Identification of New PNEPs Indicates a Substantial Non-PEXEL Exportome and Underpins Common Features in Plasmodium falciparum Protein Export

Abstract

Malaria blood stage parasites export a large number of proteins into their host erythrocyte to change it from a container of predominantly hemoglobin optimized for the transport of oxygen into a niche for parasite propagation. To understand this process, it is crucial to know which parasite proteins are exported into the host cell. This has been aided by the PEXEL/HT sequence, a five-residue motif found in many exported proteins, leading to the prediction of the exportome. However, several PEXEL/HT negative exported proteins (PNEPs) indicate that this exportome is incomplete and it remains unknown if and how many further PNEPs exist. Here we report the identification of new PNEPs in the most virulent malaria parasite Plasmodium falciparum. This includes proteins with a domain structure deviating from previously known PNEPs and indicates that PNEPs are not a rare exception. Unexpectedly, this included members of the MSP-7 related protein (MSRP) family, suggesting unanticipated functions of MSRPs. Analyzing regions mediating export of selected new PNEPs, we show that the first 20 amino acids of PNEPs without a classical N-terminal signal peptide are sufficient to promote export of a reporter, confirming the concept that this is a shared property of all PNEPs of this type. Moreover, we took advantage of newly found soluble PNEPs to show that this type of exported protein requires unfolding to move from the parasitophorous vacuole (PV) into the host cell. This indicates that soluble PNEPs, like PEXEL/HT proteins, are exported by translocation across the PV membrane (PVM), highlighting protein translocation in the parasite periphery as a general means in protein export of malaria parasites.

Identity of a Plasmodium lactate/H+ symporter structurally unrelated to human transporters

“…PfFNT resides in the plasmodial plasma membrane. Finally, we expressed a PfFNT-GFP (green fluorescent protein) fusion protein for localization in P. falciparum parasites 30,31 . Fluorescence was restricted to the intraerythrocytic parasite and concentrated at the parasite's periphery where it co-localized with the plasma membrane protein merozoite surface protein 1 (MSP1; Fig.…”

“…For expression of GFP-tagged PfFNT in P. falciparum parasites, the coding sequence was cloned into pARL1-PF08_0003-GFP via KpnI and AvrII replacing PF08_0003 (ref. 30). Parasites were cultured with O þ erythrocytes in RPMI 1640 plus 0.5% Albumax, transfected 37 , selected using 4 nM WR99210 (Jacobus Pharmaceuticals) and imaged with a Zeiss Axioskop M1 microscope at  100/1.4 oil immersion 31 .…”

Maurer's clefts, the enigma of Plasmodium falciparum

“…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). Interestingly, processed N termini of PEXELpositive proteins promote the export of a reporter gene, like unprocessed N termini of PEXEL-negative proteins (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). 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.…”

Toxoplasmaexports dense granule proteins beyond the vacuole to the host cell nucleus and rewires the host genome expression

“…Of note, GRA24 does not contain any PEXEL-like motif and the latter does not share primary sequence signature with GRA16 strengthening the idea that, like in Plasmodium, the sequences promoting export are non-specific. Indeed in Plasmodium, a substantial number of PEXELnegative exported proteins (PNEPs) have been already characterized (Heiber et al, 2013). In general, PNEPs do not contain the classical N-terminal SP either but the latter is somehow functionally compensated by the presence of a single internal hydrophobic stretch for targeting to the ER (Boddey and Cowman, 2013b;Marti and Spielmann, 2013).…”