An Apicoplast Localized Ubiquitylation System Is Required for the Import of Nuclear-encoded Plastid Proteins

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.


Introduction
Apicomplexans are eukaryotic pathogens and responsible for important human and animal diseases including malaria and toxoplasmosis. The Apicomplexa evolved from single-celled photosynthetic algae, and their adaptation to animal parasitism likely predates the emergence of animals from water to land. The presence of a plastid, the apicoplast, is the most important remnant of this evolutionary past [1,2]. While no longer photosynthetic, the organelle synthesizes isoprenoids and fatty acids [3]. The apicoplast is essential for parasite survival, and its metabolism, biogenesis and maintenance are important targets for anti-parasitic drug treatment. The apicoplast was derived by secondary endosymbiosis, where a unicellular red alga was incorporated into a heterotrophic protist. As a consequence of this secondary endosymbiosis the apicoplast is surrounded by four membranes. The organelle carries a genome, yet most of its proteins are nuclear-encoded and imported into the organelle after translation. Targeting depends on a bipartite leader peptide, the first section of which mediates co-translational import into the endoplasmic reticulum, and the second part mediates delivery to the apicoplast, likely through fusion of endosomal vesicles with the outermost membrane of the organelle [4]. Three translocons breaching successive membranes have been proposed to act in the further transport of proteins into the stroma of the apicoplast [5]. The two inner membranes of the apicoplast are homologous to the membranes of the primary chloroplast and protein transport depends on systems derived from the chloroplast TIC and TOC machinery [6,7,8,9]. Insight into the third translocon emerged first in cryptomonads, an algal group that like Apicomplexa harbors a secondary plastid. The secondary plastids of cryptomonads retained a remnant of the algal nucleus, the nucleomorph. Analysis of the gene content of the nucleomorph led to the discovery of plastid proteins that resembled components of the endoplasmic reticulum associated degradation (ERAD) machinery [10]. ERAD is a quality control mechanism that retro-translocates misfolded secretory proteins across the ER membrane [11]. Sommer and colleagues proposed that this mechanism has been adapted for protein import in secondary plastids [10]. There is now significant support for this hypothesis. Homologs of ERAD proteins have been identified and localized to plastids in various algal and apicomplexan species including a core of the membrane protein Der1, the AAA ATPase Cdc48 and its cofactor Ufd1 [10,12,13,14,15]. Recombinant plastid proteins can complement yeast ERAD mutants [14]. Importantly, genetic ablation of the ERAD component Der1 Ap in T. gondii blocks apicoplast protein import, producing a phenotype that closely resembles ablation of the apicoplast TIC component Tic20 [6,15].
During classical ERAD, protein translocation coincides with ubiquitylation, a process that typically employs a cascade of three enzymes: ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2), and ubiquitin ligase (E3) [16,17]. Consuming ATP, the E1 enzyme adenylates ubiquitin at the C-terminus, creating a mixed anhydride. The sulfhydryl group of the E1 active-site cysteine then attacks the anhydride, which results in the formation of a highenergy thio-ester linking ubiquitin to E1. Ubiquitin is then passed to the active-site cysteine of the E2 enzyme. Lastly, with the aid of an E3 ligase, ubiquitin is transferred from E2 and covalently attached to the e-amino group of a lysine in the target protein. Although clearly important in mediating ERAD, the role of ubiquitylation in protein import into secondary plastids is unclear. Interestingly, some ERAD-like ubiquitylation factors are observed in the plastids of cryptomonads, diatoms, and Apicomplexa [12,18,19].
While protein degradation is the key function of classical ERAD this could seem counterintuitive in the context of apicoplast protein import. However ubiquitin's functions are not limited to proteasomal degradation and extend to a variety of cellular protein trafficking systems [20]. Furthermore, ubiquitylation may be a critical mechanistic requirement of protein transport via the ERAD translocon [11,21]. Some authors now view the ERAD associated E3 ligase Hrd1 as a favored candidate for the actual protein-conducting pore [22].
In this study, we elucidate the function of ubiquitylation in the apicoplast. We identify and localize a comprehensive set of ubiquitylating components in the apicomplexan parasites P. falciparum and T. gondii. Using recombinant apicoplast enzymes from P. falciparum we reconstitute ubiquitylation in vitro using a variety of heterologous and homologous cofactors. By genetic analysis in T. gondii we demonstrate that loss of the apicoplastlocalized ubiquitin-conjugating enzyme leads to loss of apicoplast protein import and parasite demise. Importantly complementation of this mutant depends on an active site cysteine required for enzymatic activity. Taken together our experiments suggest an essential mechanistic role for the ERAD-like ubiquitylation machinery in apicoplast protein import.

Results
Ubiquitylation factors localize to the apicoplast in Toxoplasma and Plasmodium Using a combination of computational approaches we identified a comprehensive set of proteins that may act as apicoplast ubiquitylation system (see Materials and Methods). The results of these analyses (summarized in Table S1 in Text S1) identified apicoplast candidates for E1, E2 and E3 enzymes in both P. falciparum and T. gondii. We next determined whether these candidates are indeed targeted to the apicoplast. We targeted the locus of T. gondii TgE1 Ap by single homologous integration and placed a haemagglutinin (HA) epitope tag at the C-terminus of the protein. Stable transgenic clones show apicoplast staining when labeled with an anti-HA antibody by immunofluorescence (Fig. 1A, the P. falciparum homolog E1 is also localized to the apicoplast [12]). Our attempts to localize the candidates for apicoplast E2 by tagging the respective genes directly in the locus did not produce viable transgenics in either T. gondii or P. falciparum. Epitope fusion close to the C-terminal active domain may interfere with function and prevent replacement of the native gene. However, the coding sequence of TgE2 Ap could be fused to an epitope tag in the context of an ectopic expression plasmid (maintaining the native locus). Parasites expressing this construct show apicoplast labeling indistinguishable from that observed for E1 when probed with an epitope specific antibody. To localize the Plasmodium homolog (and to aid subsequent biochemical analysis) we also expressed a portion of Mal13P1.227 fused to an affinity tag in E. coli and used the purified recombinant protein to raise a specific antiserum. Immunofluorescence assays on P. falciparum parasites with this serum produced labeling that coincides with labeling for the apicoplast marker ACP (Fig. 1 C).
Two putative apicoplast E3 ubiquitin ligases were identified in Plasmodium, PfE3c Ap (PFC0740c -PF3D7_0316900) and PfE3w Ap (PFC0510w -PF3D7_0312100), and two in Toxoplasma (TGME49_226740 and TGME49_304460). We attempted to tag the proteins by placing different epitopes at the C-terminus through homologous gene targeting but were not successful. In case of PfE3c Ap transgenics that showed initial locus targeting were quickly lost upon selection ( Fig. S1A in Text S1). However, we recovered viable transgenic parasites tagged in the PfE3w Ap locus. Targeted integration of the cassette and transcription of PfE3w Ap -GFP was confirmed by PCR and RT-PCR (Fig. S1B-C in Text S1). Immunofluorescence assays showed PfE3w Ap -GFP to localize to the apicoplast (Fig. 1D). Finally, using an episomal expression vector, we found that the first 167 amino acids of PfE3c Ap target a GFP reporter to the apicoplast (Fig. 1E).
Apicoplast proteins are often processed at the N-terminus removing a leader peptide [4]. We analyzed processing for TgE1 Ap , TgE2 Ap and PfE2 Ap for which suitable reagents were available. TgE1 Ap produces the pattern typical for apicoplast proteins, two major bands likely corresponding to the precursor (heavier band) and mature protein (lighter band) Fig. 1F. Interestingly both TgE2 Ap and PfE2 Ap blots showed additional bands potentially arising from further post-translational modification ( Fig. 1 G, H).

Author Summary
The apicoplast is an essential parasite organelle derived from an algal endosymbiont. Most apicoplast proteins are nuclear encoded and post-translationally imported. Part of this journey utilizes the endoplasmic reticulum associated degradation or ERAD system of the algal endosymbiont. Typically, the ERAD system is ubiquitylation-dependent and acts in the retrotranslocation across the ER membrane and proteasomal destruction of misfolded secretory proteins. In the apicoplast, this system has been retooled into a protein importer. The apicoplast ERAD system is broadly conserved between most apicomplexans and surprisingly retains the ubiquitylation machine typically associated with destruction. This study brings together biochemical studies in Plasmodium and genetic studies in Toxoplasma. Together they provide significant mechanistic insight into the process of protein import into the apicoplast. We provide evidence that ubiquitylation may be a mechanistic requirement for import and demonstrate it to be essential to the parasite, thus providing new opportunities for drug development.
While the immunofluorescence assays indicate apicoplast localization of the ubiquitylation enzymes, overlap with luminal markers is only partial (see enlarged insert in Fig. 1A). We fixed and processed TgE2 AP -HA parasites for electron microscopy and incubated cryosections with an anti-HA antibody. Note that gold particles are found in the membranous periphery of the apicoplast ( Fig. 2 and Fig.  S4 in Text S1). This labeling is indistinguishable from that previously observed for the apicoplast ERAD-like proteins Der1 Ap and Cdc48 Ap [15] and the periplastid protein PPP1 [23]. We conclude that the apicoplast has a full complement of E1, E2 and E3 ubiquitylation enzymes localized to the periphery of the organelle, most likely the periplastid compartment as observed for the ERADlike system in the diatom Phaeodactylum tricornitum [14,18,19].
In vitro reconstitution of ubiquitylation using recombinant and native Plasmodium apicoplast enzymes We next sought to establish whether the candidate apicoplast ubiquitylation system is capable of activating and ligating ubiquitin. We amplified or synthesized sequences encoding full length PfE1L Ap and PfE2 Ap , or the RING domains of PfE3w Ap and PfE3c Ap respectively, and engineered them to be expressed as recombinant fusion proteins carrying an N-terminal glutathione Stransferase (GST) and/or six-histidine (HIS) affinity tag. Proteins of the expected size could be purified for all four constructs (Fig. 3A, B). We established biochemical ubiquitylation assays using combinations of parasite enzymes and commercially available heterologous components (Fig. 3C, recombinant human factors are shown in red, Plasmodium enzymes in green). Enzymes were incubated with recombinant human ubiquitin in a buffer containing an ATP regenerating system. When analyzed by Western blot, ubiquitin chains can be detected as ladders of high molecular weight bands [24]. Among the numerous human ubiquitin-activating enzymes tested, UBCH5a and UBCH13 were found to be suitable partners for PfE3c Ap and PfE3w Ap leading to robust ubiquitylation. Note that this activity is strictly dependent on the recombinant parasite E3 and absent in controls (Fig. 3D, E). The pattern obtained differed between the two E2 enzymes and suggested ubiquitylation of the RING domain in the context of only UBCH5a, while interaction with UBCH13 appeared to produce free poly-ubiquitin. Variation of ubiquitylation pattern depending on the E2 partnered with the ligase is frequently observed [25]. To test this independently we probed the in vitro reaction with anti-GST antibody to visualize the E3 and its higher molecular weight ubiquitin adducts. Consistently, this revealed shifts in molecular weight of PfE3c Ap and PfE3w Ap only when incubated with UBCH5a ( Fig. 3F) as free polyubiquitin is not detected in this assay format.
Next we tested whether ubiquitylation activity can be reconstituted entirely with parasite enzymes. When recombinant PfE1L Ap and PfE2 Ap were incubated with ubiquitin alone (Fig. 3G, left lane), no ubiquitylation was detected. However, upon addition of recombinant E3 ligase PfE3w Ap or PfE3c Ap , ubiquitylation was readily observed. Lastly we wished to evaluate the activity for native parasite enzymes. Among the reagents generated and tested in this study a custom-made antibody to PfE2 Ap was found suitable for immunoprecipitation under native conditions. Often the conjugating and ligating enzymes form a complex and can be co-precipitated and detected by their combined activity [26,27]. We incubated pull down fractions from parasite lysates with recombinant human UBA1, and biotinylated-ubiquitin (using tagged ubiquitin enhances sensitivity and focuses the assay on only newly ubiquitylated proteins). We observed significant ubiquitylation that was dependent on the immunoprecipitate and UBA1 (Fig. 3H). Taken together our observations provide biochemical support for the notion that the apicoplast ERAD-like system is capable of mediating ubiquitylation.
E2 Ap is required for parasite growth and protein import into the apicoplast The apicoplast ERAD system has a critical role in protein import into the organelle [5,18]. We tested whether ubiquitylation is a mechanistic requirement of this process by genetic ablation of the apicoplast ERAD-like ubiquitylation enzymes. We attempted disruption of the loci of PfE3c Ap , PfE3w Ap , and PfsUBA1. We isolated strains bearing drug marker insertions in the PfE3w Ap gene and documented loss of associated transcription (Fig. S2B, S3C in Text S1). However, we also noted multiple genomic duplications in these strains complicating interpretation (Fig. S3D in Text S1). We did not obtain viable parasites with disrupted PfE3cAP or PfsUBA1 loci. This is consistent with a potentially essential role for these proteins, and we therefore turned to T. gondii where the construction of conditional mutants is feasible.   We engineered a parasite strain where the endogenous promoter of the TgE2 Ap gene was replaced by a regulatable promoter in the following referred to as (i)DTgE2 Ap (Fig. 4A, [23]). This was accomplished by double cross over in the T. gondii TATiDKu80 background, a parasite line that favors homologous recombination and expresses a transactivator that can be modulated using anhydrotetracycline (ATc). Drug resistant parasite clones were tested by PCR and integration of the promoter was confirmed by Southern blot. We monitored the level of TgE2 Ap mRNA in response to ATc by quantitative PCR. Fig. 4D shows downregulation of the transcript below 10% of its normal level at day four of ATc treatment. We asked whether loss of TgE2 Ap affects parasite growth and performed plaque and real-time fluorescence assays. Parasites grow normally in the absence of ATc indicated by formation of plaques, however in the presence of ATc, plaque formation is severely attenuated (Fig. 4F). Similarly, (i)DTgE2 Ap parasites show significant growth reduction in the fluorescence assay in the presence of ATc (Fig. 4E), preincubation of parasites in ATc abolished growth entirely. We conclude, that TgE2 Ap is critical for parasite growth.
We next tested the ability of (i)DTgE2 Ap parasites to import apicoplast proteins in the absence or presence of ATc and measured the import-dependent lipoylation of the apicoplast pyruvate dehydrogenase E2 subunit [6]. (i)DTgE2 Ap parasites were treated with ATc for different periods and pulse-labeled for one hour with [ 35 S] methionine/cysteine. For the chase samples the radioactive isotope was removed, and cells were incubated for two additional hours in normal media. The samples were then used for immunoprecipitation with an anti-lipoic acid antibody followed by separation on SDS-PAGE. Treatment of cells with ATc for 2 days resulted in attenuation of import, leading to complete loss after 4 days (Fig. 4G, H). Lipoylation of two mitochondrial enzymes remained unaffected. We also monitored apicoplast loss, a frequent consequence of interference with apicoplast protein import [6,15]. We observed a drop over time, but note that loss of import significantly precedes plastid loss. Loss of apicoplast protein import has also been shown to result in loss of leader peptide removal and backing up of precursor protein into the ER and other elements of the secretory pathway [6,15,28] We therefore measured the levels of precursor and processed mature form of the apicoplast reporter protein FNR-RFP [29].. We grew parasites for 0 to 4 days on ATc and performed Western blots using parasite protein extracts from each day. Probing these blots with an antibody against RFP revealed that precursor levels of FNR-RFP remained unchanged throughout the 4 days, while the mature protein was no longer detected after 2 days on ATc further supporting a strong import defect (Fig. 4I). We also monitored the localization of FNR-RFP in treated and untreated (i)DTgE2 Ap parasites by immunofluorescence assay. In untreated parasites FRN-RFP is restricted to the apicoplast (Fig. 4K). After 48 hours of ATc treatment 38% of parasite vacuoles also show significant labeling outside of the apicoplast surrounding the nucleus likely representing the ER (Fig. 4J, untreated TgE2 Ap or ATc treated wild type parasites showed such labeling in ,3% of counted four cell vacuoles, n = 200). We conclude that apicoplast protein import is impaired in the absence of TgE2 Ap .
A conserved cysteine residue in the active site of TgE2 Ap is required for its function Apicoplast ubiquitylation enzymes are capable of synthesizing ubiquitin chains in vitro, but is this activity required in vivo? To test this we established a complementation assay. The coding sequence of the TgE2 Ap gene driven by a constitutive promoter was introduced into the uracil-phosphoribosyltransferase (UPRT) locus of the (i)DTgE2 Ap mutant (Fig. 5C). Parasites were selected for the loss of UPRT activity using 5-fluorodeoxyuridine [30] and a clonal cell line that now constitutively expressed a second copy of TgE2 Ap in the conditional knock down background was isolated. We confirmed correct integration by PCR (Fig. 5D). We tested the ability of this strain to form plaques when expression from the native locus is ablated by ATc treatment, and found that genetic complementation fully rescued growth (Fig. 5E).
Multiple sequence alignment of TgE2 Ap and E2 enzymes from a wide range of eukaryotes showed that TgE2 Ap shares conserved features, reported earlier to be critical for this class of enzymes. We therefore modelled the C-terminal domain of TgE2 Ap onto the structure of UBC4, a well characterize yeast ubiquitin conjugating enzyme [31]. Multiple sequence alignment and homology modelling identified C573 as the presumptive active site cysteine (Fig. 5A, B, see Fig. S3 in Text S1). Most E2 enzymes possess a signature HPN triad proximal to the active site cysteine [32]. The histidine has been previously suggested to be dispensable for E2catalyzed ubiquitylation, but is important for the folding of the active site in other systems [33]. The asparagine residue on the other hand was consistently found to be important for RING-E3/ E2-dependent ubiquitin conjugation [34]. A conserved HXH triad is found at this position in apicomplexans (Fig. 5B). We engineered a series of point mutants in TgE2 Ap replacing C573, H563, and H565 with alanine respectively. These genes were then introduced into the (i)DTgE2 Ap mutant as described above and tested for their ability to complement loss of TgE2 Ap upon ATc treatment using plaque assay. Expression of the H563A point mutant fully complemented loss of native TgE2 Ap (Fig. 5E) and parasites now grow even in the presence of ATc. In contrast, despite numerous attempts we were unable to establish a stable parasite line expressing H565A, which may suggest dominant effects of this mutation. We were able to isolate mutants expressing C573A, however these strains show no complementation, and are still fully susceptible to ATc treatment (Fig. 5E). We conclude that enzymatic activity is a requirement for TgE2 Ap function in vivo and that C573 and H565 residues are critical for the function of the enzyme while H563 is likely dispensable.

Discussion
Endosymbiosis is a key evolutionary mechanism underlying the emergence and diversification of eukaryotes -in particular for photosynthetic eukaryotes. The acquisition of a eukaryotic red algal symbiont led to the chromalveolates, a large super-phylum of tremendous ecological diversity that includes apicomplexan parasites. The descendent of the algal symbiont, the apicoplast, maintains a highly compartmentalized organization, and nuclear encoded proteins have to overcome four membranes on their journey to the stroma. An apicoplast-localized ERAD-like system appears to play an important role in apicoplast protein import. Recent reports have identified and characterized components of this ERAD-like system in different algal and parasite species [7,10,12,13,14,15]. In this study we provide significant biochemical and genetic evidence for the hypothesis that an apicoplast localized ubiquitylation cascade is an essential element of this protein import system. We identify apicoplast ubiquitin activating, conjugating and ligating enzymes in two important apicomplexan parasites, P. falciparum and T. gondii. We show in vitro and in vivo that these proteins have conserved biochemical activities and are capable of ubiquitin transfer. Finally, in genetic studies, we show that TgE2 Ap , for which we were able to isolate a conditional mutant, is essential for apicoplast protein import, organellar maintenance and parasite growth. Overall these observations support a direct mechanistic role of ubiquitylation in protein translocation independent of ubiquitin's function in proteasomal degradation [11]. The classical ERAD system is believed to recognize and respond to the folding state of secretory proteins. Interestingly, recent studies show that the transit peptide of apicoplast proteins is primarily unstructured and that this conformation may be critical for proper transport to the organelle [35]. This model would need a distinguishing element to avoid elimination of apicoplast proteins by the classical ERAD. Specific chaperone sets could potentially provide such specificity, but remain to be discovered. A recent study in Arabidopsis has identified a role for ubiquitylation also in primary plastids, however this role appears to be distinct from secondary plastids. In this case ubiquitylation results in degradation of the components of the TOC complex and is thought to more globally regulate chloroplast biogenesis during plant development [36].
The identity of the apicoplast ubiquitin or ubiquitin-like modifier remains a significant unresolved question. Our results demonstrate that apicoplast enzymes are capable of acting on archetypical ubiquitin (recombinant human protein), studies in P. tricornitum show similar activity for a E3 ligase found in the diatom secondary chloroplast [18]. However whether the apicoplast system actually utilizes ubiquitin in vivo remains to be established. As shown in Fig. 1G and H Western blots for TgE2 Ap and PfE2 Ap show additional bands. It is conceivable that these bands represent ubiquitin or a ubiquitin-like protein covalently bound to the active site of the apicoplast localized E2. However we note that, for TgE2 A , none of the bands was affected by reduction of the protein or point mutation of the active site cysteine (data not shown). Alternatively this may indicate an ubiquitin-like protein bound to a residue different from the active site of the enzyme or multiple processing steps as have been observed for some apicoplast membrane proteins [37]. Our efforts to demonstrate ubiquitin bound to apicoplast ubiquitylation enzymes purified from P. falciparum or T. gondii so far did not result in robust detection (using either antibodies or mass spectrometry, data not shown). Furthermore ubiquitylation of plastid-bound cargo proteins is not readily observed in apicomplexans or diatoms. A reasonable candidate for which apicoplast localization has been suggested [12] is an atypical, large ubiquitin-like protein (PF08_0067). Curiously, this protein lacks the di-glycine motif typically required for the formation of the isopeptide bond and a homolog has yet to be identified in the Toxoplasma genome. Similarly, plastid ubiquitin candidates from algae show lack of sequence motifs typically required for polyubiquitylation [19]. It is conceivable that this ubiquitin-like protein could be processed and/or ligated in a novel fashion that does not depend on a di-glycine sequence. Alternatively, its function may resemble that of the HERP protein in the classical ERAD pathway. Like PF08_0067, HERP has an ubiquitinlike domain at the N-terminus followed by transmembrane domains at the C-terminus [38]. HERP is believed to interact with HRD1 and to regulate the ubiquitylation activity of the ERAD translocon in response to folding stress [39]. In that case PF08_0067 is likely not the main substrate for the apicoplast ubiquitylation system and the modifier is yet to be discovered.
Studying the apicoplast ubiquitin faces technical obstacles that so far prevented direct tagging of the candidate ubiquitin and subsequent detection of modified cargo. There are several strong candidates for plastid-localized deubiquitylation enzyme in apicomplexans and diatoms (Table S1 in Text S1, [13,18]). The activity of these enzymes may dramatically shorten the time ubiquitin remains on proteins and thus prevent the robust detection of ubiquitin adducts [22]. Isolation of mutants lacking apicoplast deubiquitylation might allow testing of this hypothesis and potentially lead to accumulation of modified cargo proteins. While a number of mechanistic details of the apicoplast ubiquitylation system remain to be elucidated, we demonstrate that the system is essential to the organelle and the parasite. Building on a longstanding effort to target ubiquitylation for the development of anti-cancer drugs [40] may potentially lead to new anti-parasitic compounds in the future.
For in vitro ubiquitylation assays recombinant P. falciparum enzymes were incubated with recombinant human or parasite factors. Typically 50-200 mM recombinant ubiquitin, 0.05-0.2 mM E1 enzyme, 1-5 mM E2 enzymes, and 1-12.5 mM of E3 ligases were incubated in 50 mM Tris-HCl, pH 7.4, 1 mM DTT in presence of a re-energizing system (BostonBiochem) containing the ATP and ATP regenerating enzymes to recycle hydrolyzed ATP needed for the assay, for 2 hours at 37uC followed by SDS-PAGE and immunoblotting.
Ex vivo ubiquitylation assays were performed by lysing 3D7 P. falciparum in 20 mM HEPES pH 7.9, 10 mM KCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 0.5 mM AEBSF (Fisher Scientific), 0.65% Igepal v/v, and protease inhibitor cocktail (Roche), or 20 mM HEPES pH 7.9, 0.1 M NaCl, 0.1 mM EDTA, 0.1 mM EGTA, 1.5 mM MgCl2, 1 mM DTT, 1 mM AEBSF and protease inhibitor cocktail (Roche). Supernatants were pooled and proteins were precipitated using the indicated antibodies and magnetic Protein A beads. Proteins bound to beads were mixed with re-energizing buffer, 0.5 mg/ml biotin-conjugated ubiquitin, 5 mM AEBSF and protease inhibitor cocktail. Reactions were incubated at 30uC with gentle agitation for two hours. Samples were eluted with 46 Laemmli buffer and analysed using biotin affinity blots. Human recombinant UBE1 and UBC enzymes, E3 ligases biotin conjugated ubiquitin and re-energizing buffer used in these assays were purchased from Boston Biochem.
T. gondii gene models were tested by 59-and 39-RACE. Note that additional exons were identified for TgE2 Ap (see genbank JX431938 for correct sequence). A conditional TgE2 Ap knock-out was generated by exchanging the native promoter for the tetracycline inducible t7s4 promoter in the TATiDKu80 parasite background. The targeting construct used 1.2 kb up-and 1.5 kb downstream of the TgE2 Ap start codon introduced into vector pDT7S4. Linearized plasmid was transfected into the parental strain followed by pyrimethamine selection. To complement the knock-out, a TgE2 Ap minigene was inserted into the UPRT locus under the control of a constitutive sag1 promoter. Transgenics were isolated in 5 mM 5-FUDR and identified by PCR. Parasite growth was measured by fluorescence and plaque assay in the presence and absence of 0.5 mm anhydrotetracycline (ATc). Please refer to the supplement materials for a more detailed description of materials and methods used in this study (including a table of all primers).

Supporting Information
Text S1 Provides supplemental materials and methods used for the Bioinformatic analysis. We also provides additional information for the T. gondii plasmids, cell line and cell culture used; the apicoplast protein import assay; the antibody based assay; the cloning and purification of recombinant P. falciparum proteins and finally the Phylogenetic analysis and homology modelling of T. gondii TgE2 Ap . Figure S1 presents the 36HA tagging strategy used for PfE3c Ap . Figure S2 shows the gene disruption strategies used for PfE1L Ap , PfE3c Ap (PFC0740c) and PfE3w Ap . Figure S3 shows the multiple sequence alignment for E2 enzymes using MUSCLE. Figure S4 shows additional cyroelectonmicroscopic images of TgE2 Ap . Table S1 presents the apicoplast candidates for E1, E2, E3 and Dub enzymes in both P. falciparum and T. gondii and lastly table S2 present a list of primers used for the cloning of recombinant proteins. Figure S1.   Figure S3. Multiple sequence alignment for E2 enzymes using MUSCLE. Figure S4. Additional cyro-electonmicroscopic images of TgE2 Ap . Sections were incubated with anti-HA antibody and gold conjugated anti-immunoglobulin as detailed in Figure 2. Table S1. Apicoplast candidates for E1, E2, E3 and Dub enzymes in both P. falciparum and T. gondii. Table S2. List of primers used for the cloning of all recombinant proteins. (DOC)