De novo mapping of the apicomplexan Ca2+-responsive proteome

Apicomplexan parasites cause persistent mortality and morbidity worldwide through diseases including malaria, toxoplasmosis, and cryptosporidiosis. Ca2+ signaling pathways have been repurposed in these eukaryotic pathogens to regulate parasite-specific cellular processes governing the transition between the replicative and lytic phases of the infectious cycle. Despite the presence of conserved Ca2+-responsive proteins, little is known about how specific signaling elements interact to impact pathogenesis. We mapped the Ca2+-responsive proteome of the model apicomplexan T. gondii via time-resolved phosphoproteomics and thermal proteome profiling. The waves of phosphoregulation following PKG activation and stimulated Ca2+ release corroborate known physiological changes but identify specific proteins operating in these pathways. Thermal profiling of parasite extracts identified many expected Ca2+-responsive proteins, such as parasite Ca2+-dependent protein kinases. Our approach also identified numerous Ca2+-responsive proteins that are not predicted to bind Ca2+, yet are critical components of the parasite signaling network. We characterized protein phosphatase 1 (PP1) as a Ca2+-responsive enzyme that relocalized to the parasite apex upon Ca2+ store release. Conditional depletion of PP1 revealed that the phosphatase regulates Ca2+ uptake to promote parasite motility. PP1 may thus be partly responsible for Ca2+-regulated serine/threonine phosphatase activity in apicomplexan parasites.


INTRODUCTION
Apicomplexan parasites cause persistent mortality and morbidity worldwide through diseases including 16 malaria, toxoplasmosis, and cryptosporidiosis (Havelaar et al., 2015). The phylum member Toxoplasma gondii 17 alone infects >2 billion people. As obligate intracellular pathogens, apicomplexans are exquisitely tuned to 18 transduce environmental signals into programs of motility, replication, and quiescence responsible for parasite 19 pathogenesis and spread (Bisio and Soldati-favre, 2019). Ca 2+ signals and their downstream effectors are a part 20 of the signaling cascade that triggers changes impacting almost every cellular function (Lourido and Moreno, 21 2015). Signaling begins with release of Ca 2+ from intracellular stores or influx through plasma membrane 22 channels, resulting in diverse downstream events central to parasite virulence, including secretion of adhesive 23 proteins, motility, and invasion into and egress from host cells. Together, these cellular processes orchestrate a 24 dramatic transition from the replicative to the kinetic phase of the life cycle that allows parasites to spread to 25 new host cells. Signal-transducing components downstream of Ca 2+ release are largely unknown yet likely 26 essential for apicomplexan viability and virulence (Lourido and Moreno, 2015;Nagamune and Sibley, 2006). fundamental Ca 2+ responses-including the channels responsible for its stimulated release-are either missing 37 from apicomplexan genomes or have diverged beyond recognition, suggesting that eukaryotic pathogens 38 evolved novel pathways for Ca 2+ mobilization and transduction (Billker et al., 2009;Lourido and Moreno, 2015). 39 In apicomplexans, Ca 2+ -dependent protein kinases (CDPKs) have garnered the most attention as the only 40 known Ca 2+ -regulated kinases in the phylum (Billker et al., 2009). These kinases possess intrinsic Ca 2+  parasite Ca 2+ signaling field, dephosphorylation has garnered comparatively little attention (Yang and 44 Arrizabalaga, 2017). The roles of the prototypical CaM and the Ca 2+ /CaM-dependent phosphatase calcineurin 45 have only been phenotypically examined in parasites, and their client proteins remain largely unknown (Paul et 46 al., 2015; Philip and Waters, 2015). Although key players are conserved and essential across the Apicomplexa, 47 no systematic efforts have been undertaken to globally map the Ca 2+ signaling pathways of these pathogens. 48 Ca 2+ signaling pathways have been repurposed in apicomplexans to regulate parasite-specific cellular processes 49 governing the transition between the replicative and kinetic phases of the infectious cycle (Brown et al., 2020;50 Pace et al., 2020). Despite the presence of conserved Ca 2+ -responsive proteins (Lourido and Moreno, 2015; 51 Nagamune and Sibley, 2006), uncovering the Ca 2+ signaling architecture of apicomplexans demands a 52 reevaluation of the entire network to understand how specific signaling elements interact to impact 53 pathogenesis. We present an atlas of Ca 2+ -regulated proteins in the model apicomplexan T. gondii, assembled 54 from high-dimensional proteomic datasets. The physiological changes associated with stimulated motility in 55 the asexual stages of parasites have been characterized for decades (Blader et al., 2015). Our approach 56 identified at once hundreds of molecular components underpinning these processes. We find numerous Ca 2+ -57 responsive proteins that are not predicted to bind Ca 2+ , yet operate at critical junctures in the parasite signaling 58 network. From this analysis, the protein phosphatase PP1 emerges as an unanticipated Ca 2+ -responsive 59 phosphatase. 60

Sub-minute phosphoproteomics reveals the topology of Ca 2+ -dependent signaling processes.
Parasite Ca 2+ fluxes can arise from different sources (Bisio et  diverging signaling states. Here, we add kinetic resolution to these signaling pathways. We quantified dynamic 73 changes in the phosphoproteome within a minute of stimulation with zaprinast and thus activation of the 74 cGMP/Ca 2+ pathway. 75 We collected five timepoints in the 60 seconds following stimulation (0, 5, 10, 30, and 60 s), as well as three 76 DMSO-treated matched timepoints (0, 10, and 30 s), in biological duplicate ( Figure 1A). Using TMTpro labeling 77 methods (Li et al., 2020), we multiplexed 16 samples, allowing us to analyze a complete time course, with 78 replicates and controls, in a single MS experiment. Phosphopeptides were enriched from the rest of the sample 79 using sequential metal-oxide affinity chromatography, which maximizes phosphopeptide capture (Tsai et al., 80 2014). Our experiments quantified 4,055 parasite proteins, none of which exhibited more than a two-fold 81 change in abundance in the 60 seconds following stimulation (Figure 1 Supplement). 82 Given the paucity of known phosphoreglatory interactions in apicomplexans compared to other organisms 83 (Weiss et al., 2020), we employed several analysis approaches to maximize the identification of changing 84 phosphosites. We first calculated phosphoregulation scores by summing peptide abundances of vehicle 85 (DMSO) and zaprinast-treated samples, taking their ratios, and standardizing the values with a modified Z 86 score ( Figure 1B). From a phosphoproteome of 11,755 unique peptides with quantification values (belonging to 87 2,690 phosphoproteins), 839 phosphopeptides increased in abundance three modified Z-scores above the 88 median, whereas 154 decreased 1.8 Z-scores below the median. Principal component analysis on the significant 89 peptides distinguished the agonist treatment and time course kinetics in the two principal components 90 accounting for the greatest variability in the data ( Figure 1C and Figure 1 Supplement). 91 Kinetically resolved clusters reveal regulatory subnetworks during zaprinast stimulation.
We leveraged the kinetic resolution of our comprehensive phosphoproteomics datasets to identify 92 subregulatory networks. A Gaussian mixture-model clustering algorithm (Invergo et al., 2017) heuristically 93 resolved four clusters for phosphopeptides arising from zaprinast treatment: three clusters increasing with 94 different kinetics, and one decreasing ( Figure 1D). On average, the 173 phosphopeptides belonging to cluster 1 95 increased sharply in abundance within 5 seconds of treatment and continued to increase in the remainder of the 96 time course (Figure 1D), suggesting that they belonged to the first wave of phosphoregulation. This cluster was 97 enriched for phosphoproteins associated with phosphodiesterase activity, phospholipid binding, and Ca 2+ 98 binding ( Figure 1E), including PDE1, PDE2, PI4,5K, PI3,4K, PI-PLC, a phosphatidylinositol-3,4,5-triphosphate 5-99 phosphatase, a putative Sec14, TgCDPK2A and TgCDPK7, and PPM2B (Table 1 and Figure 1F). 100 Peptides in clusters 2 and 3 (173 and 527, respectively) increased more gradually and exhibited lower fold-101 changes than cluster 1 sites ( Figure 1D). Cluster 2 was notably enriched in proteins functioning in transport of 102 monovalent ions and lipids, cyclase activity, and Ca 2+ binding ( Figure 1E). This set includes two putative Ca 2+ -103 activated K + channels and the sodium-hydrogen exchangers NHE1 and NHE3 ( ATPase G subunit, TgATP4, and the guanylyl cyclase (Table 1), which has a P-type ATPase domain with 125 unknown ion specificity. Small-molecule transporters included a putative nucleoside transporter, ApiAT5-3, and 126 a MFS family transporter. Cluster 4 was the only class of peptides that was not functionally enriched in Ca 2+ 127 binding proteins ( Figure 1E). 128 We identified phosphoproteins with peptides belonging to several clusters ( Figure 1G). Such proteins may have  129  multiple phosphosites regulated with different kinetics by the same enzyme; or by different enzymes that  130  alight upon the target at varying spatiotemporal scales. For example, SCE1 and TGGT1_309910 have  131  phosphopeptides belonging to all three increasing clusters, likely resulting from phosphorylation by TgCDPK3  132 and PKG, respectively. Indeed, SCE1 was implied to be a  (Herneisen et  171 al., 2020). As measured by immunoblot band intensity, TgCDPK1 was strongly stabilized by Ca 2+ (Figure 2A), 172 suggesting that our experimental system is sensitive to Ca 2+ -dependent stability changes. The calculated EC 50 173 using this approach was in the low µM range, consistent with previous studies using recombinant enzymes 174 (Ingram et al., 2015;Wernimont et al., 2010). 175 The effect of Ca 2+ on the global thermostability of the proteome has not been assessed. Therefore, we first 176 generated thermal profiles of the T. gondii proteome without or with 10 µM Ca 2+ , which is representative of the 177 resting and stimulated Ca 2+ concentrations of cell cytoplasms (Lourido and Moreno, 2015). A thermal challenge 178 between 37 and 67°C induced denatured aggregates, which were separated from stable proteins by 179 ultracentrifugation. The soluble fraction was digested and labeled with isobaric mass tags, pooled, 180 fractionated, and analyzed with an orbitrap mass spectrometer ( Figure 2B) without and with Ca 2+ , respectively. The distribution of melting temperatures was largely overlapping in the 185 two conditions (Figure 2D), suggesting that Ca 2+ -dependent changes in protein stability were restricted to a 186 subset of proteins. We additionally calculated an area under the curve (AUC) metric by numerical integration 187 using the trapezoidal rule (Herneisen and Lourido, 2021) to compare the stabilities of proteins with atypical 188 melting behavior (Figure 2 Supplement), such as components of the tubulin cytoskeleton or parasite conoid. 189 To discover proteins with Ca 2+ -dependent stability shifts in the initial temperature range experiment, we rank-190 ordered proteins by euclidean distance (ED) scores (Dziekan et al., 2020) quantifying the shift in thermal 191 profiles with and without Ca 2+ (Figure 2B and 2E). The majority of proteins, such as DNA polymerase β 192 (TGGT1_233820), exhibited similar melting behavior in both conditions ( Figure 2F). Our analysis identified as 193 Ca 2+ -responsive parasite-specific proteins with EF hands, including TgCDPK7 and the calmodulin-like proteins 194 CAM1 and CAM2 (Table 1, Figure 2F and The data also inform hypotheses about Ca 2+ homeostasis and energetics in the parasite mitochondrion and 203 apicoplast. A divergent subunit of the mitochondrial complex III, TgQCR9, as well as components of the ATP 204 synthase complex, were destabilized by Ca 2+ (Figure 2 Supplement). These include the ATP synthase subunits 205 alpha, gamma, 8/ASAP-15, and f/ICAP11/ASAP-10; and the ATP synthase-associated proteins ASAP-206 18/ATPTG14 and ATPTG1 (Table 1). TGGT1_209950, a conserved alveolate thioredoxin-like protein suggested 207 to localize to the apicoplast by spatial proteomics (Barylyuk et al., 2020), was similarly destabilized by Ca 2+ 208 (Figure 2

Determining the specificity and sensitivity of Ca 2+ -responsive proteins
The temperature range experiment has the advantage of generating complete thermal stability profiles, but 222 does not inform the relative affinities of the Ca 2+ -dependent stability change. To address this gap, we examined 223 protein stability across 10 Ca 2+ concentrations (EGTA and 10 nM to 1 mM). Based on the temperature-range 224 experiments, we selected thermal challenge temperatures of 50, 54, and 58°C to target protein with a range of 225 thermal stabilities in these dose-response experiments (Figure 2 Supplement). We hypothesized that Ca 2+ -226 responsive proteins would exhibit sigmoidal dose-response trends in thermal stability, similarly to TgCDPK1 227 ( Figure 2B). We performed four independent concentration-range experiments on two mass spectrometers 228 using different separation methods (ultracentrifugation or filtration) to capture different types of aggregates. 229 The concentration range thermal-profiling experiments provide insight into the magnitude of the Ca 2+ -230 dependent change (AUC parameter) and its dose-dependency (EC 50 ) ( Figure 2B). Clear responses were 231 observed for several known Ca 2+ -binding enzymes with EF hands, including calmodulin, calcineurin, and several 232 parasite Ca 2+ -dependent protein kinases (CDPKs; Figure 3A). These parameters can be used to classify proteins 233 as stabilized (AUC > 1; e.g., TgCDPK1, TgCDPK2A, and TgCDPK3) or destabilized (AUC < 1; e.g., calmodulin and 234 calcineurin B) by Ca 2+ (Figure 3A). Furthermore, EC 50 measurements may inform specific hypotheses about a 235 protein's involvement in signaling or homeostasis, based on the Ca 2+ concentration at which the protein is 236 predicted to change. that such Ca 2+ -responsive proteins were significantly enriched in Ca 2+ -binding functions, protein kinase activity, 242 and metal affinity ( Figure 3C). We examined each of the 40 EF hand domain-containing proteins detected in 243 our mass spectrometry experiments ( Figure 3D) although the magnitude of the change was small (Figure 3 Supplement). The atypical kinase ERK7 was 257 destabilized at high Ca 2+ concentrations (Figure 3 Supplement), similarly to other Ca 2+ -responsive proteins 258 detected at the parasite apex. Rhoptry and dense granule kinases had EC 50 values in between 100 µM and 1 259 mM, consistent with the high concentration of Ca 2+ in the secretory pathway ( Figure 3E). We also searched for 260 Ca 2+ -responsive phosphatases. The known Ca 2+ -regulated phosphatase subunit, CnB, was destabilized 261 ( Figure 3F). Protein-phosphatase 1 (PP1) responded to Ca 2+ more consistently than many known Ca 2+ -binding 262 proteins (Figures 3F and 5A), although the catalytic subunit is not an intrinsic Ca 2+ sensor. Our resource thus 263 places proteins without previously characterized Ca 2+ responses within the broader Ca 2+ signaling network. 264 Our search and annotation of Ca 2+ -responsive proteins also identified candidates that link Ca 2+ and ion 265 homeostasis. This list includes several transporters and channels (Table 1): TgCAX, a protein with structural 266 homology to LDL receptors (TGGT1_245610), two proteins with structural homology to the mitochondrial Ca 2+ uniporter (TGGT1_257040 and TGGT1_211710), and an apicoplast two-pore channel. Several Ca 2+ -responsive 268 hydrolases were also found, including SUB1, a metacaspase with a C2 domain, and a Ca 2+ -activated apyrase. 269 These proteins may function during the intracellular, replicative phase of the lytic cycle, for which the function 270 of Ca 2+ is relatively unexplored. 271 Several divalent cations regulate protein structure and activity in addition to Ca 2+ , including Mg 2+ and Zn 2+ . Such 272 metal-binding proteins might appear Ca 2+ -responsive in our thermal profiling approach. We buffered our 273 parasite extracts with excess Mg 2+ (1 mM) to mitigate non-specific changes caused by the concentration of 274 divalent cations. Nevertheless, to determine whether our approach identified additional metal-binding 275 proteins, we compared the EC 50 values of candidates that bind different divalent cations, as cataloged via the 276 presence of Interpro domains and through manual annotation (Figure 3

Validation of Ca 2+ -dependent thermal stability
We selected five proteins exhibiting consistent Ca 2+ -responsive behavior by MS for validation: an EF hand 285 domain-containing protein (Eps15), two kinases (RON13 and PKA-C1), and two uncharacterized putative metal-286 binding proteins (TGGT1_286710 and TGGT1_309290). The first three candidates were selected for potential 287 involvement in dynamic Ca 2+ -regulated processes. Eps15 (TGGT1_227800) was recently shown to mediate 288 endocytosis in P. falciparum (Birnbaum et al., 2020) and localized to puncta bridging the inner membrane 289 complex (IMC) and cytoskeleton in T. gondii (Chern et al., 2021). PKA-C1 is thought to antagonize Ca 2+ signaling 290 in T. gondii (Jia et  We appended epitope tags to the endogenous locus of each candidate. These Ca 2+ -responsive proteins 296 localized to diverse structures ( Figure 4B). To validate the Ca 2+ -dependent stability of individual candidates, we 297 prepared parasite extracts as described earlier, but relied on immunoblot readout instead of MS. In these five 298 cases, stabilization of the candidates was confirmed in multiple biological replicates. Several controls (TUB1,  299 MIC2, GAP45, and SAG1) could be shown to be stable across all Ca 2+ concentrations tested ( Figure 4C and 300 Figure 4 Supplement). In the case of PKA-C1, Eps15, and both putative metal-binding proteins, the 301 immunoblot experiments revealed an even higher EC 50 than was measured in the MS experiments. We 302 conclude that the stability changes detected by our global proteomics methods are robust, although the 303 precise features of Ca 2+ -dependent stability may differ based on the method used to assess them. 304

A PP1 holoenzyme serves Ca 2+ -responsive functions required for parasite spread
Our orthogonal proteomics approaches map Ca 2+ -responsive phosphorylation and thermal stability. 305 Unexpectedly, the catalytic subunit of protein phosphatase 1 (PP1, TGGT1_310700) exhibited consistent 306 stabilization by Ca 2+ in our mass spectrometry experiments (Figures 2B and 5A). The contribution of 307 phosphatases to Ca 2+ signaling in apicomplexans is poorly understood (Yang and Arrizabalaga, 2017) and the 308 Ca 2+ -responsive behavior of PP1 has not been reported in other eukaryotes. The function of the phosphatase 309 has not been directly studied in T. gondii, although PP1 inhibitors have been shown to block invasion of host 310 cells (Delorme et al., 2002), and recent experiments in P. falciparum suggest that PP1 is required for the 311 merozoite egress-to-invasion transition (Paul et al., 2020). Intriguingly, PP1 was recently observed to relocalize 312 to the apical complex in highly motile Plasmodium berghei ookinetes (Zeeshan et al., 2021), suggesting that the 313 enzyme may serve apicomplexan-specific, Ca 2+ -responsive functions in remodeling the parasite 314 phosphoproteome. 315 To track localization during the T. gondii lytic cycle, we tagged the endogenous C terminus of PP1 with an mNG 316 fluorophore and Ty epitope. We confirmed the Ca 2+ -dependent stability of PP1 by immunoblotting ( Figure 5B). 317 Live microscopy revealed a diverse array of PP1 localizations in parasites ( Figure 5C). In accordance with 318 imaging in Plasmodium (Zeeshan et al., 2021), PP1-mNG was distributed diffusely in the cytoplasm, as well as in 319 foci resembling the nucleus, centrosome, and in some parasites, the periphery. These diverse localizations may 320 arise from the association of PP1 with distinct regulatory subunits forming different functional holoenzymes, as 321 characterized in metazoans (Brautigan and Shenolikar, 2018). To determine whether PP1 exhibits Ca 2+ -322 dependent relocalization, we treated parasites with zaprinast and A23187. The PP1 signal intensity increased at 323 the apical cap and pellicle following zaprinast stimulation ( Figure 5C and Video 1). However, Ca 2+ ionophore 324 treatment failed to induce the same relocalization patterns ( Figure 5C and Video 2). The dynamics of PP1 325 activity thus appear specific to cGMP signaling within the parasites, which is nevertheless upstream of Ca The parasite apex and pellicle are hotspots for the signaling that potentiates motility and invasion, so the 328 relocalization of PP1 suggests it may function in these processes. We created strains expressing the PP1 329 catalytic subunit with an endogenous C-terminal auxin-inducible degron (AID) for rapid conditional knockdown 330 ( Figure 5D). Conditional degradation of PP1 was confirmed by immunofluorescence and immunoblotting 331 ( Figure 5E and 5F). Parasites depleted of PP1 failed to form plaques ( Figure 5G), implicating the phosphatase in 332 one or more essential functions during the lytic cycle (Sidik et al., 2016a). Even in the absence of IAA, the PP1-333 AID strain formed small plaques, indicating substantial hypomorphism. PP1-AID parasites exhibited slower 334 replication than untagged parasites, and this effect was exacerbated with IAA treatment ( Figure 5H). Parasites 335 depleted of PP1 had a reduced invasion efficiency ( Figure 5I), although this effect was modest and subject to 336 technical variation, likely arising from hypomorphism of the C-terminal tagged strain. Parasites treated with 337 auxin egressed more slowly than untreated parasites when stimulated by zaprinast; however, egress kinetics 338 were indistinguishable with a Ca 2+ ionophore agonist ( Figure 5J). Together, these results suggest that PP1 339 holoenzymes function at multiple steps in the lytic cycle. At least one holoenzyme relocalizes when parasite 340 cGMP/Ca 2+ pathways are stimulated. Because parasites lacking PP1 exhibit delays specifically in zaprinast-341 induced egress, we hypothesize that the peripheral holoenzyme enhances Ca 2+ signaling; however, its 342 requirement can be bypassed with nonspecific Ca 2+ influx. 343 A PP1 holoenzyme dephosphorylates signaling enzymes at the parasite periphery PP1 is one of the major serine/threonine phosphatases in eukaryotic cells (Brautigan and Shenolikar, 2018). As 344 the catalytic subunit relocalizes during cGMP/Ca 2+ -stimulated transitions in apicomplexans, we hypothesized 345 that PP1 dephosphorylates crucial targets during egress and motility. To identify putative PP1 holoenzyme 346 targets, we first treated intracellular PP1-AID parasites with IAA or vehicle for 3 hours prior to mechanically 347 releasing parasites for analysis. We performed sub-minute phosphoproteomics by resuspending the 348 extracellular parasites in a zaprinast solution followed by denaturation in SDS to stop enzymatic activity after 0, 349 10, 30, and 60 seconds. Multiplexing with TMTpro reagents followed by phosphopeptide enrichment allowed us 350 to compare the zaprinast time courses with or without PP1-depletion in biological duplicate ( Figure 6A).

351
Analysis of an unenriched fraction of the proteome revealed significant depletion of PP1, which we confirmed in 352 parallel by immunoblot (Figure 6 Supplement). The hypomorphism of the PP1-AID strain and reduced parasite 353 yield resulted in a phosphoproteome with lower coverage: 6,916 phosphopeptides with quantification values. 354 To identify peptides exhibiting PP1-dependent regulation, we selected peptides exhibiting a 2-fold difference in 355 abundance between vehicle-and IAA-treated samples in both replicates for at least one time point. In total, 757 356 peptides passed this threshold. 357 To focus on zaprinast-dependent changes, we clustered these peptides on the basis of their abundance relative 358 to the earliest time point of stimulation. The peptides fit into five clusters with respect to PP1-dependence and 359 kinetics ( Figure 6B). Clusters 1, 2, and 3 contained peptides increasing in abundance upon stimulation in PP1-360 depleted parasites. The peptides belonging to Cluster 1 generally increase in abundance with zaprinast 361 treatment, which occurs more rapidly with PP1 depletion. By contrast, the abundances of the 614 peptides 362 belonging to clusters 2 and 3 were elevated in the absence of PP1, suggesting that PP1 antagonizes these 363 phosphorylation events. Under normal conditions, peptides in cluster 4 decreased sharply in abundance 364 between 10 and 30 seconds of zaprinast treatment and recovered by 60 seconds; however, these peptides did 365 not change in abundance when PP1 was depleted. Peptides in cluster 5 increased gradually between 10 and 30 366 seconds and decreased to basal levels by 60 seconds; when PP1 is depleted, these peptides exhibit a delay. 367 The effect of PP1 disruption on the phosphoproteome was pervasive, which may reflect disruption of numerous 368 PP1 holoenzymes. Many of the examined phosphopeptides exhibited substantial basal hyperphosphorylation in 369 the absence of PP1 ( Figure 6C). Given the likely pleiotropy of catalytic subunit depletion, we focused our 370 analysis on perturbed pathways rather than individual targets ( Figure 6D). Cluster 1 did not possess enough 371 peptides for enrichment analysis. Cluster 2 was enriched in phosphoproteins functioning in transmembrane 372 transport, including P-type ATPases and ABC transporters. Cluster 3 contained both the guanylyl and adenylyl 373 cyclases and was further enriched in putative sodium-hydrogen exchangers and tubulin-tyrosine ligases. Cluster 374 4 was solely enriched for proteins involved in ubiquitin transfer, including TGGT1_280660, an uncharacterized 375 HECT domain-containing protein ( Figure 6C). Cluster 5 phosphoproteins were notably involved in cytoskeletal 376 motor activity, actin binding, and RNA binding. Numerous apical proteins exhibited PP1-dependent 377 phosphorylation (Table 1), including AC7, CIP1, and two hypothetical proteins-TGGT1_258090 and 378 TGGT1_320030, localizing to the conoid base and the second apical polar ring, respectively (Koreny et al., 379 2021). The protein abundances did not vary between vehicle and IAA treatment, indicating that the 380 phosphopeptide abundance changes arose from dynamic covalent modifications. The PP1-dependent 381 phosphoproteome therefore supports the existence of apical and peripheral PP1 holoenzymes, as observed by 382 live microscopy. 383

PP1 activity is important for Ca 2+ entry to enhance the kinetics of egress
The phosphoproteomics data pointed to ion homeostasis dysregulation in the absence of PP1 ( Figure 6D). 384 Numerous transporters are hyperphosphorylated when PP1 is depleted, including Ca 2+ ATPases, proton 385 transporters, and MFS and ABC-family transporters. We hypothesized that aberrant Ca 2+ mobilization may 386 underlie the phenotypes observed in the motile stages of the lytic cycle upon PP1 depletion (  which are widespread eukaryotic human parasites whose signaling pathways remain largely unmapped due to 498 their evolutionary divergence from model organisms (Lourido and Moreno, 2015). In principle, this approach 499 can be applied to other post-translational modifications, natural ligands (Lim et al., 2018;Sridharan et al., 500 2019), and organisms (Jarzab et al., 2020) to establish-and in some cases re-evaluate-the topology of 501 complex signaling pathways. 502

Parasite transfection and strain construction
Genetic background of parasite strains 507 Existing T. gondii RH strains were used as genetic backgrounds for this study. All strains contain the 508 ∆ku80∆hxgprt mutations to facilitate homologous recombination (Huynh and Carruthers, 2009 transfected with 20 µg gRNA/Cas9-expression plasmid pBM041 (GenBank MN019116.1) and 10 µg repair 519 template. GFP-positive clones were isolated by limiting dilution following fluorescence-activated cell sorting. 520 521

Endogenous tagging of PP1 538
A cutting unit specific to the C-terminus of PP1 (TGGT1_310700; P8) was assembled into the pALH086 HiT 539 vector backbone. Approximately 50 µg of this vector was BsaI-linearized and co-transfected with 50 μg of the 540 pSS014 Cas9-expression plasmid into TIR1/RH or Parasite populations were selected for 1 week in standard 541 media with 25 µg/mL mycophenolic acid and 50 µg/mL xanthine and were then subcloned by limiting dilution. 542 Single clones were screened for tag expression by immunofluorescence, immunoblot and sequencing of the 543 junction spanning the 3 CDS and the tag. 544

Sub-minute phosphoproteomics experiments
Parasite harvest and treatment 545 T. gondii tachyzoites from the RH strain were infected onto confluent HFF monolayers in 4-8 15-cm dishes. 546 After the parasites had completely lysed the host cell monolayer (40-48 hours post-infection), extracellular 547 parasites were passed through 5 µm filters into 50 ml conical vials. The samples were spun at 1000 x g for 7 548 minutes. The supernatant was decanted, and the parasite pellet was resuspended in 1 ml FluoroBrite DMEM 549 lacking serum and transferred to a 1.5 ml protein low-bind tube. The sample was spun in a mini centrifuge at 550 1000 x g for 7 minutes. The supernatant was aspirated, and the parasite pellet was resuspended in 800 µl 551 FluoroBrite DMEM. The sample was split into aliquots of 300 and 500 µl, which were spun at 1000 x g for 7 552 minutes followed by aspiration of the supernatant. The pellet containing ⅝ of the parasite harvest was  The fractions were lyophilized and resuspended in 10-20 µl of 0.1% formic acid for MS analysis and were 702 analyzed on a Q-Exactive HF-X Orbitrap mass spectrometer connected to an EASY-nLC chromatography 703 system using 0.1% formic acid as Buffer A and 80% acetonitrile/0.1% formic acid as Buffer B. Peptides were 704 separated at 300 nl/min on a gradient of 6-9% B for 3 minutes, 9-31% B for 100 minutes, 31-75% B for 20 705 minutes, and 75 to 100% B over 15 minutes. 706 The orbitrap was operated in positive ion mode. Full scan spectra were acquired in profile mode with a scan 707 range of 375-1400 m/z, resolution of 120,000, maximum fill time of 50 ms, and AGC target of 3 × 10 6 with a 15 s 708 dynamic exclusion window. Precursors were isolated with a 0.8 m/z window and fragmented with a NCE of 32. 709 The top 20 MS2 spectra were acquired over a scan range of 350-1500 m/z with a resolution of 45,000, AGC 710 target of 8 × 10 3 , and maximum fill time of 100 ms, and first fixed mass of 100 m/z. Protein abundances were loaded into the R environment (version 4.0.4) and were analyzed using the 723 mineCETSA package (Dziekan et al., 2020), which performed normalization, generated log-logistic fits of the 724 temperature profiles, and calculated a euclidean distance score (Supplementary File 4). Individual curve plots 725 were generated using custom scripts, which are available upon request. 726 Thermal profiling concentration range: Experiment 1

Parasite harvest and treatment 727
Concentration range thermal profiling experiments were performed in biological duplicate on different days. 728 Confluent HFF cells in 15-cm dishes were infected with 2-5 × 10 7 RH tachyzoites each. When the parasites had 729 fully lysed the host cell monolayer (40-48 hours later), the extracellular parasites passed through a 5 µm filter. 730 The parasite solution was concentrated by centrifugation for 10 minutes at 1000 x g. Parasites were 731 resuspended in 1 ml of wash buffer (5 mM NaCl, 142 mM KCl, 1 mM MgCl 2 , 5.6 mM glucose, 25 mM HEPES pH 732 7.2) and spun again for 10 minutes at 1000 x g. 733 The parasite pellet was resuspended in 1200 µl of lysis buffer (5 mM NaCl, 142 mM KCl, 1 mM MgCl 2 , 5.6 mM 734 glucose, 25 mM HEPES pH 7.2 with 0.8% IGEPAL CA-630, 1X Halt Protease Inhibitors, and 250 U/ml benzonase) 735 and subjected to three freeze-thaw cycles. An equivalent volume of parasite lysate was combined with 2X 736 [Ca 2+ ] free buffers to attain the final concentrations: 0 nM, 10 nM, 100 nM, 250 nM, 500 nM, 750 nM, 1 µM, 10 µM, 737 100 µM, and 1 mM. The solutions were aliquoted into two PCR tubes and were incubated for 5 minutes at 5% 738 CO 2 /37°C. The tubes were placed on heat blocks pre-warmed at 54°C or 58°C for 3 minutes and were then 739 immediately placed on ice. The lysates were transferred to a TLA-100 rotor and were spun at 100,000 x g for 20 740 minutes at 4 °C in a Beckman Ultra MAX benchtop ultracentrifuge. The solution, containing the soluble protein 741 fraction, was removed for further processing. 742

MS Sample Preparation and Data Acquisition 744
Samples were prepared for mass spectrometry as described in the Temperature Range methods. MS data was 745 acquired using the same instrument and methods as described above. Raw files were searched in Proteome 746 Discoverer 2.4 using the search parameters described in the Temperature Range methods. Arginine HCl/ 13 C 6 15 N 2 L-Lysine-2HCl (Thermo Fisher 89990 and 88209). The parasites were harvested, lysed, and treated as described in the Concentration range: Experiment 1 procedure. The lysates were incubated on 757 heat blocks pre-warmed at 50°C, 54°C or 58°C for 3 minutes and were then immediately placed on ice. 758 Insoluble aggregates were removed by filtration as described in (Herneisen and Lourido, 2021). In brief, the 759 lysates were applied to a pre-equilibrated 96-well filter plate (Millipore MSHVN4510) and were spun at 500 x g 760 for 5 minutes. The concentrations of the filtrates containing soluble fractions were quantified with a DC assay. 761 The heavy and light samples were combined at 1:1 wt/wt, yielding an estimated 50 µg total per concentration. 762 763 Protein cleanup and digestion 764 Proteins were reduced with 5 mM TCEP for 20 minutes at 50°C. Alkylation of cysteines was performed with 15 765 mM MMTS for 10 minutes at room temperature. The samples were precipitated in 80% ethanol and were 766 washed using the SP3 protocol as described in the Temperature range procedure. After the final wash, the 767 samples were resuspended in 35 µl of digest buffer (50 mM TEAB) and 1 µg Trypsin). Digestion proceeded 768 overnight at 37°C on a thermomixer shaking at 1,000 rpm. The beads were magnetically separated, and the 769 peptide eluatse were removed from further analysis. were analyzed using the mineCETSA package (Dziekan et al., 2020). The data were not further normalized 809 using the package, as normalization had already been performed by the Proteome Discoverer software. Log-810 logistic fitting of the relative abundance profiles was performed using default settings. The AUC reflects the 811 mean stability change when proteins were detected in both replicates of an experiment. If proteins were not 812 detected, the AUC was calculated from one replicate. Proteins were considered Ca 2+ -responsive if the curve-fit 813 parameters had an R 2 > 0.8 and AUC two modified Z-scores from the median. The two concentration range 814 experiments were analyzed separately. Individual curve plots were generated using custom scripts, which are 815 available upon request. 816

Enrichment Analysis
Sets of gene ontology terms from the differentially regulated (or Ca 2+ -responsive) and background proteome 817 (all proteins with quantification values in each mass spectrometry experiment) were downloaded from 818 ToxoDB.org (Molecular Function, Computed evidence, P-Value cutoff set to 1). Gene ontology terms were 819 tested for enrichment across all gene ontology terms identified in the background proteome. A p-value for the 820 likelihood of a given enrichment to have occurred by chance was obtained using a hypergeometric test. 821 Immunoblotting Samples were prepared as described in the thermal profile concentration range or phosphoproteomics 822 procedures prior to proteomics sample preparation. The samples, which had already been treated with 823 benzonase, were combined with 5X laemmli sample buffer (10% SDS, 50% glycerol, 300 mM Tris HCl pH 6.8, 824 0.05% bromophenol blue, 5% beta-mercaptoethanol) and were incubated at 37°C for 10 minutes. The samples 825 were then run on precast 4-15% SDS gels (BioRad) and were transferred overnight onto nitrocellulose 826 membranes at 4°C and 30 mA in 25 mM TrisHCl, 192 mM glycine, and 20% methanol. Blocking and antibody 827 incubations were performed in 5% milk in TBS-T for 1 hour at room temperature. The membrane was washed 828 three times with TBS-T between antibody incubations. Imaging was performed with the LICOR Odyssey CLx. 829

Immunofluorescence assays
Confluent HFFs seeded onto coverslips were infected with extracellular parasites and were grown at 37°C/5% 830 CO 2 . Approximately 21 hours later, IAA or a vehicle solution of PBS were added to the wells to a final 831 concentration of 500 µM where indicated. At 24 hours post-infection, the media was aspirated, and coverslips 832 were fixed in 4% formaldehyde in PBS. Following three washes in PBS, the fixed cells were permeabilized with 833 0.25% triton for 10 minutes at room temperature. Residual permeabilization solution was removed with three 834 washes of PBS. The coverslips were incubated in blocking solution (5% IFS/5% NGS in PBS) for 10 minutes at 835 room temperature, followed by a 60-minute incubation in primary antibody solution. An anti-CDPK1 antibody 836 (Covance) was used as a parasite counterstain (Waldman et al., 2020). After three washes with PBS, the 837 coverslips were incubated in blocking solution at room temperature for 5 minutes, followed by a 30-minute 838 incubation in secondary antibody solution. The coverslips were washed three times in PBS and once in water. 839 Coverslips were mounted with Prolong Diamond and were set for 30 minutes at 37°C. Imaging was performed 840 with the Nikon Ti Eclipse and NIS Elements software package. 841

Invasion assays
Confluent HFFs seeded onto coverslips were incubated with 5 × 10 6 extracellular parasites for 60 minutes at 842 37°C/5% CO 2 . The coverslips were washed four six times with PBS and were fixed for 10 minutes at room 843 temperature with 4% formaldehyde in PBS. The coverslips were incubated in blocking solution (1% BSA in PBS) 844 for 10 minutes. Extracellular parasites were stained with mouse anti-SAG1 for 30 minutes at room temperature. 845 Following permeabilization with 0.25% triton-X100 for 10 minutes, the coverslips were incubated with guinea-846 pig anti-CDPK1 as a parasite counterstain for 30 minutes at room temperature. The coverslips were incubated 847 with a secondary antibody solution containing Hoechst and were mounted on coverglass with Prolong 848 Diamond. The number of parasites invaded was calculated by normalizing the number of intracellular, invaded 849 parasites to host cell nuclei in a field of view. Five random fields of view were imaged per coverslip. Each 850 experiment was performed in technical duplicate. 851 852

Egress assays
Automated, plate-based egress assays were performed as previously described (Shortt and Lourido, 2020). In 853 brief, HFF monolayers in a clear-bottomed 96-well plate were infected with 7.5 × 10 4 or 1 × 10 5 parasites of the 854 TIR1 or PP1-AID strains, respectively. IAA or PBS were added to a final concentration of 500 µM 20 hours later. 855 After 3 hours, the media was exchanged for FluoroBrite supplemented with 3% calf serum. Three images were 856 taken before zaprinast (final concentration 500 μM) or A23187 (final concentration 8 µM) and DAPI (final 857 concentration 5 ng /mL) were injected. Imaging of DAPI-stained host cell nuclei continued for 9 additional 858 minutes before 1% Triton X-100 was injected into all wells to determine the total number of host cell nuclei. 859 Imaging was performed at 37°C and 5% CO 2 using a Biotek Cytation 3. Results are the mean of two wells per 860 condition and are representative of three independent experiments. 861

Replication assays
Parasites were inoculated onto coverslips containing HFFs. After 1 hour, the media was aspirated and replaced 862 with media containing 500 µM IAA or PBS vehicle. At 24 h post-IAA addition, the coverslips with intracellular 863 parasites were fixed, permeabilized, and stained with CDPK1 antibody and Hoechst as described under 864 "Immunofluorescence assays". For each sample, multiple fields of view were acquired with an Eclipse Ti 865 microscope (Nikon) and the number of nuclei per vacuole were calculated from the full field of view (at least 100 866 vacuoles). Results are the mean of three independent experiments. 867 Plaque assays 500 parasites were inoculated into 12-well plates of HFFs maintained in D10 and allowed to grow undisturbed 868 for 7 days. IAA or vehicle (PBS) was added to a final concentration of 100 µM. Plates were washed with PBS and 869 fixed for 10 min at room temperature with 100% ethanol. Staining was performed for 5 min at room 870 temperature with crystal violet solution, followed by two washes with PBS, one wash with water, and drying. 871 Live microscopy PP1-mNG parasites were grown in HFFs in glass-bottom 35 mm dishes (Ibidi) for 24 hours. The media was 872 decanted and the dish was washed once with 1 ml Ringer's buffer (155 mM NaCl, 2 mM CaCl 2 , 3 mM KCl, 1mM 873 MgCl 2 , 3 mM NaH 2 PO 4 , 10 mM HEPES, 10 mM glucose). Parasites were stimulated to egress with 500 μM 874 zaprinast or 4 µM A23187 in Ringer's buffer (155 mM NaCl, 2 mM CaCl 2 , 3 mM KCl, 1mM MgCl 2 , 3 mM NaH 2 PO 4 , 875 10 mM HEPES, 10 mM glucose) supplemented with 1% FBS (v/v) and recorded every 2 seconds for 300 seconds 876 using an Eclipse Ti microscope (Nikon) with an enclosure maintained at 37 °C. 877

Cytosolic Ca 2+ measurements with FURA-2AM
Fura-2 AM loading of T. gondii tachyzoites was done as described previously (Moreno and Zhong, 1996) Glucose) by centrifugation (706 x g for 10 min) and re-suspended to a final density of 1 x l0 9 parasites/ml in 881 loading buffer (BAG plus 1.5% sucrose, and 5 μM Fura-2 AM). The suspension was incubated for 26 min at 26 °C 882 with mild agitation. Subsequently, parasites were washed twice by centrifugation (2000 x g for 2 min) with BAG 883 to remove extracellular dye, re-suspended to a final density of 1x10 9 parasites per ml in BAG and kept on ice. 884 Parasites are viable for a few hours under these conditions. For fluorescence measurements, 2 x 10 7 885 parasites/mL were placed in a cuvette with 2.5 mL of BAG. Fluorescence measurements were done in a Hitachi 886 F-7100 fluorescence spectrometer using the Fura 2 conditions for excitation (340 and 380 nm) and emission (510 887 nm). The Fura-2 fluorescence response to Ca 2+ was calibrated from the ratio of 340/380 nm fluorescence values 888 after subtraction of background fluorescence at 340 and 380 nm as described previously (Grynkiewicz et al., 889 1985  spanning 37-67°C. In the concentration range experiment, parasite lysates were combined with 10 different 931 [Ca 2+ ] free (nM-mM range) and heated at 50, 54, or 58°C. Temperature-range shifts were quantified by the 932 euclidean distance (ED) score, a weighted ratio of thermal stability differences between treatments and 933 replicates. Concentration-range shifts were summarized by pEC 50 , area under the curve (AUC), and goodness of 934 fit (R 2 ). (C) Heat map of protein thermal stability relative to the lowest temperature (37°C) in 0 or 10 µM Ca 2+ . 935 The mean relative abundance at each temperature was calculated for 2,381 proteins. Proteins are plotted in the 936 same order in both treatments. (D) Raincloud plots summarizing the distribution of T m in lysates with EGTA 937 (gray) or 10 µM [Ca 2+ ] free (blue). The average melting temperatures of proteins identified in two replicates were 938 plotted. (E) Proteins rank-ordered by euclidean distance score quantifying the Ca 2+ -dependent shift in thermal 939 stability. Solid and dotted lines represent the median ED score and two modified Z-scores above the median, 940 respectively. Highlighted proteins have EF hand domains (blue) or are conserved in apicomplexans (pink). (F) 941 Thermal profiles of individual proteins: DNA polymerase β (TGGT1_233820); the EF hand domain-containing 942 proteins CDPK7 (TGGT1_228750) and the calmodulin-like protein CAM2 (TGGT1_262010); potential Ca 2+ -leak 943 channels TGGT1_255900 and TGGT1_206320); and AKMT (TGGT1_216080). 944 against the sensitivity (pEC 50 ) for protein abundances exhibiting a dose-response trend with an R 2 > 0.8. Point 961 size is scaled to R 2 . Summary parameters for the different separation methods (ultracentrifugation or filtration) 962 are plotted separately. Colors identify candidates with Ca 2+ -responsive behavior validated in Figure 4. (C) Gene 963 ontology (GO) terms enriched among candidate Ca 2+ -responsive proteins (AUC greater than two modified Z 964 scores and R 2 dose-response > 0.8). Fold enrichment is the frequency of Ca 2+ -responsive proteins in the set 965 relative to the frequency of the GO term in the population of detected proteins. Significance was determined 966 with a hypergeometric test; only GO terms with p < 0.05 are shown. (D-F) EF hand domain proteins (D), protein 967 kinases (E), and protein phosphatases (F) detected in the thermal profiling mass spectrometry datasets. The 968 top rows indicate if a protein passed the AUC cutoff (orange) or R 2 cutoff (blue) for dose-response behavior. The 969 opacity of the band represents the number of experiments in which the protein exhibited the behavior (out of 970 five). The five rows below summarize the pEC 50 of each experiment in which the protein exhibited a dose-971 response trend with R 2 > 0.8. Kinases are loosely grouped as CDPK's (included as a reference), non-rhoptry 972 kinases, and secretory pathway kinases. 973