There were approximately 247 million malaria cases in 2006, resulting in close to a million deaths, mostly of children under 5 [1
]. The malaria parasite, Plasmodium
, is a unicellular eukaryote, and transmitted to humans via mosquito bites. Symptoms of the disease occur when the parasite enters the bloodstream, where it invades and replicates inside erythrocytes. Within the erythrocyte, Plasmodium
resides in a membrane-bounded vacuole, the parasitophorous vacuole (PV). By exporting proteins across its own plasma membrane as well as the PV membrane, and into the cytoplasm and to the surface of the infected erythrocyte, the parasite is able to alter the solute permeability, cytoskeleton, and adhesion properties of its host cell [2
]. Specific soluble proteins are exported across the PV membrane into the erythrocyte cytoplasm. Certain membrane proteins, some of which are linked to disease pathology [4
], are also exported and become integrated into the host cell plasma membrane and vesicular membrane structures, called Maurer’s clefts, within the infected erythrocyte’s cytoplasm.
The first step in the export pathway involves targeting of a protein to the parasite endoplasmic reticulum (ER), either by an N-terminal signal sequence or a transmembrane domain. Translocation into the ER lumen is likely mediated by the conserved ER co-translational translocation pathway [5
]. Several lines of evidence indicate that exported proteins are initially targeted to the parasite endoplasmic reticulum; firstly, soluble exported proteins are characterized by the presence of an N-terminal hydrophobic signal sequence that can be replaced by the signal sequence of an ER targeted protein, without compromising export [6
], and secondly, fusion of an ER retention sequence to the C-terminus of an exported protein blocks its export [7
After targeting to the parasite ER, exported proteins are likely trafficked by a vesicle-mediated pathway to the parasite plasma membrane where they are released into the PV lumen. Most proteins that are transported across the PV membrane also contain a HT motif (also referred to as a PEXEL) [8
], which is generally located less than 32 residues downstream of the predicted signal sequence cleavage site [10
]. The HT motif corresponds approximately to the consensus RxLxE/D/Q (x is any amino acid) and is required for efficient export of most proteins [8
]. Mutation of residues within the HT motif prevents protein export across the PV membrane and leads to accumulation of protein within the parasite secretory pathway and in the parasitophorous vacuole lumen [8
]. Additionally, a linker of 11 residues or more must separate the HT motif from any folded C-terminal domain. Placing the HT motif too close to a C-terminal folded domain, such as GFP, blocks export [6
Recent reports show that the HT motif is cleaved by a protease and that the newly generated N-terminus is N-acetylated. It is unclear where in the cell this processing occurs or indeed whether the cleaved or uncleaved HT motif targets a protein for export [13
]. In all exported proteins examined, the HT motif is cleaved after the leucine residue (in the sequence RxLxE/D/Q). Surprisingly, in a protein where the HT motif is placed close to a folded GFP domain, preventing export, the HT motif is still cleaved and acetylated [14
]. This suggests that processing occurs within the parasite secretory pathway or in the PV lumen, before a protein is exported. Furthermore, treatment of parasites with Brefeldin A for 24 hours, which leads to an accumulation of exported proteins in the parasite ER, does not prevent their cleavage and acetylation [14
]. This suggests that processing of the HT motif occurs in the parasite ER. However, interpretation of this experiment is complicated as even brief treatment of mammalian cells with Brefeldin A causes Golgi resident proteins to be relocalized to ER [15
] and results in certain ER resident proteins being abnormally modified with o-linked glycans [16
]. It is therefore possible that a parasite protease, normally localized to the Golgi apparatus, is mislocalized to the ER where it mediates cleavage of the HT motif upon treatment with Brefeldin A. The consequences of prolonged 24-hour incubation in Brefeldin A, used to block protein export [14
], have not been characterized. They likely include an accumulation of non-ER proteins in the parasite ER, as newly synthesized proteins cannot exit from the organelle normally. After such treatment the parasite ER may contain significant amounts of non-resident ER proteins that would normally be localized to later compartments in the secretory pathway, the PV lumen, or even exported to the red cell cytoplasm. Therefore, based on this experiment it is difficult to conclude where in the parasite secretory pathway the HT motif is normally cleaved.
To establish whether the HT motif is cleaved in the parasite ER we have generated parasites that express HT motif containing proteins that are localized to different cellular compartments. We find that, as previously shown, the HT motif in an exported protein is cleaved and acetylated. This cleavage does not occur during cell lysis, as it is not seen in a control protein. Using quantitative mass spectrometry we show that an ER resident protein, containing a HT motif, is cleaved with similar efficiency to an exported protein, indicating that cleavage of the HT motif occurs in the parasite ER. Significantly, this experiment is done without blocking general protein trafficking from the parasite ER or disruption of the parasite secretory pathway, avoiding any potential artifacts associated with use of drugs such as Brefeldin A.