Formation of ER-lumenal intermediates during export of Plasmodium proteins containing transmembrane-like hydrophobic sequences.

During the blood stage of a malaria infection, malaria parasites export both soluble and membrane proteins into the erythrocytes in which they reside. Exported proteins are trafficked via the parasite endoplasmic reticulum and secretory pathway, before being exported across the parasitophorous vacuole membrane into the erythrocyte. Transport across the parasitophorous vacuole membrane requires protein unfolding, and in the case of membrane proteins, extraction from the parasite plasma membrane. We show that trafficking of the exported Plasmodium protein, Pf332, differs from that of canonical eukaryotic soluble-secreted and transmembrane proteins. Pf332 is initially ER-targeted by an internal hydrophobic sequence that unlike a signal peptide, is not proteolytically removed, and unlike a transmembrane segment, does not span the ER membrane. Rather, both termini of the hydrophobic sequence enter the ER-lumen and the ER-lumenal species is a productive intermediate for protein export. Furthermore, we show in intact cells, that two other exported membrane proteins, SBP1 and MAHRP2, assume a lumenal topology within the parasite secretory pathway. Although the addition of a C-terminal ER-retention sequence, recognised by the lumenal domain of the KDEL receptor, does not completely block export of SBP1 and MAHRP2, it does enhance their retention in the parasite ER. This indicates that a sub-population of each protein adopts an ER-lumenal state that is an intermediate in the export process. Overall, this suggests that although many exported proteins traverse the parasite secretory pathway as typical soluble or membrane proteins, some exported proteins that are ER-targeted by a transmembrane segment-like, internal, non-cleaved hydrophobic segment, do not integrate into the ER membrane, and form an ER-lumenal species that is a productive export intermediate. This represents a novel means, not seen in typical membrane proteins found in model systems, by which exported transmembrane-like proteins can be targeted and trafficked within the lumen of the secretory pathway. Author summary: Symptoms of malaria occur during the blood stage of infection when the malaria parasite resides inside human red blood cells. Hundreds of proteins, synthesized by the parasite, are exported into the host cell where they modify its properties. Some exported proteins become embedded in membranes and are referred to as membrane proteins. Despite their importance in disease pathology, how these membrane proteins are transported into the red blood cell is poorly understood. Some exported membrane proteins are thought to become membrane embedded in the parasite endoplasmic reticulum and are subsequently extracted from the parasite plasma membrane before being transported into the red blood cell where they are then re-inserted into the appropriate membrane. Contrary to this, we find that a subset of membrane proteins enter into the lumen of the endoplasmic reticulum and either do not insert into the parasite endoplasmic reticulum membrane or insert and are rapidly extracted. This behaviour is very different from the behaviour of non-exported membrane proteins that have been studied in model systems such as human or yeast cells and suggests that the trafficking of exported membrane proteins is a mechanistically distinct process that may represent a unique drug target. [ABSTRACT FROM AUTHOR]