Development of a conditional localization approach to control apicoplast protein trafficking in malaria parasites

: Secretory proteins are of particular importance to apicomplexan parasites and comprise over 15% of the genomes of the human pathogens that cause diseases like malaria, toxoplasmosis and babesiosis as well as other diseases of agricultural significance. Here, we developed an approach that allows us to control the trafficking destination of secretory proteins in the human malaria parasite Plasmodium falciparum. Based on the unique structural requirements of apicoplast transit peptides, we designed three conditional localization domains (CLD1, 2 and 3) that can be used to control protein trafficking via the addition of a cell permeant ligand. Studies comparing the trafficking dynamics of each CLD show that CLD2 has the most optimal trafficking efficiency. To validate this system, we tested whether CLD2 could conditionally localize a biotin ligase called holocarboxylase synthetase 1 (HCS1) without interfering with the function of the enzyme. In a parasite line expressing CLD2-HCS1, we were able to control protein biotinylation in the apicoplast in a ligand-dependent manner, demonstrating the full functionality of the CLD tool. We have developed and validated a novel molecular tool that may be used in future studies to help elucidate the function of secretory proteins in malaria parasites.