Dually localized proteins found in both the apicoplast and mitochondrion utilize the Golgi-dependent pathway for apicoplast targeting in Toxoplasma gondii

Like other apicomplexan parasites, Toxoplasma gondii harbours a four-membraned endosymbiotic organelle - the apicoplast. Apicoplast proteins are nuclear-encoded and trafficked to the organelle through the endoplasmic reticulum (ER). From the ER to the apicoplast, two distinct protein trafficking pathways can be used. One such pathway is the cells secretory pathway involving the Golgi, while the other is a unique Golgi-independent pathway. Using different experimental approaches, many apicoplast proteins have been shown to utilize the Golgi-independent pathway, while a handful of reports show that a few proteins use the Golgi-dependent pathway. This has led to an emphasis towards the unique Golgi-independent pathway when apicoplast protein trafficking is discussed in the literature. Additionally, the molecular features that drive proteins to each pathway are not known. In this report, we systematically test eight apicoplast proteins, using a C-terminal HDEL sequence to assess the role of the Golgi in their transport. We demonstrate that dually localised proteins of the apicoplast and mitochondrion (TgSOD2, TgTPx1/2 and TgACN) are trafficked through the Golgi while proteins localised exclusively to the apicoplast are trafficked independent of the Golgi. Mutants of the dually localised proteins that localised exclusively to the apicoplast also showed trafficking through the Golgi. Phylogenetic analysis of TgSOD2, TgTPx1/2 and TgACN suggested that the evolutionary origins of TgSOD2, TgTPx1/2 lie in the mitochondrion while TgACN appears to have originated from the apicoplast. Collectively, with these results, for the first time, we establish that the driver of the Golgi-dependent trafficking route to the apicoplast is the dual localisation of the protein to the apicoplast and the mitochondrion.