Analysis of targeting sequences demonstrates that trafficking to the Toxoplasma gondii plastid branches off the secretory system

Apicomplexan parasites possess a plastid-like organelle called the apicopla st. Most proteins in the Toxoplasma gondii apicoplast are encoded in the nu cleus and imported post-translationally. I: gondii apicoplast proteins ofte n have a long N-terminal extension that directs the protein to the apicopla st. It can be modeled as a bipartite targeting sequence that contains a sig nal sequence and a plastid transit peptide. We identified two nuclearly enc oded predicted plastid proteins and made fusions with green fluorescent pro tein to study protein domains required for apicoplast targeting. The N-term inal 42 amino acids of the apicoplast ribosomal protein S9 directs secretio n of green fluorescent protein, indicating that targeting to the apicoplast proceeds through the secretory system. Large sections of the S9 predicted transit sequence can be deleted with no apparent impact on the ability to d irect green fluorescent protein to the apicoplast. The predicted transit pe ptide domain of the S9 targeting sequence directs protein to the mitochondr ion in vivo. The transit peptide can also direct import of green fluorescen t protein into chloroplasts in vitro. These data substantiate the model tha t protein targeting to the apicoplast involves two distinct mechanisms: the first involving the secretory system and the second sharing features with typical chloroplast protein import.