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

Abstract

Background InformationLike 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 cell's secretory pathway involving the Golgi, whereas the other is a unique Golgi‐independent pathway. Using different experimental approaches, many apicoplast proteins have been shown to utilize the Golgi‐independent pathway, whereas 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.ResultsIn 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/IRP) are trafficked through the Golgi, whereas 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/IRP suggested that the evolutionary origins of TgSOD2 and TgTPx1/2 lie in the mitochondrion, whereas TgACN/IRP appears to have originated from the apicoplast.Conclusions and SignificanceCollectively, 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.

Knock-sideways by inducible ER retrieval enables a novel approach for studyingPlasmodiumsecreted proteins

“…In this study, we demonstrate knockER simultaneously allows for trafficking and loss-of-function studies. The ability to determine whether a protein traffics from the ER through the cis Golgi is an important consideration for nuclear-encoded apicoplast proteins since Golgi-dependent and -independent trafficking routes have been reported (32, 34, 71). While these apicoplast trafficking studies were carried out with reporters and chimeras, knockER allows for monitoring cis Golgi transit of endogenous proteins.…”

Recognition of Two Distinct Pathways for Trafficking of Proteins to the Apicoplast

“…As SNAREs are involved in the vesicular trafficking pathway, it is important to note that multiple routes exist for apicoplast trafficking in Toxoplasma gondii and Plasmodium falciparum . One route leads directly from the endoplasmic reticulum (ER) to the apicoplast ( 2 , 3 ), and the other route leads from the ER to the apicoplast via the Golgi pathway ( 4 , 5 ).…”

“…A broad statement about dependency on the Golgi pathway is surprising not only because it fails to acknowledge the substantial literature on Golgi pathway-independent trafficking of apicoplast proteins but also because in an article from our lab, we use an HDEL-mediated ER retention strategy to show that proteins localized to the apicoplast alone use the Golgi-independent trafficking route from the ER to the apicoplast ( 5 ). Of the apicoplast markers chosen by Cao and colleagues, TgACP ( 6 ), TgATrx2, and TgATrx1 ( 5 ) have been tested using the HDEL strategy and are unambiguously demonstrated to bypass the Golgi pathway. In contrast to proteins localized to the apicoplast alone, proteins that are targeted to both the apicoplast and the mitochondrion (TgTPx1/2, TgSOD2, or TgACN) use the Golgi-dependent trafficking route from the ER to the apicoplast ( 5 ).…”

“…Of the apicoplast markers chosen by Cao and colleagues, TgACP ( 6 ), TgATrx2, and TgATrx1 ( 5 ) have been tested using the HDEL strategy and are unambiguously demonstrated to bypass the Golgi pathway. In contrast to proteins localized to the apicoplast alone, proteins that are targeted to both the apicoplast and the mitochondrion (TgTPx1/2, TgSOD2, or TgACN) use the Golgi-dependent trafficking route from the ER to the apicoplast ( 5 ). Mutant versions of these proteins that are targeted only to the apicoplast also use the Golgi pathway.…”

Reply to Prasad and Patankar, “Recognition of Two Distinct Pathways for Trafficking of Proteins to the Apicoplast”

“…However, the results are complicated. A couple of studies documented that the proteins were still found in apicoplasts and not observed in the ER in either T. gondii ( 2 , 3 ) or Plasmodium falciparum ( 4 ), while a separate study reported that the addition of an SDEL sequence to the S9(S+T)-GFP caused reduced delivery to the apicoplast and retention in the ER in P. falciparum ( 5 ). The slight difference among these studies is that the expression of the proteins was driven by different promoters.…”