Summary Toxoplasma gondii parasites rapidly exit their host cell when exposed to calcium ionophores. Calcium‐dependent protein kinase 3 (TgCDPK3) was previously identified as a key mediator in this process, as TgCDPK3 knockout (∆cdpk3) parasites fail to egress in a timely manner. Phosphoproteomic analysis comparing WT with ∆cdpk3 parasites revealed changes in the TgCDPK3‐dependent phosphoproteome that included proteins important for regulating motility, but also metabolic enzymes, indicating that TgCDPK3 controls processes beyond egress. Here we have investigated a predicted direct target of TgCDPK3, ApiAT5‐3, a putative transporter of the major facilitator superfamily, and show that it is rapidly phosphorylated at serine 56 after induction of calcium signalling. Conditional knockout of apiAT5‐3 results in transcriptional upregulation of most ribosomal subunits, but no alternative transporters, and subsequent parasite death. Mutating the S56 to a non‐phosphorylatable alanine leads to a fitness cost, suggesting that phosphorylation of this residue is beneficial, albeit not essential, for tyrosine import. Using a combination of metabolomics and heterologous expression, we confirmed a primary role in tyrosine import for ApiAT5‐3. However, no significant differences in tyrosine import could be detected in phosphorylation site mutants showing that if tyrosine transport is affected by S56 phosphorylation, its regulatory role is subtle.

N-myristoylation is a ubiquitous class of protein lipidation across eukaryotes and N-myristoyl transferase (NMT) has been proposed as an attractive drug target in several pathogens. Myristoylation often primes for subsequent palmitoylation and stable membrane attachment, however, growing evidence suggests additional regulatory roles for myristoylation on proteins. Here we describe the myristoylated proteome of Toxoplasma gondii using chemoproteomic methods and show that a small-molecule NMT inhibitor developed against related Plasmodium spp. is also functional in Toxoplasma. We identify myristoylation on a transmembrane protein, the microneme protein 7 (MIC7), which enters the secretory pathway in an unconventional fashion with the myristoylated N-terminus facing the lumen of the micronemes. MIC7 and its myristoylation play a crucial role in the initial steps of invasion, likely during the interaction with and penetration of the host cell. Myristoylation of secreted eukaryotic proteins represents a substantial expansion of the functional repertoire of this co-translational modification.

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22Toxoplasma gondii parasites rapidly exit their host cell when exposed to calcium 23 ionophores. The calcium-dependent protein kinase 3 (TgCDPK3) was previously 24 identified as a key mediator in this process, as TgCDPK3 knockout (∆cdpk3) (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint . http://dx.doi.org/10.1101/339671 doi: bioRxiv preprint first posted online Jun. 5, 2018; 3 allow the parasites to sense and respond rapidly to their environment. While some 46 key kinases and secondary messengers within these pathways have been 47 identified, functional analysis of non-kinases has been very limited. This is 48 especially true for candidates that are not predicted to play a role in active motility 49 or are not known to function in established signalling pathways. Here we have 50 followed up on an unexpected target of the T. gondii calcium-dependent kinase 3 51 (TgCDPK3), a plant-like calcium dependent kinase, that was previously shown to 52 play an important role in calcium-mediated exit from the host cell. We show that, 53 in addition to controlling motility of the parasite (as previously shown), TgCDPK3 54 phosphorylates an essential tyrosine transporter in the plasma membrane. 55Mutational analysis of the phosphorylation sites demonstrates an important role 56 in maintaining parasite fitness, thus demonstrating that TgCDPK3 plays a 57 pleiotropic role in controlling both egress and metabolism. 58 59

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