Sequence requirements for the export of the Plasmodium falciparum Maurer's clefts protein REX2

Abstract

SummaryA short motif termed Plasmodium export element (PEXEL) or vacuolar targeting signal (VTS) characterizes Plasmodium proteins exported into the host cell. These proteins mediate host cell modifications essential for parasite survival and virulence. However, several PEXEL-negative exported proteins indicate that the currently predicted malaria exportome is not complete and it is unknown whether and how these proteins relate to PEXEL-positive export. Here we show that the N-terminal 10 amino acids of the PEXEL-negative exported protein REX2 (ringexported protein 2) are necessary for its targeting and that a single-point mutation in this region abolishes export. Furthermore we show that the REX2 transmembrane domain is also essential for export and that together with the N-terminal region it is sufficient to promote export of another protein. An N-terminal region and the transmembrane domain of the unrelated PEXEL-negative exported protein SBP1 (skeleton-binding protein 1) can functionally replace the corresponding regions in REX2, suggesting that these sequence features are also present in other PEXEL-negative exported proteins. Similar to PEXEL proteins we find that REX2 is processed, but in contrast, detect no evidence for N-terminal acetylation.

Development and host cell modifications of Plasmodium falciparum blood stages in four dimensions

“…On rupture, merozoites rapidly dispersed (Supplementary Movie 1). To visualize the formation of merozoites, we used parasites expressing a GFP tagged protein residing in the parasitophorous vacuole (REX2∆TM + SP GFP 37 ). This revealed that merozoite formation is a fast process: the first parasite internal traces of GFP suggestive of invagination of the parasite plasma membrane were visible 3-4 h before rupture, and within 2 h before rupture the parasitophorous vacuole delineated individual merozoites (Fig.…”

Maurer's clefts, the enigma of Plasmodium falciparum

“…In contrast, GFP-tagged Maurer's clefts resident proteins were found only in a punctuate manner at Maurer's clefts, indicating distinct, independent structures. In the case of a continuous network, fluorescent signal would have been observed throughout the network (38)(39)(40)(41)(42)(43)(44)(45), but in photo-bleaching experiments no lateral diffusion of Maurer's clefts resident proteins or lipids (41,44,75,76) was observed. In contrast, the erythrocyte membrane showed a fast recovery after photo-bleaching when BODIPYceramide was used (44).…”

“…Maurer's clefts look highly convoluted and interconnected in whole-cell imaging (21) (21). Fluorescence microscopy of erythrocytes infected with transfectant parasites expressing GFPchimera of resident proteins shows discrete puncta (38)(39)(40)(41)(42)(43)(44)(45), which cannot be replenished by lateral diffusion (41).…”

“…It has been suggested that all these proteins enter the classic secretory pathway (41-43, 48, 105), and lately several research groups tried to identify sequence requirements for export, yet no common motif or perceptible sequence has been identified. A transmembrane domain or a signal peptide seems to be essential, most likely for entering the ER and the secretory pathway (41,43,48,106). Unfolding appears to be equally required for PEXELpositive proteins, suggesting similar mode of translocation across the PVM (104,107).…”

“…This group of proteins includes some wellcharacterized proteins known to be located to the Maurer's clefts, including Maurer's clefts resident proteins MAHRP1, SBP1, REX1, and REX2 (40,43,83,102,103), as well as the tether protein MAHRP2 (48). Heiber et al recently based their prediction on rather general criteria and demonstrated that, most likely, many more PEXEL-negative proteins exist with various hydrophobic domains (104).…”