Characterisation of the Toxoplasma gondii tyrosine transporter and its phosphorylation by the calcium-dependent protein kinase 3 Bethan A Wallbank Caia S Dominicus Malgorzata Broncel Nathalie Legrave James I MacRae Henry M Staines Moritz Treeck 10779/crick.11410050.v1 https://crick.figshare.com/articles/journal_contribution/Characterisation_of_the_Toxoplasma_gondii_tyrosine_transporter_and_its_phosphorylation_by_the_calcium-dependent_protein_kinase_3/11410050 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. 2019-12-19 17:17:35 Treeck FC001189 MET CB-ack AS-ack PC-ack PRT-ack FC-ack 06 Biological Sciences 11 Medical and Health Sciences 07 Agricultural and Veterinary Sciences Microbiology