Biochemical mutagenesis signatures systematically identify functional palmitoylation post-translational modifications in Toxoplasma gondii

Apicomplexan parasites are obligatorily intracellular, with motility and invasion of host cells necessitating the discharge of a multitude of conserved proteins from the parasite"s apical organelles. Post-translational modifications (PTM) such as palmitoylation regulate the function of apical organelle-associated proteins. Palmitoylation has been shown to be critical for the parasite"s lytic cycle with inhibition of its addition and removal found to disrupt parasite motility and invasion1,2. Additionally, mutation of palmitoylated cysteines in proteins such as the Toxoplasma protein TgARO resulted in the disruption of rhoptry organisation and secretion3,4. Despite this knowledge, the full extent to which palmitoylation regulates organelle secretion is not fully understood. While the writers and erasers for this PTM are known and palmitoylated proteins globally profiled, these datasets do not indicate which cysteines are palmitoylated5, and more importantly, which contribute to protein function.

We hypothesised that 1) palmitoylation is an important and general regulator apical organelle secretion, and 2) since enzymatic addition of palmitate onto cysteine thiols is chemoselective, should a palmitoylation event be functionally important, the target cysteine should be intolerant to mutagenesis.

We used CORe6 to generate saturated mutagenesis profiles, revealing biochemical signatures of functional palmitoylation modifications. We have tested the function of 38 predicted cysteine targets of palmitoylation distributed across 23 palmitoylated proteins, profiling a total of 798 individual amino acid substitutions in a single experiment, in live parasites, and in high throughput. These data have allowed us to identify candidate cysteine residues on apical organelle secretion-associated proteins that are tolerant and intolerant to substitution, confirming our hypothesis that biochemical mutagenesis profiles serve as signatures for functional PTMs.

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