The Skp1 Protein from Toxoplasma Is Modified by a Cytoplasmic Prolyl 4-Hydroxylase Associated with Oxygen Sensing in the Social Amoeba Dictyostelium*

Background: A cytoplasmic prolyl 4-hydroxylase of Dictyostelium contributes to O2 sensing by modifying Skp1. Results: The corresponding protein of Toxoplasma exhibits related functions but has high affinity for O2. Conclusion: Hydroxylation of Skp1 is conserved in protists but may sense O2 indirectly. Significance: The putative evolutionary precursor of animal prolyl 4-hydroxylases that modify HIFα potentially remodeled the proteome via degradation rather than transcriptionally. In diverse types of organisms, cellular hypoxic responses are mediated by prolyl 4-hydroxylases that use O2 and α-ketoglutarate as substrates to hydroxylate conserved proline residues in target proteins. Whereas in metazoans these enzymes control the stability of the HIFα family of transcription factor subunits, the Dictyostelium enzyme (DdPhyA) contributes to O2 regulation of development by a divergent mechanism involving hydroxylation and subsequent glycosylation of DdSkp1, an adaptor subunit in E3SCF ubiquitin ligases. Sequences related to DdPhyA, DdSkp1, and the glycosyltransferases that cap Skp1 hydroxyproline occur also in the genomes of Toxoplasma and other protists, suggesting that this O2 sensing mechanism may be widespread. Here we show by disruption of the TgphyA locus that this enzyme is required for Skp1 glycosylation in Toxoplasma and that disrupted parasites grow slowly at physiological O2 levels. Conservation of cellular function was tested by expression of TgPhyA in DdphyA-null cells. Simple gene replacement did not rescue Skp1 glycosylation, whereas overexpression not only corrected Skp1 modification but also restored the O2 requirement to a level comparable to that of overexpressed DdPhyA. Bacterially expressed TgPhyA protein can prolyl hydroxylate both Toxoplasma and Dictyostelium Skp1s. Kinetic analyses showed that TgPhyA has similar properties to DdPhyA, including a superimposable dependence on the concentration of its co-substrate α-ketoglutarate. Remarkably, however, TgPhyA had a significantly higher apparent affinity for O2. The findings suggest that Skp1 hydroxylation by PhyA is a conserved process among protists and that this biochemical pathway may indirectly sense O2 by detecting the levels of O2-regulated metabolites such as α-ketoglutarate.

In diverse types of organisms, cellular hypoxic responses are mediated by prolyl 4-hydroxylases that use O 2 and ␣-ketoglutarate as substrates to hydroxylate conserved proline residues in target proteins. Whereas in metazoans these enzymes control the stability of the HIF␣ family of transcription factor subunits, the Dictyostelium enzyme (DdPhyA) contributes to O 2 regulation of development by a divergent mechanism involving hydroxylation and subsequent glycosylation of DdSkp1, an adaptor subunit in E3 SCF ubiquitin ligases. Sequences related to DdPhyA, DdSkp1, and the glycosyltransferases that cap Skp1 hydroxyproline occur also in the genomes of Toxoplasma and other protists, suggesting that this O 2 sensing mechanism may be widespread. Here we show by disruption of the TgphyA locus that this enzyme is required for Skp1 glycosylation in Toxoplasma and that disrupted parasites grow slowly at physiological O 2 levels. Conservation of cellular function was tested by expression of TgPhyA in DdphyA-null cells. Simple gene replacement did not rescue Skp1 glycosylation, whereas overexpression not only corrected Skp1 modification but also restored the O 2 requirement to a level comparable to that of overexpressed DdPhyA. Bacterially expressed TgPhyA protein can prolyl hydroxylate both Toxoplasma and Dictyostelium Skp1s. Kinetic analyses showed that TgPhyA has similar properties to DdPhyA, including a superimposable dependence on the concentration of its co-substrate ␣-ketoglutarate. Remarkably, however, TgPhyA had a significantly higher apparent affinity for O 2 . The findings suggest that Skp1 hydroxylation by PhyA is a conserved process among protists and that this biochemical pathway may indirectly sense O 2 by detecting the levels of O 2 -regulated metabolites such as ␣-ketoglutarate.
All cells must be able to detect oxygen and rapidly respond to changes in its availability. Over the past decade prolyl 4-hydroxylases (P4Hs) 3 have emerged as key cellular O 2 sensors. The P4Hs (1) consume ␣-ketoglutarate (␣KG) and O 2 to form succinate and 4(trans)-hydroxyproline (Hyp) on the target protein. The K m toward O 2 of known cytoplasmic P4Hs lies near or above the atmospheric level (21%), which allows them to sense acute changes in O 2 . Under conditions of normoxia and sufficient metabolic availability of ␣KG, cytoplasmic animal P4Hs (known as PHDs, for prolyl hydroxylase domain containing) hydroxylate the HIF␣ family of transcriptional factor subunits, resulting in recognition by the von Hippel-Lindau tumor suppressor protein (2). von Hippel-Lindau tumor suppressor protein, as a subunit of a VBC-class E3-Ub ligase, directs the polyubiquitination of HIF-1␣ and its subsequent degradation in the 26 S proteasome. In hypoxia, unhydroxylated HIF␣ accumulates and dimerizes with HIF-1␤ (ARNT), leading to the transcriptional activation of hypoxia-response genes.
The HIF␣-specific PHDs have been implicated to be direct O 2 sensors because of their high K m values toward O 2 . However, other mechanisms may also contribute to PHD-dependent O 2 sensing because low O 2 affects levels of Krebs cycle intermediates, including ␣KG, which is a PHD substrate, and others that inhibit PHD activity (3)(4)(5). Changes in O 2 availability also affect reactive oxygen species production, which might influence the oxidation state of iron in the enzyme active site, although evidence on this point is controversial (6,7). The availability of iron mediated via chaperones (8) or other metals that compete for iron binding may also be regulatory. Alternatively, oxidants like H 2 O 2 or other metals may influence PHD activity via effects on the ascorbate pool, thus, indirectly affect-ing the redox state of iron (9). Finally, PHDs have been proposed to be regulated by other gasses such as NO (10).
Dictyostelium discoideum is a social soil amoeba that undergoes starvation-induced aggregation and development to form fruiting bodies at the soil surface to support dispersal of the aerial spores to new locations. The migratory slug, an intermediate stage in the developmental program, uses O 2 as a guide to move to the soil surface and as a trigger to culminate there into fruiting bodies. In Dictyostelium, the animal PHD homolog DdPhyA (11) mediates the 4(trans)-hydroxylation of Pro-143 of DdSkp1 (12), which is a subunit of the SCF class of E3 Ub ligases (13). In contrast to HIF␣, whose hydroxylation dictates its recognition by von Hippel-Lindau tumor suppressor protein and its stability, Hyp143 can then be modified by a novel pentasaccharide by the action of five glycosyltransferase activities encoded by three genes (14 -16). The sequential steps of Skp1 glycosylation modulate O 2 control of culmination (17)(18)(19). Substantial sequence (20) and biochemical (12) data indicate that despite the differences in target protein recognition, the animal and Dictyostelium P4Hs share a similar catalytic mechanism and affinities for O 2 , ␣KG, Fe ϩ2 , and inhibitors. Development (18) and Skp1 hydroxylation 4 are O 2 -regulated in Dictyostelium, and genetic evidence shows that DdPhyA hydroxylation of Skp1 (17)(18)(19)21) at least partially mediates the O 2 effect. But as for animals, whether DdPhyA directly or indirectly senses O 2 is unclear.
Bioinformatic analyses predict that orthologues of the DdPhyA and the Skp1-modifying glycosyltransferases exist in the protozoan pathogen Toxoplasma gondii (22), which is an important infection in immune compromised people and in developing fetuses. Toxoplasma has a complex life cycle in which the sexual stage occurs in the felid gut and the resulting sporozoites are shed in fecal material within oocysts (23)(24)(25)(26). Upon ingestion of oocysts, the parasites are released and infect intestinal cells and convert into the disease-causing tachyzoite form. Tachyzoite infection of the intestine triggers the recruitment of innate immune cells that are in turn infected by the tachyzoites and used by the parasite to disseminate to various tissues including the brain, retina, and muscle. While the resulting immune response kills the majority of the parasites, some escape destruction by developing into encysted bradyzoites. The discovery that DdSkp1 modification pathway genes are conserved in Toxoplasma raised the unexplored possibility that related O 2 or metabolic sensing may play a role in allowing the parasite to survive in the wide range of O 2 tensions encountered by the parasite.
Here we demonstrate that Toxoplasma PhyA (TgPhyA) modifies TgSkp1 in tachyzoites and contributes to adaptation to low O 2 in a growth assay. Furthermore, using Dictyostelium as a surrogate expression system and in studies of purified recombinant proteins, we show evidence that TgPhyA is uniquely dependent on ␣KG and thus may act as an indirect O 2 sensor. These findings open a new arena for studying the role of metabolic regulation of Toxoplasma biology and virulence.

Expression and Purification of Recombinant TgHis 6 PhyA-
The predicted coding region for TgPhyA was initially identified by BLAST analysis of T. gondii genomes that yielded the 8-exon gene model TGGT1_114560 from strain GT-1 (ToxoDB Release 7.1). TgPhyA cDNA was amplified from RNA isolated from RH strain tachyzoites using the oligonucleotides PHbS and PHbAS (supplemental Table S1), cloned into pCR2.1TOPO vector (Invitrogen), excised using NdeI and XhoI (sites underlined), and ligated into similarly digested pET15b (Novagen). The insert was sequenced using vectorbased primers and found to match that of the gene model. This vector, pET15b-His 6 TgPhyA, was expected to encode the fulllength of TgPhyA preceded by a His 6 tag and a tobacco etch virus cleavage site from pET15bTEV (supplemental Fig. S1). pET15b-TgHis 6 PhyA was transformed into competent BL21-Gold (DE3) (Invitrogen) cells. A single clone was grown overnight on a shaker in 3 ml of LB medium containing 100 g/ml ampicillin at 37°C, collected by centrifugation at 5000 g ϫ 5 min, resuspended in 1 liter of LB medium with ampicillin, and shaken at 37°C. After attaining an A 590 of 0.4 -0.6, isopropyl 1-thio-␤-D-galactopyranoside was added to 0.5 mM, and incubation was continued for 16 h at 22°C. Cells were collected as above at 4°C, rinsed with ice-cold 20 mM Tris-HCl (pH 8.2) buffer, resuspended in 40 ml of Escherichia coli extraction buffer (0.1 M Tris-HCl, pH 8.2, 5 mM benzamidine, 0.5 g/ml pepstatin A, 5 g/ml leupeptin, 5 g/ml aprotinin, 0.5 mM PMSF, 1 mg/ml lysozyme), and passed through a French press cell at 18,000 p.s.i. An S100 fraction was prepared by centrifugation at 100,000 g ϫ 1 h, and the supernatant was loaded onto a 5-ml HisTrap HP column (GE Healthcare) pre-equilibrated in binding buffer (20 mM Tris-HCl (pH 7.9), 0.5 M NaCl, 10% (v/v) glycerol, 5 mM imidazole). After washing with the same buffer containing 60 mM imidazole, the column was eluted with ascending linear gradient of 60 -1000 mM imidazole in the same buffer.
Expression and Purification of Recombinant TgSkp1-Tg-Skp1 cDNA was amplified from and cloned as for TgPhyA using the primers SkpS and SkpAS (supplemental Table S1). Sequencing showed a perfect match with gene model TGGT1_020570. The cDNA was excised with NcoI and BamHI and cloned into similarly digested pET15b (Invitrogen) designed to express the native TgSkp1 sequence without tags. TgSkp1 was purified from E. coli by sequential DEAE-Sepharose, phenyl-Sepharose, Q-Sepharose, and Superdex-200 chromatography as described for DdSkp1 (11).
Toxoplasma Growth and Fitness-Toxoplasma was grown in HFF cells and maintained in DMEM medium supplemented with 10% heat inactivated fetal bovine serum (27). Plaque assays were performed by infecting confluent HFF monolayers in 24-well plates with 500 parasites/well. The cells were grown 5-6 days and then methanol-fixed and stained with crystal violet to detect plaques (27), which were measured using Metamorph software (Molecular Devices, Sunnyvale, CA). For fitness studies, confluent HFFs in 25-cm 2 flasks were inoculated with equal numbers of parasites (2 ϫ 10 5 parasites of each strain). After 3 days, the flasks were scraped and syringe-lysed, and parasites were counted in a hemacytometer. Then 500 parasites were added to 24-well plates containing confluent HFFs and grown in the absence (allows all parasites to grow) or presence (only mutant cells grow) of 25 g/ml mycophenolic acid (Sigma) and 25 g/ml xanthine (Sigma). After 5-6 days, the plates were fixed, and plaques were counted by crystal violet staining. Replication rates were determined by infecting HFFs on glass coverslips for 24 h as described (27). The cells were then fixed, stained with SAG1 antisera, and at least 50 randomly selected vacuoles were counted for parasite content for each sample.
Construction of TgPhyA Disruption Strains-To generate the TgPhyA knock-out vector, exon 1 was deleted by double-crossover homologous recombination. 5Ј-Flank and 3Ј-flank targeting sequences were amplified from RH strain genomic DNA by PCR using primer pairs g and h and pairs i and j (supplemental Table S1). A stop codon (TGA) was inserted into primer i to block translation of exon 2 of a potential transcript. The 5Ј-fragment was inserted into pminiGFP.ht (from Dr. Gustavo Arrizabalaga, University of Idaho) between its KpnI and HindIII sites, and the 3Ј flank was inserted between its XbaI and NotI sites (see Fig. 1A). The plasmid was linearized with KpnI and electroporated (28) into the type 1 strain RH⌬hxgprt⌬ku80 (KU80⌬⌬), which is impaired in non-homologous end-joining DNA repair, resulting in higher frequency of homologous recombination (29). Drug-resistant transformants were selected for in the presence of mycophenolic acid/xanthine. Tachyzoites were cloned by limiting dilution and screened by PCR to identify strains harboring the desired replacement (see "Results").
Overexpression of TgPhyA and TgPhyA(R252A) in Dictyostelium-To express TgPhyA in Dictyostelium, its codon usage was modified to be similar to that of DdphyA without changing the amino acid sequence, based on analysis using the online software GCUA (30). The modified TgphyA nucleotide sequence, named TgphyA*, is compared with the original in supplemental Fig. S1. This sequence and flanking sequences designed to facilitate construction of the transgenic replacement DNA at the DdphyA locus (see below) was chemically synthesized by Genscript Co. Ltd., and inserted into pUC57 between its EcoRI and HindIII sites. Synthetic pUC57-TgphyA* was digested with KpnI and BamHI, and the released TgphyA* fragment was ligated between the KpnI and BamHI sites of the pVSC and pVSE vectors (18), designed to drive expression in prespore or prestalk cells, respectively. The ligation products pVSC-TgphyA* and pVSE-TgphyA* were electroporated into Dictyostelium strain Ax3 or HW288 (phyA Ϫ ). G418-resistant clones were isolated as described (11), and slug stage cells were screened for expression of TgPhyA by Western blotting or prolyl hydroxylase enzyme activity.
To express an enzymatically inactive form of TgPhyA, an R252A substitution, corresponding to the R276A mutation that inactivates DdPhyA (18), was introduced by site-directed mutagenesis. pVSC-TgphyA*R252A was amplified from pUC57-TgphyA* using primers R252A-S and R252A-AS (supplemental Table S1). The mutagenesis products were digested by DpnI and transformed into TOP10 cells. Positive clones were screened for the introduced NsiI restriction enzyme site.
The resulting construct TgphyA*R252A was digested with KpnI and BamHI, and the purified fragment was inserted into pVSC, predigested with KpnI and BamHI, and then transformed into strain HW288 as above.
Transgenic Replacement of DdphyA with TgphyA-The DdphyA upstream genomic sequence (1240 bp) was amplified from D. discoideum genomic DNA with primers m and n (supplemental Table S1), and the resulting PCR product was cloned into pCR4TOPO (Invitrogen). After sequencing to confirm fidelity, the inserted fragment was released with EcoRI and KpnI and inserted into pUC57-TgphyA predigested with same restriction enzymes, resulting in pUC57-UP-TgphyA. The downstream genomic sequence (1615 bp) was amplified by primers o and p and similarly cloned into pCR4TOPO. After sequencing, the inserted fragment was released with SpeI and HindIII and ligated into pUC57-UP-TgphyA predigested with same restriction enzymes, yielding pUC57-UP-TgphyA-DOWN. The blasticidin S cassette was released from pLPBLP (31) using XmaI and inserted into pUC57-UP-TgphyA-DOWN pre-linearized with XmaI, resulting in pUC57-UP-TgphyA-BSR-DOWN. The final plasmid was digested with EcoRI and HindIII to retrieve the desired disruption DNA, which was electroporated into strain Ax3 strain. Clones growing in 10 g/ml blasticidin S in HL5 ϩ medium were screened by PCR for homologous-directed or ectopic insertions as described under "Results." Prolyl Hydroxylase Activity Assays-For purified enzyme proteins, prolyl hydroxylase activity was quantitated using either the 14 CO 2 release assay, which detects evolution of 14 CO 2 in the presence of [1-14 C]␣KG, or the one-step tandem 3 H incorporation assay, which detects the appearance of 3 H in HO-Skp1 in the presence of DpGnt1 and UDP-[ 3 H]GlcNAc, as described previously (12). Reactions were typically conducted at 22°C at ambient (21%) O 2 . O 2 was varied as described (11). P4H activity was assayed in crude extracts as described (11,12). Briefly, S100 preparations were desalted by gel filtration, and the transfer of [ 3 H]GlcNAc from UDP-[ 3 H]GlcNAc into the hydroxylated product of exogenous Skp1 in the presence of 100 g of extract protein and DpGnt1 in 20 l was assayed using the one-step 3 H assay.
MALDI-TOF MS Analysis of TgSkp1-TgSkp1 was modified by TgPhyA or DdPhyA in 25 mM Tris-HCl (pH 7.4), 2.5 mM DTT, 1.5 mM ␣KG, 5.0 M FeSO 4 , 1.0 mM ascorbate, and 0.1 mg/ml catalase (Sigma) at 29°C overnight and supplemented with additional aliquots of enzyme after 4 and 16 h. The pool of TgSkp1 was denatured, reduced in 8 M urea, 10 mM DTT, 0.4 M NH 4 HCO 3 , and alkylated in 30 mM iodoacetamide at room temperature in the dark for 1 h. After the addition of DTT to a final concentration to 40 mM, TgSkp1 was purified on a Supelco Discover C8 Biowide 5-m 2.1/10 reverse phase column with an acetonitrile gradient (5-90%) in 0.1% trifluoroacetic acid. The TgSkp1 pool was concentrated by vacuum centrifugation, brought to 0.1 M Tris-HCl (pH 9.2) and 2 M urea and then 0.69 g/ml Endo-lys-C (Waco), and incubated overnight at 37°C. Alternatively, NH 4 HCO 3 was added to a concentration of 100 M (pH 8.0 final), and the reaction was supplemented with trypsin to 0.05 g/ml and incubated at 37°C overnight. The resulting peptides were analyzed directly after adsorption to and release from a C18 zip-tip on a Bruker Ultraflex II MALDI-TOF/TOF-MS or first fractionated on a RPC C2/C18 2.1/10 column (GE Healthcare ) with a gradient of acetonitrile from 5 to 40% and trifluoroacetic acid from 0.1 to 0.085%.
Culture and Development of Dictyostelium Cells-Dictyostelium strains were grown and developed on filters in different O 2 /N 2 atmospheres as described (18). Cells were digitally imaged using a SPOT FLEX camera on a stereomicroscope after 36 h, and sporulation was quantitated by transferring filters into 15-ml tubes, vortexing in the presence of 0.5% Nonidet P-40, 5 mM EDTA in PDF buffer (33 mM NaH 2 PO 4 , 10.6 mM Na 2 HPO 4 , 20 mM KCl, 6 mM MgSO 4 , pH 5.8) and counting in a hemacytometer.
Soluble extracts were combined with 4ϫ Laemmli sample buffer, boiled for 1 min, and electrophoresed on a 4 -12% gradient SDS-PAGE gel (NuPAGE Novex, Invitrogen). Gels were transferred to nitrocellulose membranes using an iBlot system (Invitrogen), probed with primary and fluorescent secondary Abs, and scanned on a Li-Cor Odyssey infrared scanner.

RESULTS
TgPhyA Sequence Characteristics-BLASTP and TBLASTN searches for DdPhyA-like sequences yielded two related genes in the three fully sequenced strains of T. gondii available at ToxoDB (ME49, GT1, and VEG) and in Neospora canium. The sequences of the predicted TgPhyA (TGGT1_114560) and TgPhyB (TGGT1_125030) proteins are 45/27 and 59%/32% similar/identical, respectively, to DdPhyA over their C-terminal 210 amino acids, which corresponds to a minimal catalytically active fragment of human PHD2 (33). In comparison, the T. gondii sequences are 46% similar and 27% identical to each other, suggesting they are only distantly related. Despite their limited similarity, the identity of catalytic amino acids associated with the active site suggested potential conservation of function with DdPhyA. TgPhyA is more similar in length (271 amino acids) to DdPhyA (284 amino acids) than TgPhyB (525 amino acids), which possesses an N-terminal extension with no significant homology to other proteins. Like PHD2 and DdPhyA (20), TgPhyA possesses sequence motifs not present in animal and plant rER P4Hs that modify collagens and plant cell wall proteins (supplemental Fig. S2). This similarity is consistent with the absence of hydrophobic organelle targeting sequences and is evidence for a line of evolutionary descent separate from the rER proteins (20). Evidence available at ToxoDB indicates that TgPhyA is expressed, with three tachyzoite EST clones, four bradyzoite EST clones, and positive RNAseq data reported. In addition, the putative promoter region is marked by increased levels of H3K4Me3 and H3K9Ac and decreased levels of H3K4Me1.
TgPhyA Is Important for T. gondii Growth at Physiological O 2 Levels-To determine if TgPhyA modifies Skp1 in T. gondii, the TgphyA locus was disrupted in the ⌬KU80 RH strain (KU80⌬⌬) by homologous recombination. To this end, the pminiGFP.ht vector was modified as depicted in Fig. 1A by the introduction of sequences flanking the first two exons of TgphyA to promote their replacement by the HXGPRT selection cassette via a double cross-over mechanism. Two GFP-negative clones and a GFP-positive clone (clone 3) were analyzed further. By PCR analysis, all three clones exhibited loss of exon 1 sequences while retaining neighboring upstream and downstream sequences as expected (Fig. 1B). Strains are listed in Table 1.
The impact of TgPhyA on Toxoplasma replication was assessed by infecting HFFs with parental (KU80⌬⌬) or TgphyA-disruption (RHphyA⌬) parasites and 24 h later counting numbers of parasites in individual vacuoles. The data indicated that the mutants had a slight but reproducible decrease in the average number of parasites/vacuole ( Fig. 2A). We then tested whether this decreased growth corresponded to a more significant growth defect by infecting confluent HFFs with parental and mutant parasites for 5 days. The cells were then fixed and stained with crystal violet, and the size of the resulting plaques was measured. Plaques formed by RHphyA⌬ parasites were ϳ50% smaller in area than the plaques formed by parental parasites (Fig. 2B). As an alternative approach to assess the impact of TgphyA disruption on replication, a competition assay was performed in which equal numbers of normal and mutant parasites were added to a monolayer of HFFs. After 3 days, the parasites were mechanically released from the host cells and counted using a hemacytometer. Five hundred parasites were then added to wells of HFFs in either the absence (allows growth of both strains) or presence (allows growth of TgphyA knock-out only) of mycophenolic acid/xanthine. The number of plaques formed was then counted, and the data indicated that RHphyA⌬ contributed to ϳ27% of the plaques (Fig.  2C). Decreased numbers of plaques formed by the mutant was not due to growth in MPA/xanthine as the expected 50:50 ratio of plaques was formed when mutant parasites were grown with wild-type RH parasites, which is the background strain of KU80⌬⌬ and contains the HXGPRT gene that enables them to grow in MPA/xanthine containing media. Similar results were obtained with an independent clone, indicating that the difference was not due to a spurious secondary effect.
TgPhyA and DdPhyA are related to the HIF␣-targeted PHDs, which are cellular hypoxia sensors. Because DdPhyA regulates development in an O 2 -dependent manner, we tested whether growth of the RHphyA⌬ mutants was more severely JULY 20, 2012 • VOLUME 287 • NUMBER 30 affected under hypoxic conditions. Thus, HFFs were infected with KU80⌬⌬ or RHphyA⌬ and the plates were grown at 0.5% O 2 , which was chosen because it represents an O 2 tension encountered by the parasites within its host. After 24 h, the cells were fixed and numbers of parasites per vacuole were counted. The data indicated that at 0.5% O 2 , RHphyA⌬ grew even more slowly than its parental strain (Fig. 2D) compared with the difference at 21% O 2 ( Fig. 2A). This defect in parasite growth was not a general result of decreased Toxoplasma growth at low O 2 because the parental strain grew equally well at either O 2 tension. These data therefore indicate that TgPhyA is important for overall Toxoplasma fitness and appears to play its most important role at decreased O 2 levels. This parallels the finding that DdPhyA is also most important for development at the lower end of the O 2 range encountered by Dictyostelium (17)(18)(19).

Toxoplasma Prolyl Hydroxylase Senses O 2 in Dictyostelium
The modification status of Skp1 was assessed by Western blotting using a rabbit antiserum generated against TgSkp1. Anti-TgSkp1 recognized Skp1 expressed in E. coli after SDS-PAGE and Western blotting (Fig. 1D). The antiserum also recognized a single band near the expected position of TgSkp1 in RH-strain tachyzoites, albeit at a slightly higher apparent M r position. A similar band was recognized in tachyzoites from the type 2 PRU strain. A similar level of Skp1 was detected in each of the drug-resistant clones, but the mobility was greater than that of the parental KU80⌬⌬ strain. The magnitude of the M r shift FIGURE 1. Disruption of TgphyA in tachyzoites. A, shown is a scheme for disrupting the TgphyA locus. B, processed extracts from wild-type and three Drug-resistant clones and HFFs were analyzed by PCR using the indicated primers, and the products were examined by agarose gel electrophoresis. Expected PCR product lengths are indicated. C, Western blot analysis shown of the soluble S16 fraction of parental and mutant tachyzoites (2 ϫ 10 6 cell eq/lane) using an anti-TgSkp1 antiserum diluted 1:1000. The bands shown were the only major bands detected in the entire gel. can be explained by loss of hydroxylation dependent glycosylation, as seen for DdSkp1 (11) and consistent with evidence for Skp1 glycosyltransferase activities in tachyzoite extracts (34) 8 . Furthermore, the apparent M r of TgSkp1 in the disruption strains does not match that of TgSkp1 expressed in E. coli, suggesting other possible modifications of tachyzoite Skp1 independent of TgPhyA. Direct characterization of TgSkp1 modifications are, however, challenging owing to the absence of known reagents that can recognize or alter the unknown putative glycan (22), and the limited amount of this intracellular pathogen that can be obtained for direct chemical analysis of Skp1. Therefore, biochemical functions of TgPhyA were investigated by expression in E. coli, and cellular functions by complementation of the Dictyostelium gene. Nevertheless, the altered gel mobility of Skp1 suggested a role for TgphyA in Skp1 prolyl hydroxylation by analogy with DdphyA in D. discoideum (11) and was consistent with effective disruption or knock-out of TgphyA. TgPhyA Activity in Vitro-To confirm the ability of TgPhyA to hydroxylate Skp1, its full length predicted coding sequence was amplified from RH tachyzoite cDNA. The sequence obtained matched that of the 8-exon gene model TGGT1_114560 from strain GT-1, another type 1 strain (supplemental Fig. S1). To evaluate the enzymatic potential of TgPhyA, the cDNA was modified to encode an N-terminal His 6 tag (Fig. 3A) and expressed in E. coli. TgPhyA was purified from the soluble fraction on an immobilized metal ion affinity chromatography column as previously described for DdPhyA. TgSkp1 was similarly cloned and found to conform to TGGT1_020570 and expressed without tags and purified to near homogeneity by conventional chromatography over four columns as previously described for DdSkp1. SDS-PAGE of the Toxoplasma and corresponding Dictyostelium proteins revealed a substantial degree of purification (Fig. 3B). TgSkp1 migrated more rapidly than DdSkp1 despite its expected larger M r . However, MALDI-TOF-MS yielded an M r value very close to the expected value (Fig. 3C), indicating that TgSkp1 and DdSkp1 migrate anomalously on SDS-PAGE. TgHis 6 PhyA hydroxylation activity was first assayed using a direct assay in which the 14 CO 2 product of the reaction with [1-14 C]␣KG was quantitated as previously done to assay DdPhyA (12). As shown in Fig. 3D, activity was linear with respect to time and amount of enzyme. The hydroxylation activity was confirmed using the more sensitive one-step tandem assay in which the product of the reaction with Skp1 was modified by [ 3 H]GlcNAc in the presence of Dp-Gnt1 and UDP-[ 3 H]GlcNAc. The data shown in panel E show that the predicted co-substrate ␣KG was required and that ascorbate promoted activity as for other Fe ϩ2 -dependent non-heme dioxygenases. The addition of Fe ϩ2 was not necessary, but because EDTA blocked the reaction (not shown), TgHis 6 PhyA may have high affinity for this metal ion as previously observed for DdPhyA and other HIF␣-targeting PHDs (11). In a direct comparison of hydroxylation activities, DdHis 6 PhyA was ϳ6-fold more active than TgHis 6 PhyA for the enzyme preparation tested, and each had similar activity toward both DdSkp1 and TgSkp1 (Fig. 3F, note that protein levels were adjusted in these assays to yield similar activities). The reason for the activity difference is unknown but possibly relates to instability during preparation of this redox sensitive enzyme. The dependence of the two enzymes on ␣KG was very similar (Fig. 3G), differing only in the greater degree of inhibition of TgHis 6 PhyA at high concentrations of ␣KG, which also occurred to a lesser extent toward DdHis 6 PhyA (11). Initial reaction rates conformed to Michaelis-Menten kinetics at the lower ␣KG concentrations and corresponded to an apparent K m of 23 M for each enzyme.
The above data were collected at ambient (21%) O 2 . As previously shown (11), DdHis 6 PhyA exhibited an approximately hyperbolic dependence on O 2 concentration over a range physiologically relevant for Dictyostelium (Fig. 3H). In comparison, activity of TgHis 6 PhyA was highest at the lowest non-zero level tested, 1.0%, and was modestly inhibited at increasing O 2 concentrations. Although activity was also detected in Ar (not shown), this likely reflected atmospheric contamination because TgPhyA but not Gnt1 (not shown) was mostly inhib- indicates that apparent M r differences between TgSkp1 and DdSkp1 are due to anomalous migration during SDS-PAGE. D, TgHis 6 PhyA activity, assayed toward DdSkp1 using the 14 CO 2 release assay, was linearly dependent on protein and time. ND, not done. Values are ϮS.E. E, TgHis 6 PhyA activity, assayed toward DdSkp1 using the tandem 3 H incorporation assay, was linearly dependent on protein (not shown) and time, dependent on ␣KG but not exogenous Fe ϩ2 , and stimulated by ascorbate. Results are representative of at least two independent trials. F, TgHis 6 PhyA (Tg) and DdHis 6 PhyA (Dd) exhibited similar activity toward either TgSkp1 (1.5 pmol) or DdSkp1 (6 pmol) in a 7.5-min reaction. TgHis 6 PhyA protein was present at a 6-fold higher concentration than DdPhyA. Note the semi-log scale. Comparable results were obtained in independent trials using different Skp1 concentrations. G, ␣KG dependence of TgHis 6  ited by the O 2 scavenger sodium dithionite at 2 mM (35). Dependence on O 2 rather than an alternative substrate is consistent with formation of the product 14 CO 2 (Fig. 3, panel D) and Hyp (see below) that could be subsequently modified by GlcNAc (panel E). The affinity of TgHis 6 PhyA for O 2 is evidently much higher than that of DdHis 6 PhyA and is reminiscent of the lower K m values exhibited by the related collagentype P4Hs (36) residing in the rER, despite its much greater sequence similarity to the PHDs.
To confirm similarity of the hydroxylation activities, the product of exhaustive reactions of TgSkp1 with either PhyA was purified by HPLC and analyzed by MALDI-TOF-MS. The mass of each reaction product was consistent with monohydroxylation (ϩ16, data not shown). To map the potential hydroxylation site, reacted and non-reacted Skp1 samples were digested with trypsin or endo-Lys-C and fractionated by HPLC. A search of the fractions for ions whose masses matched the predicted m/z values of unmodified or Met-oxidized peptides yielded almost complete coverage (90%) for the unreacted sample. A search of the TgSkp1 sample reacted with DdHis 6 PhyA yielded a similar set of masses except for the apparent replacement of a single-charged ion with an m/z of 2020.7 by a larger ion with an m/z of 2036.6 or an increase of 16 corresponding to that expected for hydroxylation (Fig. 3I). The m/z of this ion uniquely matches that of a peptide containing a single Proresidue, Pro-148, which is the equivalent of the modification target Pro-143 in DdSkp1. The same shift was observed for the same peptide from the reaction with TgHis 6 PhyA, suggesting identical target specificity. A similar finding was observed in a search of peptide ions from a digest of the TgSkp1 samples with endo Lys-C in which an MH ϩ ion with an m/z of 3402.7 was replaced by an ion of 3418.7 after reaction with either PhyA (data not shown). This matches the TgSkp1 peptide TPEEIR-RIFNIVNDFTPEEEAQVREENK, which includes Pro-148 and the rest of the tryptic peptide described above. All of the Procontaining peptides were detected for each of the proteases, with m/z values observed only for the unmodified isoform (except for those containing Pro-148). This result confirms the conclusion that Pro-148 is the hydroxylation target and excludes all other Pro residues.
Function of TgPhyA in Dictyostelium-TgPhyA was heterologously overexpressed in a phyA Ϫ strain of Dictyostelium to test for conservation of cellular function. With the goal of expressing a transcript whose translational efficiency was similar to that of DdPhyA, a TgPhyA cDNA was chemically synthesized with codon usage matching that of DdPhyA (supplemental Fig. S1). The new cDNA was cloned into standard prespore-or prestalk-specific expression vectors (Fig. 4A), which direct strong, rapid induction during starvation-induced development, beginning at the tipped aggregate stage and continuing through slug formation and culmination into fruiting bodies. These promoters have undetectable activity during growth and selection. TgPhyA was expressed without a tag in case of a deleterious effect on protein function and was detected with a rabbit antiserum raised against the recombinant protein.
In addition, a point mutation, R252A, was introduced to test the function of the enzymatically inactive protein. The corresponding DdPhyA mutant was inactive in vivo (18). Clones expressing TgPhyA in prestalk cells (ecmA::TgPhyA) or prespore cells (cotB::TgPhyA) in a phyA Ϫ background (see Table 1) were compared with identically prepared and previously described strains complemented with DdPhyA (18).
Western blot analysis confirmed expression of the PhyA proteins as expected. The anti-DdPhyA and anti-TgPhyA antisera exhibited negligible cross-species reactivity (Fig. 4B). The TgPhyA enzyme was highly overexpressed based on comparison with the endogenous enzyme protein, which was difficult to detect, and the ϳ20-fold increase in PhyA-like hydroxylation activity in the cotB::TgPhyA/phyA Ϫ strain (Fig. 4C) and the other strains (data not shown). Expression in either prestalk or prespore cells led to the similar accumulation of a more slowly migrating species of Skp1 corresponding to the isoform expressed in the normal strain Ax3 (Fig. 4B). The results could be attributed to hydroxylation by PhyA as the catalytically inactive mutant (R252A) had no effect. The results were indistinguishable from that of overexpressing DdPhyA, suggesting that, as expected from the in vitro data (Fig. 3), TgPhyA had an ability to hydroxylate DdSkp1 similar to that of DdPhyA in the living Dictyostelium cell, at least when overexpressed.
The ability of overexpressed TgPhyA to substitute for DdPhyA in O 2 sensing was assessed by morphological appearance and counting spore numbers at the conclusion of starvation-induced development. The results shown for the control strains in Fig. 4D are consistent with and extend those of the previous report (18). The normal strain Ax3 formed fruiting bodies at 12% O 2 and at all higher levels, whereas phyA Ϫ cells required Ն21%. phyA Ϫ strains complemented with DdPhyA were similar to the normal strain, showing fruiting bodies at 12% O 2 albeit with slightly distinct appearances. In comparison, strains complemented with TgPhyA were most similar to the DdPhyA-complemented strain, with substantial fruiting body formation at 15% O 2 . The rescue depended on the catalytic activity of the expressed protein based on comparison with the catalytically inactive point mutant. Similar results were obtained for all of the TgPhyA-complemented strains ( Fig. 4D and data not shown), indicating under these conditions of overexpression, indistinguishable activity of the two enzymes. Quantitation of the extent of culmination into fruiting bodies by counting spores formed yielded a similar interpretation (Fig.  4E) and also confirmed proper execution of the later sporulation step that can be affected by pathway mutants.
As a more sensitive measure of TgPhyA function, the DdphyA chromosomal locus coding region was replaced with the codon-modified TgphyA cDNA without inclusion of an epitope tag, as shown in Fig. 5A. The modified gene was designed to drive expression of native levels of a codon-adapted version of TgphyA under native DdphyA transcriptional regulation. PCR analyses of genomic DNA from blasticidin-resistant clones yielded strains with the desired genetic exchange (denoted phyA::TgPhyA/phyA Ϫ or homologous) based on formation of appropriate amplification products from primer pairs positioned in the flanking DNA and within the disruption DNA. Other clones incorporated the transgene ectopically (denoted phyA::TgPhyA/phyA ϩ or ectopic) in unknown locations (an example of each is shown Fig. 5B). Strains are listed in Table 1. Analysis of slug stage extracts confirmed expression of JULY 20, 2012 • VOLUME 287 • NUMBER 30

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PhyA-like enzyme activity (Fig. 5C), albeit at about 50% that of the expected level, and TgPhyA protein (Fig. 5D) in the homologous recombined clones. In comparison, higher than normal enzyme activity (6-fold in the example shown), presumably reflecting the sum of the endogenous and transfected enzymes, was measured in the ectopic insertion clones. Despite evidence for TgPhyA activity in the homologous recombined clones, little or no Skp1 was hydroxylated based on Western blotting (Fig.  5D). Therefore, TgPhyA activity at a level corresponding to 50% that of DdPhyA was insufficient to modify Skp1.
Morphological analysis of two homologous inserted clones revealed an appearance that was similar to phyA Ϫ cells (data not shown), not only confirming disruption of the endogenous phyA locus but also indicating that the single copy of TgPhyA was insufficient to compensate for its absence. Spore counting showed that a high level of O 2 was required for culmination of the transgenic strains, confirming their similarity to phyA Ϫ cells (Fig. 5E). Ectopic expression of TgPhyA with endogenous DdPhyA had no apparent effect, as the slightly reduced O 2 requirement observed in this trial was similar to that seen when a catalytically inactive form of TgPhyA was expressed instead (Fig. 5E). The inability of a single copy of TgPhyA to substitute for DdPhyA might be due to subtle evolutionary specialization toward TgSkp1, other differences in regulation that could include dependence on Fe ϩ2 or ascorbate (1), or differences in how accessibility to Skp1 in cells is modulated (12).

DISCUSSION
P4Hs are encoded by an ancient gene lineage that extends from prokaryotes through unicellular eukaryotes and their viruses on to plants, Metazoa, and humans (37,33,20). Very early in eukaryotic evolution, if not in prokaryotes, the lineage divided into the well known rER-associated enzymes that modify secreted structural glycoproteins including plant extensins and animal collagens and the more recently discovered cytoplasmic/nuclear enzymes that in metazoans modify HIF␣ and potentially other proteins to mediate responses to O 2 and other factors (1,2). Enzymes in the HIF1␣-targeting clade generally exhibit low O 2 affinity, consistent with their ability to be affected by physiological changes in O 2 , but TgPhyA did not conform to this rule (Fig. 3H). P4Hs in the rER-type clade generally exhibit high O 2 affinity, although genetic interactions with O 2 sensing implicate the involvement of some in O 2 regulation as well (38). DdPhyA and TgPhyA along with a sequence from the protist Perkinsus olseni, an oyster parasite, belong to the HIF␣-targeting clade based on sequence (supplemental Fig.  S2). But protists lack HIF␣-like genes (39), and there is considerable sequence divergence among the unicellular eukaryote members, necessitating validation of the biochemical and cellular functions of the predicted homologs.
TgPhyA is an authentic Skp1 P4H-like DdPhyA based on (a) its ability to hydroxylate Skp1 in a manner equivalent to that of DdPhyA in vitro (Fig. 3), (b) its necessity for modification of Skp1 in tachyzoites (Fig. 1C), and (c) its ability when overexpressed to complement the absence of PhyA in Dictyostelium with respect to Skp1 hydroxylation and glycosylation and O 2 sensing during development (Fig. 4). The hydroxylation site was mapped to a peptide of TgSkp1 containing Pro-148, the equivalent of the target Pro-143 of DdSkp1. By analogy to DdPhyA, TgPhyA is anticipated to modify Pro-148 with a 4(trans)-hydroxyl moiety (12). The Toxoplasma genome encodes other genes required to assemble E3 SCF Ub ligases (40), allowing for effects on polyubiquitination as hypothesized in Dictyostelium (22).
The occurrence of the Skp1 P4H PhyA in both Dictyostelium and Toxoplasma suggests that this activity was present in ancestral protists. This is supported by the occurrence of related sequences in numerous other protists that also harbor Skp1 glycosyltransferase-like genes (22), reinforcing a potential joint function for the encoded enzymes in processing Skp1.
However, the genome of the protist P. olseni apparently lacks Skp1 glycosyltransferase-like genes, leaving the identity of its target substrate open to speculation. Its expression is responsive to O 2 levels (41), and its sequence conserves an N-terminal MYND domain present in mammalian PHD2s, although not the Dictyostelium and Toxoplasma enzymes, suggesting functional conservation in O 2 signaling. Because PhyA mediates partial O 2 signaling in the absence of glycosylation in Dictyostelium (17), the occurrence of a Skp1-dependent process in Perkinsus is not excluded. Although the properties of protist phyA genes may justify creation of a third category of P4Hs, we suggest inclusion with the HIF␣-targeting P4Hs and refer to the group as cytoplasmic P4Hs, to be contrasted with the rER-associated class of collagen and plant wall protein P4Hs.
The absence of HIF␣-like genes in protists suggests that Skp1 was the primordial cytoplasmic P4H substrate. HIF␣ and Skp1 appear to be unrelated in sequence and structure, so the mechanism of the proposed shift in target specificity is unknown. Whereas animal cytoplasmic P4Hs are able to modify HIF␣ peptides, all tested peptides of Skp1 are essentially inactive as substrates for DdPhyA (12), suggesting a distinct mechanism of substrate selection. The transition may have allowed for selection of multiple targets by animal P4H paralogs (2,1,42). However, the fact that the HIF␣ product of animal PHDs is subject to polyubiquitination by an E3 Ub ligase whose elongin C subunit is homologous to Skp1 suggests that the evolutionary transition conserved a basic mechanism regulating protein stability, although the final effect became transcriptional regulation in animals. Indeed, the stability of Skp1 is not affected by its hydroxylation (17), and there is no evidence for its polyubiquitination. The possibility that vertebrate Skp1 could be a substrate for endogenous PHDs was excluded by replacement of the equivalent of Pro-143 with Glu. However, Skp1 from Caenorhabditis elegans is an excellent substrate for DdPhyA and TgPhyA (12), which is concordant with evolutionary evidence for early animal cytoplasmic P4Hs with transitional specificity (39). The sequences of the protist and animal enzymes are so distinct that structural studies are needed to elucidate the basis for their altered specificity.
A major biochemical difference between the two protist enzymes is the high affinity of purified TgPhyA for O 2 on which is superimposed in vitro a gradient of inhibition at higher O 2 levels (Fig. 3H) that might be due to inhibitory auto-hydroxylation (37). In comparison, no difference in affinity was observed for the co-substrate ␣KG (Fig. 3G). The high O 2 affinity is reminiscent of collagen-type prolyl 4-hydroxylases whose K m for O 2 is 1 ⁄ 6 that of the HIF␣-targeting PHD2 (36), although the TgPhyA sequence is of the cytoplasmic P4H type. This potentially relates to differences in O 2 levels encountered during their FIGURE 5. Replacement of the DdphyA coding region with TgphyA. A, shown is a schematic of the gene replacement strategy of DdphyA on chromosome 2 using a synthetic cDNA. B, extracts from parental Ax3 and two blasticidin-resistant clones were analyzed by PCR using the indicated primer pairs as denoted in panel A. Based on the amplification of DNA products of the expected sizes (in bp) as indicated, the DNA integrations were assigned as ectopic or homologous. C, analysis of PhyA enzyme activity in desalted S100 extracts from 16-h slugs of the transgenic strains was as in Fig. 4C. D, shown is analysis of TgPhyA expression and status of Skp1 modification by Western blotting of S100 fractions of 16-h slugs formed under ambient (21%) O 2 . Sample wells were loaded with 2 ϫ 10 5 cell equivalents, except for the right-most lane, which was 20-fold under-loaded as evidenced by the loss of the two nonspecific bands denoted with an *, to avoid saturation of the TgPhyA signal. TgPhyA was detected using anti-TgPhyA, and Skp1 was detected using mAb 4E1. E, numbers of spores formed after 42 h at various O 2 levels are shown. The presence of spores corresponded to fruiting body formation as assessed morphologically. All data are representative of at least two independent trials. life cycles. Amoebae reside in the soil where development is responsive to O 2 levels compatible with plant root growth: 5-21%. Toxoplasma spends most of its life cycle proliferating as tachyzoites or residing as bradyzoites in cysts within mammalian host cells, which likely present O 2 levels down to Ͻ1% due to consumption of O 2 by cytochrome oxidase and other enzymes (43). Furthermore, Toxoplasma differentiates into oocysts in the feline gut under conditions approaching anoxia. Thus the higher affinity of TgPhyA may be adaptive to lower O 2 levels experienced by Toxoplasma, and the difference suggests that the P4Hs are evolutionarily tunable to changing O 2 needs.
Despite the difference in O 2 affinities, overexpression of TgPhyA decreased the O 2 requirement for Dictyostelium culmination to about the same level as did overexpressed DdPhyA in a phyA Ϫ background. Similar rescue was seen using either the prespore or prestalk cell-specific promoter, which as previously discussed may be due to cell type switching (18). The similar O 2 thresholds, despite the much higher apparent O 2 affinity of TgPhyA in vitro, raises the possibility that the effect of O 2 is indirect and that TgPhyA activity is directly regulated by another factor. The indistinguishable concentration dependences of the PhyA activities toward another substrate, ␣KG (Fig. 3G), makes this metabolite an attractive candidate. ␣KG has been implicated in regulation of animal PHD2 based on the effects of manipulating ␣KG levels genetically and biochemically (1)(2)(3)(4)(5), and O 2 availability is likely to influence levels of mitochondrial and cytoplasmic ␣KG as well as the levels of other Krebs cycle intermediates that can inhibit PHD2 by interacting with its ␣KG binding site. The implication of this comparison is that TgPhyA and DdPhyA may be sensors of ␣KG as an indirect measure of O 2 level. However, it is also possible that TgPhyA is regulated by a different factor such as ascorbate or an oxidation state of iron, which might also be dependent on O 2 availability. Studies in Dictyostelium suggest that whereas low O 2 slows the rate of Skp1 hydroxylation, the steady-state fraction of modified Skp1 remains high. 4,5 Testing these hypotheses in Toxoplasma will be the subject of future studies.
This proposed mechanism may be suited to the biphasic life cycle of Toxoplasma (23)(24)(25), which switches from the anoxic environment in the gut of the determinate feline host and tissue beds of indeterminate hosts where O 2 levels are relatively low, which likely impacts ␣KG concentrations. The proliferation studies clearly reveal an advantage for parasites containing TgPhyA to grow, especially at low O 2 conditions, in a cell monolayer model (Fig. 2). However, TgPhyA is likely not the only factor enabling the parasite to grow at decreased O 2 tensions. These factors will likely include other parasite proteins as well as host cell HIF-1␣, which we demonstrated is also required for Toxoplasma growth at decreased O 2 levels (27).
In Dictyostelium, the action of PhyA is modulated by subsequent glycosylation of the Hyp, and the specificity of the responsible glycosyltranferases toward Skp1 reinforces the conclusion that Skp1 is the functional mediator of PhyA action (17,19). Similar mechanisms may operate in T. gondii. The magnitude of the SDS-PAGE mobility shift of Skp1 caused by disruption of TgphyA is similar to that observed in Dictyostelium when its PhyA is disrupted, suggestive of hydroxylationdependent glycosylation in this organism as well. The apparent glycosylation may be mediated by homologs of the first two of the three Dictyostelium glycosyltransferase genes, gnt1 and pgtA, that are apparent in the genomes of the same apicomplexans that harbor the phyA gene. Indeed, extracts of tachyzoites exhibit Gnt1-like activity (34). Additional studies are needed to explore the function of these glycosyltransferase-like genes.
Although other apicomplexans, including the related Neospora, possess phyA-and glycosyltransferase-like genes, some major groups such as Cryptosporidium and Plasmodium, which include agents of cryptosporidiosis and malaria respectively, lack them. Although a sequence homolog of the distantly related TgphyB is found in Plasmodium genomes, its function is unknown. Considering the broad yet eclectic distribution of this gene set across unrelated protists (22), which include many species in major groups such as diatoms, brown algae, ciliates, green algae, oomycetes (Phytophthora plant pathogens), and other amoebae (e.g. Acanthamoeba), it is likely that these genes were retained in select unicellular organisms where rapid direct changes of the proteome via polyubiquitination by E3 SCF Ub ligases in response to environmental and metabolic factors were advantageous.