Click Chemistry for Imaging in-situ Protein Palmitoylation during the Asexual Stages of Plasmodium falciparum

Palmitoylation refers to the modification of the cysteine thiols in proteins by fatty acids, most commonly palmitic acid, through `thioester bond' formation. In vivo, palmitoylation of proteins is catalyzed by palmitoyl acyltransferases (PATs or DHHC-PATs). Palmitoylation has recently emerged as a crucial post-translational modification in malarial parasites. The expression and activity of palmitoyl transferases vary across different developmental stages of the malarial parasite's life cycle. The abundance of palmitoylated proteins at a given stage is a measure of overall PAT activity. The PAT activity can also change in response to external signals or inhibitors. Here, we describe a protocol to `image' palmitoyl-transferase activity during the asexual stages using Click Chemistry and fluorescence microscopy. This method is based on metabolic labeling of a clickable analog of palmitic acid by parasitic cells, followed by CuAAC (Copper-catalyzed Alkyne-Azide Cycloaddition reaction) Click Chemistry to render palmitoylated proteins fluorescent. Fluorescence allows the quantitation of intracellular palmitoylation in parasite cells across various development stages. Using this method, we observed that intracellular palmitoylation increases as the parasite transitions from ring to schizont stages and appears to be most abundant during the schizont stages in Plasmodium falciparum.