hour of labeling (Pulse, P) was followed by a two hour of chase (C) resulting in import and lipoylation of PDH-E2 in non treated parasites. Lipoylation is lost upon ATc treatment. Data shown are representative of four independent experiments. Bands labeled with an asterisk represent lipoylated host cell proteins (note that these bands are observed in host-only controls (Host, C). (E) Bands in D were quantified by densitometry and the ratio of lipoylated PDH-E2 over mito-E2 (upper mitochondrial band) is shown. (F) (Tet)PPP1(HA) parasite were grown in ATc as indicated and plastids were counted in 100 four-parasites vacuoles for each sample. Y-axis shows the percent of parasites that show a clearly identifiable apicoplast.