SPARK regulates AGC kinases central to the Toxoplasma gondii asexual cycle

  1. Alice L Herneisen
  2. Michelle L Peters
  3. Tyler A Smith
  4. Emily Shortt
  5. Sebastian Lourido  Is a corresponding author
  1. Whitehead Institute for Biomedical Research and Department of Biology, Massachusetts Institute of Technology, United States
7 figures, 2 tables and 6 additional files

Figures

Figure 1 with 1 supplement
SPARK interacts with an Elongin C-like protein.

(A) Protein abundances from immunopurified SPARK-mNG lysates or an untagged control strain. Dotted lines correspond to one modified z-score. Only proteins quantified by greater than one peptide are shown. Proteins identified in only one IP were assigned a pseudo-abundance of 104.5. (B) TGGT1_291010 SPARK elongin-like protein (SPARKEL) gene model. (C) Neighbor-joining phylogenetic tree of SKP1/BTB/POZ domains of TGGT1_291010 orthologs in apicomplexan species along with human and arabidopsis proteins as outgroups. Bootstrap values determined from 1000 replicates. (D) Protein abundances from immunopurified SPARKEL-Ty lysates or an untagged control strain. Dotted lines correspond to one modified z-score. Only proteins quantified by greater than one peptide are shown. Proteins identified in only one IP were assigned a pseudo-abundance of 104.5. (E) Protein abundances following biotinylation and streptavidin enrichment of samples derived from parasites expressing mNG- or SPARKEL-TurboID fusion constructs. A pseudocount of 104.5 was assigned to proteins identified in only one sample. Point colors correspond to significance thresholds. Dotted lines correspond to one median absolute deviation. (F) Immunofluorescence microscopy of intracellular parasites co-expressing SPARKEL-mNG and SPARK-mCherry. Parasites and nuclei were stained with GAP45 and Hoechst 33342, respectively. Scale bar, 5 µm.

Figure 1—figure supplement 1
Additional data supporting the interaction between SPARK and SPARKEL.

(A) Protein abundances from immunopurified SPARK-mNG-mAID (Smith et al., 2022) lysates or an untagged control strain from n = 2 biological replicates. Dotted lines correspond to one modified z-score. Only proteins quantified by greater than one peptide are shown. Proteins identified in only one IP were assigned a pseudo-abundance of 104.5. (B) Alignment of the SKP1/BTB/POZ domains of Homo sapiens elongin C, S. cerevisiae elongin C, and SPARKEL. The elongin B interface, cullin binding sites, and target protein binding sites based on the Homo sapiens annotation are shown. (C) Protein abundances following biotinylation and streptavidin enrichment of samples derived from parasites expressing mNG- or TurboID-SPARKEL fusion constructs from n = 2 biological replicates. A pseudocount of 104.5 was assigned to proteins identified in only one sample. Point colors correspond to significance thresholds. Dotted lines correspond to one median absolute deviation.

Figure 2 with 3 supplements
SPARKEL depletion phenocopies the loss of SPARK.

(A) Plaque assays of 500 TIR1 or SPARKEL-AID parasites inoculated onto host cell monolayers and allowed to undergo repeated cycles of invasion, replication, and lysis for 7 days in media with or without 500 µM IAA. (B) Immunoblot of parasites expressing SPARKEL-V5-AID and SPARK-V5 after the indicated h with IAA. TUB1 serves as a loading control. (C) Immunoblot of parasites expressing SPARK-V5-AID and SPARKEL-V5 after the indicated h with IAA. CDPK1 serves as loading control. (D) The number of parasites per vacuole measured for SPARKEL-AID and the parental strain after 24 hr of 500 µM IAA treatment. Mean counts (n=8) are expressed as a percentage of all vacuoles counted. SEM is shown as shaded area. No comparisons yielded significant p-values using ANOVA and Tukey’s test. (E) Invasion assays SPARKEL-AID, SPARK-AID or TIR1 parental strains treated with IAA or vehicle for 3 or 24 hr. Parasites were incubated on host cells for 20 min prior to differential staining of intracellular and extracellular parasites. Parasite numbers were normalized to host cell nuclei for each field. Different shapes correspond to means of n=3 biological replicates. For clarity, only significant comparisons (Welch’s one-sided t-test) are shown. (F) The time to egress of individual intracellular vacuoles following zaprinast treatment. Points correspond to different vacuoles; shapes to different biological replicates (n = 3). Black shapes are the mean for each replicate. p-Values were calculated from a one-tailed t-test. (G, H) Selected frames from live video microscopy of zaprinast-treated SPARK-AID and SPARKEL-AID parasites, respectively, expressing the calcium indicator GCaMP6f, and the corresponding parental strain, 25 hr after infection and with the indicated IAA treatment period. See also Figure 2—video 1 and Figure 2—video 2. (I, J) Relative GCaMP fluorescence of SPARK-AID or SPARKEL-AID vacuoles, respectively, 60 s following zaprinast treatment. Points correspond to different vacuoles; shapes to different biological replicates (n = 3). Black shapes are the mean for each replicate. p-Values were calculated from a one-tailed t-test.

Figure 2—source data 1

This file contains source data that was used to generate the blot in Figure 2B.

V5, LICOR.

https://cdn.elifesciences.org/articles/93877/elife-93877-fig2-data1-v1.zip
Figure 2—source data 2

This file contains source data that was used to generate the blot in Figure 2B.

TUB1, LICOR.

https://cdn.elifesciences.org/articles/93877/elife-93877-fig2-data2-v1.zip
Figure 2—source data 3

This file contains source data that was used to generate the blot in Figure 2C.

V5, LICOR.

https://cdn.elifesciences.org/articles/93877/elife-93877-fig2-data3-v1.zip
Figure 2—source data 4

This file contains source data that was used to generate the blot in Figure 2C.

CDPK1, LICOR.

https://cdn.elifesciences.org/articles/93877/elife-93877-fig2-data4-v1.zip
Figure 2—figure supplement 1
Extended data related to Figure 2.

(A) Primers specific to the 3′ terminus of SPARK and the V5-mAID-HA tagging payload amplified a product in the SPARK-V5-mAID-HA strain but not the untagged parental strain. (B) Confirmation of SPARK-V5-mAID-HA depletion via immunoblot using the V5 epitope. ALD1 was used as a loading control. (C) SPARK-V5-mAID-HA depletion was visualized in formaldehyde-fixed intracellular parasites using the HA epitope and GAP45 staining as a parasite marker. DNA was visualized with Hoechst. HA signal intensity was normalized relative to the TIR1 parental strain. (D) Uncropped immunoblots corresponding to Figure 2B. (E) Uncropped immunoblots corresponding to Figure 2C. (F, G) Normalized GCaMP6f fluorescence of individual SPARK-AID and SPARKEL-AID vacuoles, respectively, after zaprinast treatment and prior to egress (transparent lines) for the indicated period of IAA treatment. The solid line represents the mean normalized fluorescence of all vacuoles across n=3 biological replicates.

Figure 2—figure supplement 1—source data 1

This file contains source data that was used to generate the blot in Figure 2—figure supplement 1B.

V5, LICOR.

https://cdn.elifesciences.org/articles/93877/elife-93877-fig2-figsupp1-data1-v1.zip
Figure 2—figure supplement 1—source data 2

This file contains source data that was used to generate the blot in Figure 2—figure supplement 1B.

ALD1, LICOR.

https://cdn.elifesciences.org/articles/93877/elife-93877-fig2-figsupp1-data2-v1.zip
Figure 2—video 1
Representative image series of SPARKEL-AID or TIR1-expresing parental parasites expressing the genetically encoded calcium indicator GcaMP following stimulation with 500 µmM zaprinast after the indicated period of IAA treatment.

Color scale as in Figure 2H.

Figure 2—video 2
Representative image series of SPARK-AID or TIR1-expresing parental parasites expressing the genetically encoded calcium indicator GcaMP following stimulation with 500 µmM zaprinast after the indicated period of IAA treatment.

Color scale as in Figure 2G.

Figure 3 with 1 supplement
Depletion of SPARK or SPARKEL leads to downregulation of AGC kinases and upregulation of chronic-stage markers.

(A) Volcano plot displaying the protein abundance ratios of SPARK-AID parasites treated with IAA or vehicle for 24 hr and adjusted p-values for n = 2 biological replicates. Proteins identified as up- or down-regulated in parasites overexpressing the driver of differentiation (BFD1) (Waldman et al., 2020) are shown in blue and vermilion, respectively. In total, 4474 proteins were quantified, 3847 of which registered more than one peptide. (B) Schematic of the phosphoproteomics experiment following SPARK depletion. Intracellular parasites expressing SPARK-AID were treated with 500 µM IAA for 24, 8, 3, or 0 hr and were harvested at 32 hpi simultaneously with the TIR1 parental strain as a control. Samples were multiplexed with tandem mass tags (TMT). The same experimental design was used for SPARKEL-AID proteomics. Each experiment included two biological replicates. (C) Average protein abundances of PKG, PKA-R, and PKA-C1 relative to the TIR1 parental strain after the indicated period of SPARK (thick lines) or SPARKEL (dotted lines) depletion. (D) Average protein abundances of up-regulated bradyzoite genes relative to the TIR1 parental strain after the indicated period of SPARK (blue lines) or SPARKEL (purple lines) depletion. Up-regulated bradyzoite proteins were defined as those significantly increased in parasites overexpressing BFD1 (Waldman et al., 2020) and two modified Z-scores above the median in the SPARK depletion proteome. Rank-ordered plots of (E) Phosphopeptide basal dysregulation score (peptide abundance in the SPARK-AID strain without IAA treatment relative to the TIR1 parental strain) or (F) IAA-dependent score (summed peptide ratios relative to the SPARK-AID t0 peptide abundance). Dotted lines correspond to 3.5 modified Z scores. Colored points correspond to phosphosites discussed in the main text. (G) Gene ontology (GO) enrichment of phosphoproteins exhibiting SPARK-dependent regulation. Gene ratio is the proportion of proteins with the indicated GO term divided by the total number of proteins. Significance was determined with a hypergeometric test; only GO terms with p<0.05 are shown. Redundant GO terms were removed. Categories discussed in the main text are highlighted with colored text. (H) Gaussian mixture modeling of SPARK-dependent peptides identified by more than one PSM revealed seven kinetically resolved clusters. Individual peptides or the median ratios in each cluster are depicted by light and dark gray lines, respectively. Clusters are numbered according to their discussion in the main text. (I) Protein abundances following biotinylation and streptavidin enrichment of samples derived from parasites expressing mNG- or SPARK-TurboID fusion constructs. A pseudocount of 104.5 was assigned to proteins identified in only one sample. Point colors correspond to significance thresholds for n = 2 biological replicates. Dotted lines correspond to one median absolute deviation.

Figure 3—figure supplement 1
Extended analysis of the SPARK-AID depletion phosphoproteome.

(A) Average protein abundance ratios of SPARK- or SPARKEL-AID parasites treated with IAA for 24 hr relative to the untreated samples. Enriched proteins identified as up-regulated in alkaline-induced bradyzoites (Waldman et al., 2020) are shown in blue. The points corresponding to PKA C1, PKA R, and PKG are highlighted in pink, orange, and green, respectively. (B, C) Principal component analysis of the SPARK-AID depletion phosphoproteome. Plots show the three components accounting for the greatest proportion of the variance (D) Average abundances of SPARK and SPARKEL peptides detected by mass spectrometry. (E) Average protein or phosphopeptide abundance ratios from SPARK-AID parasites treated with IAA for the indicated number of h relative to untreated samples. Values corresponding to PKA C1, PKA R, and PKG are color-coded.

Figure 4 with 1 supplement
PKA C1 levels are down-regulated upon SPARK depletion.

(A–B) Average protein and phosphopeptide abundances of PKA C1 (A) and PKA R (B) following SPARK depletion. (C) Schematic of the genetic strategy used to monitor PKA C1 and PKA R abundances following SPARK (or SPARKEL) down-regulation with IAA. (D) Immunofluorescence microscopy of parasites expressing SPARK-AID, PKA C1-mNG, and PKA R-mCherry after 0 or 24 hr of IAA treatment to degrade SPARK. Parasites and nuclei were stained with GAP45 and Hoechst 33342, respectively. GAP45 staining and mNG or mCherry staining were normalized to vehicle-treated tagged samples. (E, F) Flow cytometry analysis of parasites expressing PKA C1-mNG, PKA R-mCherry, and SPARK-AID or SPARKEL-AID, respectively, after the indicated period of IAA treatment. The dotted line centers the mode of the vehicle-treated sample. Traces were normalized by unit area. (G) Violin plots displaying the distribution of phosphopeptide abundance values following SPARK depletion. The distributions of candidate PKA C1 targets, as defined in the text and methods, are shown in green. The distributions and p-values (KS test) were derived from the overlapping subset of phosphopeptides identified in each dataset. (H) Heat map of the abundance ratios of candidate PKA C1 targets following SPARK depletion. PKA R depletion results in up-regulation of PKA C1, and candidate PKA C1 targets therefore have positive abundance ratios following PKA R down-regulation.

Figure 4—figure supplement 1
Transcriptional and phosphoproteomic effects of SPARK depletion.

(A) Transcript abundances for PKG, PKA C1, and PKA C3 were compared by qRT-PCR in TIR1 and SPARK-AID, treated with IAA for 0, 3, or 24 hr. Expression levels were normalized to the parental TIR1 strain across n=4 biological replicates. No comparisons yielded significant p-values using a Kruskall-Wallis test. (B) Overlap between the SPARK and PP1 phosphoproteomes. Violin plots displaying the distribution of phosphopeptide abundance values following SPARK depletion. The distributions of candidate PP1 targets, as defined in the text and methods, are shown in green. The distributions and p-values (KS test) were derived from the overlapping subset of phosphopeptides identified in each dataset. PP1 proteome data was obtained from Herneisen et al., 2022.

Characterization of PKG function during SPARK depletion.

(A) Protein and phosphopeptide abundances of PKG following SPARK depletion. (B) A23187-stimulated egress assays performed at different concentrations of compound 1 after TIR1 and SPARK-AID parasites had been treated with IAA for 24 hr. Curves were fit to the average values of six replicates and were compared with an extra sum of squares F test. (C) The IC50 values of each strain for compound 1; each point represents a biological replicate (n = 6). Significance was assessed with a two-tailed t-test. (D) Violin plots displaying the distribution of phosphopeptide abundance values following SPARK depletion. The distributions of candidate PKG targets are shown in green. The distributions and p-values (KS test) were derived from the overlapping subset of phosphopeptides identified in each dataset. (E) Heat map of the abundance ratios of candidate PKG targets following SPARK depletion.

Figure 6 with 1 supplement
SPARK, SPARKEL, and PKA C3 are negative regulators of differentiation.

(A) Immunofluorescence differentiation assays following knockdown of the indicated AID strains for 48 hr under standard growth conditions (unstressed) or alkaline stress. GAP45 was used to stain parasite vacuoles. Differentiated vacuoles were stained with biotinylated DBA/streptavidin-APC. Nuclei were stained with Hoechst. (B) Quantification of the number of DBL +vacuoles expressed as a percentage of the total stained vacuoles is shown for parasites grown under unstressed or stressed conditions. One-sided t-test, n=3 biological replicates. (C) Fixed, intracellular PKA C3-mNG-AID parasites visualized by immunofluorescence microscopy using the mNG epitope after 1 hr of vehicle or IAA treatment. The mNG signal was internally normalized to the TIR1 parental strain. (D) Immunoblot of PKA C3-AID parasites following the addition of vehicle or 500 µM IAA for 1, 3, or 24 hr. TIR1 was included as an untagged control. PKA C3-AID was detected with V5, and ALD1 was probed as a loading control. (E) Immunofluorescence differentiation assays following knockdown of the indicated ME49/AID-tagged strains under unstressed conditions for 48 hr. Staining was performed as described above, except CDPK1 was used as a parasite vacuole marker. (F) Quantification of the number of DBL +vacuoles expressed as a percentage of the total stained vacuoles in (E). Two-sided t-test, n=5 biological replicates. (G) Immunofluorescence differentiation assays of parasite strains with or without BFD1, depleted of PKA C3 or SPARK with 500 µM IAA and grown under unstressed conditions or alkaline stress for 48 hr. (H) The percentage of DBL +vacuoles corresponding to (G). One-sided t-test. Three biological replicates were quantified under unstressed conditions; five replicates were performed under alkaline stress (I) Effects of 24 hr of PKA C3 knockdown on the transcriptome relative to the untagged strain. Dotted lines correspond to an absolute log2 change of 1. Genes significantly affected by BFD1 overexpression p-value <0.001 as previously defined (Waldman et al., 2020) are colored according to log2 change in the chronic-stage transcriptome. Highlighted points are discussed in the text. P-values were determined from a Wald test implemented in DESeq2.

Figure 6—source data 1

This file contains source data that was used to generate the blot in Figure 6D.V5, LICOR.

https://cdn.elifesciences.org/articles/93877/elife-93877-fig6-data1-v1.zip
Figure 6—source data 2

This file contains source data that was used to generate the blot in Figure 6D.

ALD1, LICOR.

https://cdn.elifesciences.org/articles/93877/elife-93877-fig6-data2-v1.zip
Figure 6—figure supplement 1
Extended data related to Figure 6.

(A) Fixed, intracellular RH/3HA-mAID-SPARKEL parasites visualized by immunofluorescence microscopy using the HA epitope after 1 hr of vehicle or IAA treatment. The HA signal was normalized to the vehicle-treated sample. (B) Quantification of the number of DBL +vacuoles expressed as a percentage of the total stained vacuoles following 48 hr of AID-SPARKEL knockdown. Two-sided t-test from n = 4 biological replicates. (C) Fixed, intracellular ME49/PKA C3-mNG-AID parasites visualized by immunofluorescence microscopy using the mNG epitope after 3 hr of vehicle or IAA treatment. The mNG signal was normalized to the vehicle-treated sample. (D) Amplification of the SPARKEL-AID and PKA C3-AID genomic loci using tag-specific primers to confirm correct integration of the tagging payload. The integration was confirmed with Sanger sequencing between the 3′ gene junction and CDPK3 3′UTR from the tag (Smith et al., 2022). (E) The strategy used to knock out and replace BFD1 with a dTomato cassette containing homology to sequenced flanking the BFD1 locus (Waldman et al., 2020). Amplification of sequences specific to the intact locus or dTom knockout for the indicated strains are shown below the schematic. Oligonucleotide sequences are listed in Supplementary file 5. (F) Live microscopy images of intact or ∆bfd1::dTom parasites showing red fluorescence arising from the knockout cassette.

Figure 7 with 1 supplement
SPARK and PKA C3 physically and genetically interact.

(A) Protein abundances from immunopurified PKA C3-mNG-AID lysates or an untagged control strain from n = 2 biological replicates. Dotted lines correspond to one modified z-score. Only proteins quantified by greater than one peptide are shown. Proteins identified in only one IP were assigned a pseudo-abundance of 104.5. Point colors correspond to significance thresholds. (B) Immunoblot of parasites expressing SPARK-V5-AID-HA and PKA C3-mNG after 24 hr of IAA treatment. ALD1 serves as a loading control. Band intensity normalized to the dual-tagged strain is shown in (C) from three replicates. (D) Flow cytometry analysis of SPARK-AID parasites expressing PKA C3-mNG treated with IAA for the indicated number of h. Traces are representative of two biological replicates. The dotted line centers the mode of the vehicle-treated sample. Traces were normalized by unit area. (E–F) Fixed, intracellular SPARK-AID/PKA C3-mNG parasites visualized by immunofluorescence microscopy using the mNG epitope after the indicated period of IAA treatment (E). The mNG signal was internally normalized to the SPARK-AID parental strain. Quantification of the PKA C3 signal intensity of three replicates is shown in (F). (G) Phosphopeptide IAA-dependent score (summed peptide ratios relative to the PKA C3-AID t0 peptide abundance). Dotted lines correspond to 3.5 modified Z scores. Colored points correspond to phosphosites discussed in the main text. (H) Gaussian mixture modeling of PKA C3-dependent peptides identified by more than one PSM revealed three kinetically resolved clusters. Individual peptides or the median ratios in each cluster are depicted by light and dark gray lines, respectively. Clusters are numbered according to their discussion in the main text. (I) Violin plots displaying the distribution of phosphopeptide abundance values following SPARK depletion. The distributions of candidate PKA C3 targets are shown in green. The distributions and p-values (KS test) were derived from the overlapping subset of phosphopeptides identified in each dataset. (J) Heat map of the abundance ratios of candidate PKA C3 targets following SPARK depletion. (K) Heat map of the abundance ratios of dense granule proteins following SPARK depletion, as discussed in the text.

Figure 7—source data 1

This file contains source data that was used to generate the blot in Figure 7D.

mNG, LICOR.

https://cdn.elifesciences.org/articles/93877/elife-93877-fig7-data1-v1.zip
Figure 7—source data 2

This file contains source data that was used to generate the blot in Figure 7D.

V5, LICOR.

https://cdn.elifesciences.org/articles/93877/elife-93877-fig7-data2-v1.zip
Figure 7—source data 3

This file contains source data that was used to generate the blot in Figure 7D.

ALD1, LICOR.

https://cdn.elifesciences.org/articles/93877/elife-93877-fig7-data3-v1.zip
Figure 7—figure supplement 1
Extended analysis of the PKA C3 depletion proteome.

(A) Volcano plot displaying the protein abundance ratios of SPARK-AID parasites treated with IAA or vehicle for 24 hr and adjusted p-values. Proteins identified as up- or down-regulated in parasites overexpressing the driver of differentiation (BFD1) (Waldman et al., 2020) are shown in blue and vermilion, respectively. (B, C) Principal component analysis of the PKA C3-AID depletion phosphoproteome. Plots show the three components accounting for the greatest proportion of the variance.

Tables

Table 1
Proteins discussed in the text.
Gene IDDescriptionContext in textModificationsReferenceDataset
TGGT1_268210AGC kinaseSPARKSmith et al., 2022
TGGT1_291010hypothetical proteinSPARKELSPARK IP-MS
TGGT1_205550AGC kinaseAGC kinaseSPARK TurboID, SPARKEL TurboID
TGGT1_310220hypothetical proteinBCC10Engelberg et al., 2022SPARKEL IP-MS
TGGT1_269460Ser/Thr phosphatase family proteinEFPP/BCC5S550Engelberg et al., 2022; Liang et al., 2021; Roumégous et al., 2022SPARKEL IP-MS, SPARK depletion timecourse phosphoproteome
TGGT1_311360protein kinase G AGC kinase family member PKGPKGBrown et al., 201724 hr SPARK depletion proteome, SPARK depletion timecourse phosphoproteome
TGGT1_226030AGC kinasePKA C1Jia et al., 2017; Uboldi et al., 201824 hr SPARK depletion proteome, SPARK depletion timecourse phosphoproteome
TGGT1_242070cAMP-dependent protein kinase regulatory subunitPKA RS27Jia et al., 2017; Uboldi et al., 201824 hr SPARK depletion proteome, SPARK depletion timecourse phosphoproteome, PKA C3 depletion proteome
TGGT1_270240MAG1 proteinMAG1S238Parmley et al., 199424 hr SPARK depletion proteome, PKA C3 depletion phosphoproteome
TGGT1_314250bradyzoite rhoptry proteinBRP1Schwarz et al., 200524 hr SPARK depletion proteome, 24 hr PKA C3 depletion proteome
TGGT1_208740putative microneme proteinWaldman et al., 202024 hr SPARK depletion proteome, 24 hr PKA C3 depletion proteome
TGGT1_264660SAG-related sequence SRS44CST1Tomita et al., 201324 hr SPARK depletion proteome, PKA C3 depletion transcriptome
TGGT1_312330hypothetical proteinCST10Tu et al., 2020; Waldman et al., 202024 hr SPARK depletion proteome, PKA C3 depletion transcriptome
TGGT1_260190microneme protein MIC13MIC13Fritz et al., 201224 hr SPARK depletion proteome, PKA C3 depletion transcriptome
TGGT1_311100zinc finger (CCCH type) motif-containing proteinBFD2S183Licon et al., 202324 hr SPARK depletion proteome, 24 hr PKA C3 depletion proteome, PKA C3 depletion phosphoproteome
TGGT1_266010phosphatidylinositol 3- and 4-kinasePI3/4 KS1439SPARK depletion timecourse phosphoproteome
TGGT1_202550NLI interacting factor family phosphataseCTD3/BCC6T384 and T386Engelberg et al., 2022SPARK depletion timecourse phosphoproteome
TGGT1_224240protein phosphatase 2 C domain-containing proteinPPM1S557Yang and Arrizabalaga, 2017SPARK depletion timecourse phosphoproteome
TGGT1_268770dual specificity phosphatase, catalytic domain-containing proteinphosphosite down-regulated with SPARK depletionS11SPARK depletion timecourse phosphoproteome
TGGT1_219682putative pyruvate dehydrogenase kinaseS633Ferrarini et al., 2021SPARK depletion timecourse phosphoproteome
TGGT1_225960STE kinaseS2905SPARK depletion timecourse phosphoproteome
TGGT1_305860calcium-dependent protein kinase 3CDPK3T40SPARK depletion timecourse phosphoproteome
TGGT1_267580cyclin2-related proteinS491SPARK depletion timecourse phosphoproteome
TGGT1_204280cell-cycle-associated protein kinase DYRKT642, S645, and S649Smith et al., 2022SPARK depletion timecourse phosphoproteome
TGGT1_239885hypothetical proteinS933 and S936Smith et al., 2022SPARK depletion timecourse phosphoproteome
TGGT1_267100protein phosphatase 2 C domain-containing proteinPPM2BS/T778-788Yang et al., 2019SPARK depletion timecourse phosphoproteome
TGGT1_311310protein phosphatase 2B catalytic subunit, calcineurin family phosphatse superfamily proteinCnAS104Paul et al., 2015SPARK depletion timecourse phosphoproteome
TGGT1_277895ubiquitin carboxyl-terminal hydrolaseUBP1S44, S52, S1691, and S1695Koreny et al., 2023; Wan et al., 2023SPARK depletion timecourse phosphoproteome
TGGT1_294360putative ubiquitin specific protease 39 isoform 2USP39S20SPARK depletion timecourse phosphoproteome
TGGT1_226050hypothetical proteinRBR E3 ligaseS319 and S322SPARK depletion timecourse phosphoproteome
TGGT1_239410hypothetical proteinputative CCR4-NOT complex subunitS80SPARK depletion timecourse phosphoproteome
TGGT1_295658zinc finger in N-recognin proteinUBR box E3 ligaseS3335SPARK depletion timecourse phosphoproteome
TGGT1_295710HECT-domain (ubiquitin-transferase) domain-containing proteinHECT E3S2166, S4313SPARK depletion timecourse phosphoproteome, PKA R depletion timecourse phosphoproteome
TGGT1_216130putative ubiquitin conjugating enzyme E2putative E2 enzymeS193SPARK depletion timecourse phosphoproteome, PKA R depletion timecourse phosphoproteome
TGGT1_293670transcription elongation factor A TFIISTFIISS356SPARK depletion timecourse phosphoproteome
TGGT1_226810histone lysine methyltransferase SET1SET1S1780 and S1781PKA C3 and SPARK depletion timecourse phosphoproteome
TGGT1_218070hypothetical proteinNOC3pS1343PKA C3 and SPARK depletion timecourse phosphoproteome
TGGT1_280800SWI2/SNF2 SRCAP/Ino80TgSRCAPS635 and S638Sullivan et al., 2003SPARK depletion timecourse phosphoproteome
TGGT1_306660RNA pseudouridine synthase superfamily proteinPUS1S506Anderson et al., 2009SPARK depletion timecourse phosphoproteome
TGGT1_312370RNA pseudouridine synthase superfamily proteinPUS proteinS951SPARK depletion timecourse phosphoproteome
TGGT1_231970pre-mRNA processing splicing factor PRP8PRP8T820SPARK depletion timecourse phosphoproteome
TGGT1_310820putative SLU7 splicing factorSLU7S[516-519]SPARK depletion timecourse phosphoproteome
TGGT1_267600FHA domain-containing proteina FHA proteinS406 and S413SPARK depletion timecourse phosphoproteome
TGGT1_216670FUSE-binding protein 2/KH type splicing regulatory proteinKH proteinS8PKA R, PKA C3, and SPARK depletion phosphoproteome
TGGT1_241170hypothetical proteinKH proteinS362, S368, and S665SPARK depletion timecourse phosphoproteome
TGGT1_235930domain K- type RNA binding proteins family proteinKH proteinS58 and S59Farhat et al., 2021SPARK depletion timecourse phosphoproteome
TGGT1_227450hydrolase, NUDIX family proteinDCP2 homologT1982 and S1985SPARK depletion timecourse phosphoproteome
TGGT1_260600Pumilio-family RNA binding repeat-containing proteinPUF1S225Joyce et al., 2013; Liu et al., 2014SPARK depletion timecourse phosphoproteome
TGGT1_246040MIF4G domain-containing proteinMIF4G domain proteinS341 and S383SPARK depletion timecourse phosphoproteome
TGGT1_249610hypothetical proteinCBP80S851 and S853Gissot et al., 2013PKA C3 and SPARK depletion timecourse phosphoproteome
TGGT1_317720putative eukaryotic translation initiation factor 3 subunit 7eIF3S587SPARK depletion timecourse phosphoproteome
TGGT1_231480putative GCN1GCN1S1100SPARK depletion timecourse phosphoproteome
TGGT1_251630slc30a2 proteinTgZnTS448Chasen et al., 2019PKA C3 and SPARK depletion timecourse phosphoproteome
TGGT1_288540nucleoside transporter proteinnucleoside transporterSPARK depletion timecourse phosphoproteome
TGGT1_260310ATP-binding cassette transporter ABC.B1ABC transporterSPARK depletion timecourse phosphoproteome
TGGT1_318710ATP-binding cassette sub-family F member 1ABC transporterSPARK depletion timecourse phosphoproteome
TGGT1_280660HECT-domain (ubiquitin-transferase) domain-containing proteinputative HECT-domain E3 ubiquitin ligaseS5275 and S5279SPARK TurboID, SPARK depletion timecourse phosphoproteome
TGGT1_286470AGC kinasePKA C3Sugi et al., 2016SPARK TurboID
TGGT1_218240hypothetical proteinIMC25S1231Wang et al., 2016PKA R and SPARK depletion phosphoproteome
TGGT1_220900hypothetical proteinAC13S101, S220, S659Back et al., 2023PKA R, PKG, PKA C3, and SPARK depletion phosphoproteome
TGGT1_225690hypothetical proteinCIP1S850 and S851Long et al., 2017PKA R and SPARK depletion phosphoproteome
TGGT1_257300hypothetical proteinCIP2S26Long et al., 2017PKA R and SPARK depletion phosphoproteome
TGGT1_308860hypothetical proteinAC3S216Chen et al., 2015PKA R and SPARK depletion phosphoproteome
TGGT1_315150putative eukaryotic initiation factor-4EeIF4ES1231PKA R and SPARK depletion phosphoproteome
TGGT1_235930domain K- type RNA binding proteins family proteinKH proteinS58 and S59Farhat et al., 2021PKA R and SPARK depletion phosphoproteome
TGGT1_251640ubiquitin-conjugating enzyme subfamily proteinE2 proteinS23PKA R and SPARK depletion phosphoproteome
TGGT1_253700transporter, major facilitator family proteinMFS transporterS144, S1287, S1288PKG and SPARK depletion phosphoproteome
TGGT1_312100plasma membrane-type Ca(2+)-ATPase A1 PMCAA1TgA1S444 and S445PKG and SPARK depletion phosphoproteome
TGGT1_314780myosin GMyoGT1300Frénal et al., 2017PKG and SPARK depletion phosphoproteome
TGGT1_295360hypothetical proteinIMC18S159, S161, and S165Chen et al., 2015PKG and SPARK depletion phosphoproteome
TGGT1_239400hypothetical proteinIMC28S1081 and S1082Chen et al., 2017PKG and SPARK depletion phosphoproteome
TGGT1_225560hypothetical proteinIMC41S440Back et al., 2023PKG and SPARK depletion phosphoproteome
TGGT1_313480hypothetical proteinAAP3S178Engelberg et al., 2020PKG and SPARK depletion phosphoproteome
TGGT1_235380hypothetical proteinAC5/TLAP3S463Chen et al., 2015; Liu et al., 2013PKG and SPARK depletion phosphoproteome
TGGT1_263520microtubule associated protein SPM1SPM1S54 and S57Tran et al., 2012PKG and SPARK depletion phosphoproteome
TGGT1_211710TB2/DP1, HVA22 family proteinTB2/DP1, HVA22 family proteinsS155 and S177PKG and SPARK depletion phosphoproteome
TGGT1_257040TB2/DP1, HVA22 family proteinTB2/DP1, HVA22 family proteinsS32PKG and SPARK depletion phosphoproteome
TGGT1_229490tetratricopeptide repeat-containing proteinTPR protein TGGT1_229490S456, S570 and S575PKG and SPARK depletion phosphoproteome
TGGT1_246600ABC1 family proteinER ABC transporterT1278PKG and SPARK depletion phosphoproteome
TGGT1_232190Sec7 domain-containing proteinSec7 proteinS180PKG and SPARK depletion phosphoproteome
TGGT1_224150hypothetical proteinTgCOG6T533 and T673Marsilia et al., 2023PKG and SPARK depletion phosphoproteome
TGGT1_203910TBC domain-containing proteinTgTBC10T254 and S256Quan et al., 2023PKG and SPARK depletion phosphoproteome
TGGT1_250870DHHC zinc finger domain-containing proteinTgDHHC1S345 and S348Frénal et al., 2013PKG and SPARK depletion phosphoproteome
TGGT1_212820ubiquitin family proteinubiquitin family protein with a C-terminal extensionS368PKG and SPARK depletion phosphoproteome
TGGT1_2909708-amino-7-oxononanoate synthaseTgSPT2Nyonda et al., 2022PKA C3 depletion transcriptome
TGGT1_259020bradyzoite antigen BAG1BAG1Behnke et al., 2008PKA C3 depletion transcriptome
TGGT1_253440cell-cycle-associated protein kinase SRPK, putativeSRPKTalevich et al., 2011PKA C3 IP-MS
TGGT1_268960putative 5'-AMP-activated protein kinase subunit beta-1 family proteinAMPK subunit betaYang et al., 2022PKA C3 IP-MS
TGGT1_209985cAMP-dependent protein kinasesecreted cAMP-dependent protein kinasesPKA C3 depletion proteome
TGGT1_356400cAMP-dependent protein kinasesecreted cAMP-dependent protein kinasesPKA C3 depletion proteome
TGGT1_277790hypothetical proteinDEP domain proteinS586-600PKA C3 depletion phosphoproteome
TGGT1_318150transporter, major facilitator family proteinTgApiAT3-1S54PKA C3 and SPARK depletion phosphoproteomes
TGGT1_259260membrane protein FtsH1FtsH1S553PKA C3 depletion phosphoproteome
TGGT1_227280dense granule protein GRA3GRA3S120, T145, S197PKA C3 depletion phosphoproteome
TGGT1_312420hypothetical proteinGRA38S457Nadipuram et al., 2016PKA C3 depletion phosphoproteome
TGGT1_204340hypothetical proteinCST8T601Tu et al., 2020PKA C3 depletion phosphoproteome
TGGT1_230180hypothetical proteinGRA24T144Braun et al., 2013PKA C3 depletion phosphoproteome
TGGT1_269180MIF4G domain-containing proteineIF4G1S1312Holmes et al., 2023PKA C3 depletion phosphoproteome
TGGT1_218300zinc finger (CCCH type) motif-containing proteinzinc finger proteinS663PKA C3 depletion phosphoproteome
TGGT1_209500hypothetical proteinDNA repair proteinS619PKA C3 depletion phosphoproteome
TGGT1_310450putative myosin heavy chainIAP2T1269PKA C3 depletion phosphoproteome
TGGT1_273050hypothetical proteinBCC8Liang et al., 2021PKA C3 depletion phosphoproteome
TGGT1_287240hypothetical proteincondensin 2PKA C3 depletion phosphoproteome
TGGT1_278660putative P-type ATPase4ATP4S83 and T166PKA C3 depletion phosphoproteome
TGGT1_233130nucleoside transporter proteinnucleoside transporterS6PKA C3 depletion phosphoproteome
TGGT1_292020GCC2 and GCC3 domain-containing proteinCRMPbS7300, S7303, S7363, and S7365Singer et al., 2023; Sparvoli et al., 2022PKA C3 depletion phosphoproteome
TGGT1_221180hypothetical proteinmicroneme proteinS788PKA C3 depletion phosphoproteome
TGGT1_304490hypothetical proteinmicroneme proteinS10PKA C3 depletion phosphoproteome
TGGT1_309590rhoptry protein ROP1ROP1T248PKA C3 depletion phosphoproteome
TGGT1_211290rhoptry protein ROP15ROP13S312PKA C3 depletion phosphoproteome
TGGT1_258580rhoptry protein ROP17ROP17T51PKA C3 depletion phosphoproteome
TGGT1_291960rhoptry kinase family protein ROP40 (incomplete catalytic triad)ROP40S98PKA C3 depletion phosphoproteome
TGGT1_308810rhoptry neck protein RON9RON9S190, S239, S327, and S375PKA C3 depletion phosphoproteome
TGGT1_310780dense granule protein GRA4GRA4S248PKA C3 depletion phosphoproteome
TGGT1_275440dense granule protein GRA6GRA6S133 and S134PKA C3 depletion phosphoproteome
TGGT1_203310dense granule protein GRA7GRA7S62, S72, S77, S135, S153PKA C3 depletion phosphoproteome
TGGT1_254720dense granule protein GRA8GRA8S198, T202, T262PKA C3 depletion phosphoproteome
TGGT1_220240hypothetical proteinGRA31S420Nadipuram et al., 2020; Young et al., 2020PKA C3 depletion phosphoproteome
TGGT1_217680hypothetical proteinGRA57S806Krishnamurthy et al., 2023; Nadipuram et al., 2020; Young et al., 2020PKA C3 depletion phosphoproteome
TGGT1_215360hypothetical proteinGRA62S319Cygan et al., 2021PKA C3 depletion phosphoproteome
TGGT1_249990hypothetical proteinGRA70S436Krishnamurthy et al., 2023; Lockyer et al., 2023PKA C3 depletion phosphoproteome
TGGT1_289540hypothetical proteinSFP1S897Young et al., 2020PKA C3 depletion phosphoproteome
TGGT1_251740AP2 domain transcription factor AP2XII-9AP2XII-9S1697PKA C3 depletion phosphoproteome
TGGT1_273870SWI2/SNF2 ISWI-like (AT hook)ISWI proteinT765PKA C3 depletion phosphoproteome
TGGT1_260240hypothetical proteinCAF1S272PKA C3 depletion phosphoproteome
TGGT1_253750PLU-1 family proteinPLU-1S3874 and S3877Wang et al., 2014PKA C3 depletion phosphoproteome
TGGT1_300330hypothetical proteinGCFCS27PKA C3 depletion phosphoproteome
TGGT1_223880zinc finger, C3HC4 type (RING finger) domain-containing proteinzinc finger proteinS3701PKA C3 depletion phosphoproteome
TGGT1_288380heat shock protein HSP90HSP90S600PKA C3 depletion phosphoproteome
TGGT1_321520hypothetical proteinp23S109PKA C3 depletion phosphoproteome
TGGT1_225450hypothetical proteinCSN3S509 and S517PKA C3 depletion phosphoproteome
TGGT1_242890hypothetical proteinPSME4S3127PKA C3 depletion phosphoproteome
TGGT1_218960AP2 domain transcription factor AP2XII-1AP2XII-1T1697, S1702, T1667, T1703Antunes et al., 2024PKA C3 depletion phosphoproteome
TGGT1_262150kelch repeat and K+channel tetramerisation domain containing proteinKelch13S139Harding et al., 2020; Koreny et al., 2023; Wan et al., 2023PKA C3 depletion phosphoproteome
TGGT1_254940MIF4G domain-containing proteineIF4G2S985 and S989Holmes et al., 2023PKA C3 and SPARK depletion phosphoproteomes
TGGT1_320020transporter, major facilitator family proteinTgApiAT2S316 and T318PKA C3 and SPARK depletion phosphoproteomes
TGGT1_233000KOW motif domain-containing proteinSpt5S1011 and S1014PKA C3 and SPARK depletion phosphoproteomes
TGGT1_279320hypothetical proteinnucleotidyltransferaseS4010PKA C3 and SPARK depletion phosphoproteomes
TGGT1_313180putative cell-cycle-associated protein kinase PRP4PRP4S316Swale et al., 2022
TGGT1_214140hypothetical proteinassociates with AP2IX4/MORC complexS205 and S212
TGGT1_309250hypothetical proteinassociates with GCN5b complexPMID: 31381949 and 24391497
Appendix 1—key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Strain, strain background RH (T. gondii)RH/TIR1PMID:
28465425
RH/TIR1/∆KU80/∆HXGPRT. Mycoplasma negative.
Strain, strain background RH (T. gondii)ME49/TIR1PMID:
37081202
ME49/TIR1/∆KU80/∆HXGPRT. Mycoplasma negative.
Strain, strain background RH (T. gondii)RH/TIR1/GCaMP6fPMID:
35484233
RH/TIR1/pTUB1-GCaMP6f/∆KU80/∆HXGPRT. Mycoplasma negative.
Strain, strain background RH (T. gondii)RH/TIR1/GCaMP6fPMID:
35976251
RH/TIR1/pMIC2-MIC2-GLuc-myc-P2A-GCaMP6f/∆KU80/∆HXGPRT. Mycoplasma negative.
Strain, strain background RH (T. gondii)RH/TIR1/mNG-TurboIDPMID:
36712004
RH/TIR1/mNG-TurboID-Ty/∆KU80/∆HXGPRT. Mycoplasma negative.
Strain, strain background RH (T. gondii)RH/TIR1/PKG-AIDPMID:
28465425
TGGT1_
311360
RH/TIR1/∆KU80/∆HXGPRT/PKG-mAID-HA/HXGPRT. Mycoplasma negative.
Strain, strain background RH (T. gondii)RH/TIR1/CDPK1-AIDPMID:
37610220
TGGT1_
301440
RH/TIR1/∆KU80/∆HXGPRT/CDPK1-V5-mNeonGreen-mAID-Ty. Mycoplasma negative.
Strain, strain background RH (T. gondii)SPARK-mNG-AIDPMID:
35484233
TGGT1_
268210
RH/TIR1/∆KU80/∆HXGPRT/SPARK-V5-mNG-mAID-Ty. Mycoplasma negative.
Strain, strain background RH (T. gondii)SPARK-mNGThis paperTGGT1_
268210
RH/∆KU80/∆HXGPRT/SPARK-V5-mNG-Ty. Mycoplasma negative. Strain construction and validation described in the Materials and Methods section. Strain available upon request.
Strain, strain background RH (T. gondii)SPARKEL-TurboIDThis paperTGGT1_
291010
RH/TIR1/∆KU80/∆HXGPRT/SPARKEL-TurboID-Ty. Mycoplasma negative. Strain construction and validation described in the Materials and Methods section. Strain available upon request.
Strain, strain background RH (T. gondii)TurboID-SPARKELThis paperTGGT1_
291010
RH/TIR1/∆KU80/∆HXGPRT/3xHA-TurboID-SPARKEL. Mycoplasma negative. Strain construction and validation described in the Materials and Methods section. Strain available upon request.
Strain, strain background RH (T. gondii)SPARKEL-TyThis paperTGGT1_
291010
RH/TIR1/∆KU80/∆HXGPRT/SPARKEL-V5-mNG-Ty. Mycoplasma negative. Strain construction and validation described in the Materials and Methods section. Strain available upon request.
Strain, strain background RH (T. gondii)SPARKEL-mNG/SPARK-mCherryThis paperTGGT1_
268210, TGGT1_
291010
RH/TIR1/∆KU80/∆HXGPRT/SPARKEL-V5-mNG-Ty/SPARK-V5-mCherry-HA/HXGPRT. Mycoplasma negative. Strain construction and validation described in the Materials and Methods section. Strain available upon request.
Strain, strain background RH (T. gondii)SPARKEL-AIDThis paperTGGT1_
291010
RH/TIR1/∆KU80/∆HXGPRT/SPARKEL-V5-HaloTag-mAID-Ty. Mycoplasma negative. Strain construction and validation described in the Materials and Methods section. Strain available upon request.
Strain, strain background RH (T. gondii)SPARKEL-AID/ME49This paperTGGT1_
291010
ME49/TIR1/∆KU80/∆HXGPRT/SPARKEL-V5-HaloTag-mAID-Ty. Mycoplasma negative. Strain construction and validation described in the Materials and Methods section. Strain available upon request.
Strain, strain background RH (T. gondii)SPARKEL-AID/GCaMPThis paperTGGT1_
291010
RH/TIR1/pMIC2-MIC2-GLuc-myc-P2A-GCaMP6f/∆KU80/∆HXGPRT/SPARKEL-V5-HaloTag-mAID-Ty. Mycoplasma negative. Strain construction and validation described in the Materials and Methods section. Strain available upon request.
Strain, strain background RH (T. gondii)SPARKEL-AID/SPARK-mCherryThis paperTGGT1_
268210, TGGT1_
291010
RH/TIR1/∆KU80/∆HXGPRT/SPARKEL-V5-HaloTag-mAID-Ty/SPARK-V5-mCherry-HA/HXGPRT. Mycoplasma negative. Strain construction and validation described in the Materials and Methods section. Strain available upon request.
Strain, strain background RH (T. gondii)SPARK-AIDThis paperTGGT1_
268210
RH/TIR1/∆KU80/∆HXGPRT/SPARK-V5-mAID-HA/HXGPRT. Mycoplasma negative. Strain construction and validation described in the Materials and Methods section. Strain available upon request.
Strain, strain background RH (T. gondii)SPARK-AID/SPARKEL-mNGThis paperTGGT1_
268210, TGGT1_
291010
RH/TIR1/∆KU80/∆HXGPRT/SPARKEL-V5-mNG-Ty/SPARK-V5-mAID-HA/HXGPRT. Mycoplasma negative. Strain construction and validation described in the Materials and Methods section. Strain available upon request.
Strain, strain background RH (T. gondii)PKA R-AIDThis paperTGGT1_
242070
RH/TIR1/∆KU80/∆HXGPRT/PKA R-V5-mAID-HA/HXGPRT. Mycoplasma negative. Strain construction and validation described in the Materials and Methods section. Strain available upon request.
Strain, strain background RH (T. gondii)AID-SPARKELThis paperTGGT1_
291010
RH/TIR1/∆KU80/∆HXGPRT/3xHA-mAID-SPARKEL. Mycoplasma negative. Strain construction and validation described in the Materials and Methods section. Strain available upon request.
Strain, strain background RH (T. gondii)SPARK-TurboIDThis paperTGGT1_
268210
RH/TIR1/∆KU80/∆HXGPRT/SPARK-TurboID-Ty. Mycoplasma negative. Strain construction and validation described in the Materials and Methods section. Strain available upon request.
Strain, strain background RH (T. gondii)PKA C1-mNG/PKA R-mCherryThis paperTGGT1_
226030, TGGT1_
242070
RH/TIR1/∆KU80/∆HXGPRT/PKA C1-mNG-3myc/PKA R-mCherry-Ty. Mycoplasma negative. Strain construction and validation described in the Materials and Methods section. Strain available upon request.
Strain, strain background RH (T. gondii)SPARKEL-AID/PKA C1-mNG/PKA R-mCherryThis paperTGGT1_
291010, TGGT1_
226030, TGGT1_
242070
RH/TIR1/∆KU80/∆HXGPRT/PKA C1-mNG-3myc/PKA R-mCherry-Ty/SPARKEL-V5-HaloTag-mAID-Ty. Mycoplasma negative. Strain construction and validation described in the Materials and Methods section. Strain available upon request.
Strain, strain background RH (T. gondii)SPARK-AID/PKA C1-mNGThis paperTGGT1_
268210, TGGT1_
226030
RH/TIR1/∆KU80/∆HXGPRT/SPARK-V5-mAID-HA/HXGPRT/PKA C1-mNG-3myc. Mycoplasma negative. Strain construction and validation described in the Materials and Methods section. Strain available upon request.
Strain, strain background RH (T. gondii)SPARK-AID/PKA C1-mNG/PKA R-mCherryThis paperTGGT1_
268210, TGGT1_
226030, TGGT1_
242070
RH/TIR1/∆KU80/∆HXGPRT/SPARK-V5-mAID-HA/HXGPRT/PKA C1-mNG-3myc/PKA R-mCherry-P2A-DHFR. Mycoplasma negative. Strain construction and validation described in the Materials and Methods section. Strain available upon request.
Strain, strain background RH (T. gondii)PKA C3-AIDThis paperTGGT1_
286470
RH/TIR1/∆KU80/∆HXGPRT/PKA C3-V5-mNG-mAID-Ty. Mycoplasma negative. Strain construction and validation described in the Materials and Methods section. Strain available upon request.
Strain, strain background RH (T. gondii)PKA C3-AID/ME49This paperTGGT1_286470ME49/TIR1/∆KU80/∆HXGPRT/PKA C3-V5-mNG-mAID-Ty. Mycoplasma negative. Strain construction and validation described in the Materials and Methods section. Strain available upon request.
Strain, strain background RH (T. gondii)SPARK-AID/∆BFD1This paperTGGT1_
268210, TGGT1_
200385
RH/TIR1/∆KU80/∆HXGPRT/∆BFD1::dTomato/SPARK-V5-mAID-HA/HXGPRT. Mycoplasma negative. Strain construction and validation described in the Materials and Methods section. Strain available upon request.
Strain, strain background RH (T. gondii)PKA C3-AID/∆BFD1This paperTGGT1_
286470, TGGT1_
200385
RH/TIR1/∆KU80/∆HXGPRT/∆BFD1::dTomato/PKA C3-V5-mNG-mAID-Ty. Mycoplasma negative. Strain construction and validation described in the Materials and Methods section. Strain available upon request.
Strain, strain background RH (T. gondii)PKA C3-mNG/SPARK-AIDThis paperTGGT1_
286470, TGGT1_
268210
RH/TIR1/∆KU80/∆HXGPRT/PKA C3-mNG-P2A-DHFR/SPARK-V5-mAID-HA/HXGPRT. Mycoplasma negative. Strain construction and validation described in the Materials and Methods section. Strain available upon request.
Cell line (Homo sapiens)Human Foreskin Fibroblasts (HFFs)ATCCSCRC-
1041
Mycoplasma negative
AntibodyRat monoclonal (16D7) anti-mCherryThermo FisherIFA (1/1,000)
AntibodyMouse monoclonal (32F6) anti-mNGChromoTekIFA (1/1,000) and WB (1/500)
AntibodyGuinea pig monoclonal anti-CDPK1CovanceCustom
antibody
WB (1/50,000)
AntibodyRabbit polyclonal anti-GAP45Lampire Biological
Laboratory
Provided by R. Drew Etheridge Lab. IFA (1/1,000). Used for differentiation assays.
AntibodyRabbit polyclonal anti-GAP45PMID:
18312842
Provided by Dominique Soldati-Favre Lab. IFA (1/10,000)
AntibodyMouse polyclonal anti-SAG1PMID:
3183382
Provided by L. David Sibley Lab. IFA (1/1,000). Used for invasion assays.
AntibodyMouse monoclonal anti-V5InvitrogenInvitrogen:
R960-25
WB (1/1000)
AntibodyMouse monoclonal anti-TUB1 (clone 12G10)Developmental Studies
Hybridoma Bank at the
University of Iowa
RRID:
AB_1157911
WB (1/5000)
AntibodyRat monoclonal (3F10) anti-HARocheRRID:
AB_390919
IFA (1/1000)
AntibodyMouse monoclonal (BB2) anti-Ty1PMID:
8813669
Protein G crosslinking (60 µg/1 mg beads)
AntibodyRabbit polyclonal clone WU1614 anti-ALDPMID:
16923803
Provided by L. David Sibley Lab. WB (1/10,000)
Antibodybiotinylated dolichosVector labs
B-1035
IFA (1/1000)
Chemical compound, drugHoechst 33342InvitrogenInvitrogen:
H3570
IFA (1/20,000)
Chemical compound, drugDAPI (4',6-Diamidino-2-Phenylindole, Dihydrochloride)InvitrogenInvitrogen:
D1306
Chemical compound, drugProlong DiamondThermo FisherThermo Fisher:
P36965
Chemical compound, drugZaprinastCalbiochemCalbiochem:
684500
Egress assay (500 µM)
Chemical compound, drugA23187CalbiochemCalbiochem:
100105
Egress assay (2 µM)
Chemical compound, drugCompound 1PMID: 12455981Egress assay (as indicated)
Chemical compound, drugBiotinSigma AldrichSigma Aldrich:
B4501-1G
TurboID (500 µM)
Chemical compound, drugTRIzol reagentThermo FisherThermo Fisher Scientific:
15596018
Commercial assay or kitS-trap microProtifiProtifi:
C02-micro-80
Commercial assay or kitPierce Quantitative Fluorometric Peptide AssayThermo Fisher
Scientific
Thermo Fisher Scientific:
23290
Commercial assay or kitTMT10plex Label Reagent SetThermo Fisher
Scientific
Thermo Fisher Scientific:
90111
Commercial assay or kitTMTpro 16plex Label Reagent SetThermo Fisher
Scientific
Thermo Fisher Scientific:
A44522
Commercial assay or kitEasyPep MS Sample Prep Kits - MaxiThermo Fisher
Scientific
Thermo Fisher Scientific:
A45734
Commercial assay or kitHigh-Select TiO2 Phosphopeptide Enrichment KitThermo Fisher
Scientific
Thermo Fisher Scientific:
A32993
Commercial assay or kitHigh-Select Fe-NTA Phosphopeptide Enrichment KitThermo Fisher
Scientific
Thermo Fisher Scientific:
A32992
Commercial assay or kitPierce High pH Reversed-Phase Peptide Fractionation KitThermo Fisher
Scientific
Thermo Fisher Scientific:
84868
Commercial assay or kitPierce Streptavidin Magnetic BeadsThermo ScientificThermo Scientific: 88817
Commercial assay or kitSep-Pak C18 Plus Short Cartridge, 360 mg Sorbent per Cartridge, 55–105 µmWatersWaters:
WAT020515
Commercial assay or kitPierce Protein G Magnetic BeadsThermo FisherThermo-Fisher:
88847
Commercial assay or kitmNeonGreen-Trap Magnetic AgaroseChromoTekChromoTek:ntma-20
Commercial assay or kitNEXTFLEX Poly(A) Beads 2.0 KitPerkin ElmerNOVA-512993
Commercial assay or kitSuperScriptIII Reverse TranscriptaseInvitrogenInvitrogen:
18080044
Commercial assay or kitPowerUp SYBR Green master mix for qPCRApplied BiosystemsA25779
Software, algorithmProteome Discoverer 4.2Thermo Fisher
Software, algorithmR version 4.0R Foundation for
Statistical Computing
Software, algorithmPrism 8GraphPad
Software, algorithmHHPREDPMID: 29258817
Software, algorithmMEGAPMID: 33892491
Software, algorithmClustalWPMID: 17846036
Software, algorithmTrimGalore version 0.6.7doi:10.5281/zenodo.
7598955
Software, algorithmSTAR version 2.7.1 aPMID: 23104886
Software, algorithmfeatureCounts version 1.6.2PMID: 24227677
Software, algorithmDESeq2 version 1.30.1PMID: 25516281

Additional files

Supplementary file 1

Protein quantification and statistical tests for immunoprecipitation-mass spectrometry experiments (SPARK, SPARKEL, and PKA C3-tagged strains), exported from the Proteome Discoverer 2.4 software.

https://cdn.elifesciences.org/articles/93877/elife-93877-supp1-v1.xlsx
Supplementary file 2

Protein quantification and statistical tests for TurboID mass spectrometry experiments (SPARKEL and SPARK TurboID-tagged strains), exported from the Proteome Discoverer 2.4 software.

https://cdn.elifesciences.org/articles/93877/elife-93877-supp2-v1.xlsx
Supplementary file 3

Protein and peptide quantification and statistical tests for SPARK, SPARKEL, and PKA C3 depletion proteomes, exported from the Proteome Discoverer 2.4 software.

https://cdn.elifesciences.org/articles/93877/elife-93877-supp3-v1.xlsx
Supplementary file 4

Peptide ratios quantified in the PKA R-AID, and PKG-AID, and SPARK-AID depletion phosphoproteomes.

Ratios are reported relative to the vehicle-treated sample within each experiment. Modified Z-scores were calculated by standardizing each value with respect to the median and median absolute deviation within each ratio.

https://cdn.elifesciences.org/articles/93877/elife-93877-supp4-v1.xlsx
Supplementary file 5

Oligonucleotides and DNA sequences used in this study.

https://cdn.elifesciences.org/articles/93877/elife-93877-supp5-v1.xlsx
MDAR checklist
https://cdn.elifesciences.org/articles/93877/elife-93877-mdarchecklist1-v1.docx

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  1. Alice L Herneisen
  2. Michelle L Peters
  3. Tyler A Smith
  4. Emily Shortt
  5. Sebastian Lourido
(2024)
SPARK regulates AGC kinases central to the Toxoplasma gondii asexual cycle
eLife 13:RP93877.
https://doi.org/10.7554/eLife.93877.3