The HT signal functions in context of a ~40 amino acid vacuolar translocation signal needed to mediate parasite protein export to the erythrocyte (Hiller et al., 2004
; Lopez-Estrano et al., 2003
). Thus the HT signal may be one (likely the most important) region necessary for selective PI(3)P recognition. In malarial HT signals, PI(3)P binding is critically dependent on the high value R in the plasmodial HT logo, providing one of the first clues that lipid binding may play a major role in targeting malaria parasite proteins to the host erythrocyte. The nanomolar affinity displayed by the HT signal of virulence determinants containing the R/KxLxE motif, including the major virulence adhesin PfEMP1, suggests a new mode of PI(3)P binding. Since nanomolar phosphoinositide (PI) binding is primarily attributed to at least four points of contact, it is likely that there are other cationic residues residing in the HT signal and flanking sequences that contribute to PI(3)P recognition. Additionally, hydrophobic residues such as L or others within the HT signal may provide further membrane anchorage through membrane penetration (Lemmon, 2008
), while the consensus E could form H-bonds with the 5-OH and discriminate against other PIs as previously reported for the FYVE domain (Dumas et al., 2001
). Further structural and biophysical studies will be necessary to fully appreciate this novel mechanism of PI(3)P recognition.
The presence of PI(3)P in the malarial ER and its affinity for endogenous HT signals, strongly suggests that HT-signal lipid interactions occur early in the ER. Importantly, with the development of specific antibody reagents, we are able to detect ER precursors carrying the HT signal, suggesting that even at steady state a detectable amount of endogenous precursor carrying the HT signal has not been processed by plasmepsin V. Our finding that PI(3)P binding is required for export even when plasmepsin V fails to cleave the oomycete HT signal, leads us to propose that the export mechanism is an efficient, early sorting event in the ER () dependent on high affinity binding to PI(3)P in the lumen of the ER. When it occurs, HT signal cleavage by plasmepsin V is expected to be restricted to an emerging and/or possibly sealed vesicle. The convergence of amino acid specificity of PI(3)P binding and plasmepsin V cleavage, may suggest that association by PI(3)P may facilitate protease cleavage by plasmepsin V. However cleavage per se (whether by plasmepsin V or signal peptidase) may not provide specificity for host targeting, rather both release protein from the ER membrane.
A model for PI(3)P dependent export from the ER of P. falciparum-infected erythrocytes
Our data also suggest that HT-independent export of parasite protein reporters to the host can occur. HT-independent pathways of export have been proposed for major parasite proteins such as P. falciparum
Skeleton binding protein 1 (PfSBP1) as well as other proteins, which lack the HT signal but which are resident in the secretory structures called Maurer's clefts in the erythrocyte cytoplasm and are thought to promote protein delivery to the host erythrocyte membrane and other destinations in the erythrocyte (Cooke et al., 2006
; Spielmann and Gilberger, 2010
). Our data suggest that non HT-dependent export to the erythrocyte is based on charge. Chaperones that interact with charged residues may well distinguish pathways of protein export to the erythrocyte relative to default secretion of well folded secretory proteins to the PV but these need not be linked to chaperones reported to be associated with plasmepsin V (Goldberg and Cowman, 2010
; Russo et al., 2010
In summary, the parasite likely sorts newly synthesized proteins into two, possibly three different export pathways that emerge from the ER, thus, separating cargo for the erythrocyte from the PV (), akin to early models of secretion proposed over a decade ago (Elmendorf and Haldar, 1993
; Wiser et al., 1997
). Wiser et al (1997)
proposed a 'secondary ER' for protein export to the erythrocyte and this was supported by rapid kinetics of secretory protein exit (Crary and Haldar, 1992
), but evidence of specialized ER domains in parasite protein export was lacking. The contribution of the Golgi to the PI(3)P/HT-signal ER exit pathway, remains unknown. A translocon mediating export of secretome proteins into the erythrocyte has been proposed (de Koning-Ward et al., 2009
). How it recognizes putative protein cargo is unclear, since the HT signal in most plasmodial proteins is largely abrogated in ER exit.
Based on the steady state distribution of secretory PI(3)P and plasmepsin V, we propose that both recycle back to the ER once HT signal sorting and cleavage are completed (), at least in early intraerythrocytic stages called ring and early trophozoite stages investigated in this study. Recent studies have reported the export of a PI3kinase into the erythrocyte and in association with membrane structures in the red cell (Vaid et al., 2010
), but how this export occurs and whether PI3kinase is recruited in the secretory pathway is not known. An alternate mechanism for concentration of PI(3)P in the ER, could be trans-bilayer import of PI(3)P synthesized on the cytoplasmic face. A recent study suggested that blood cell infection increases PI(3)P levels (Tawk et al., 2010), although the overall ratio of PI(3)P in the ER to total cellular PI(3)P is unknown.
The presence of PI(3)P in the lumen of ER regions and its binding to HT signals, is consistent with a sorting function for this lipid in malaria parasites. Given that malarial PI(3)P is utilized by the oomycete HT signal to target proteins to the host erythrocyte and malarial signals and oomycete signals are functionally equivalent in both Plasmodium and Phytophthora, it is possible that PI(3)P also functions in the P. infestans ER. Thus PI(3)P binding in the ER may be a generalized mechanism for pathogenic secretion in eukaryotic pathogens, aspects of which may be targeted to disrupt pathogen-host interactions that underlie disease.