microneme proteins released in response to calcium-mediated signals are not only essential for parasite entry into host cells but also for gliding motility. Accordingly many parasite cell lines deficient in microneme proteins such as MIC2, M2AP, AMA1, MIC8 are severely impaired in invasion and virulence in mouse infections. Hence studying the synthesis, transport, storage, secretion and functioning of microneme proteins might reveal new players that could act as novel targets for intervention. Recent studies on microneme trafficking in T. gondii
have revealed that cytoplasmic domains of transmembrane microneme proteins do not necessarily contain sufficient signals for trafficking (22
). This view is also supported by the findings that propeptide domains play a crucial role in correct targeting of microneme proteins (19
). In this study we take a step further and show that propeptides within T. gondii
and also in the related parasite Eimeria tenella
can substitute for one another and hence appear to function in a context independent manner. We also define at the single residue level that aliphatic amino acids near the N-terminus of microneme propeptides are crucial for correct targeting to the micronemes.
We found that the fates of M2AP and MIC5 are different in trafficking defective parasites, suggesting that propeptides contribute to trafficking at distinct places within the secretory pathway. Several studies have shown that T. gondii
regulated secretory pathways to the rhoptries and micronemes involve the parasite endosomal system including early and late endosomes (14
). As depicted in a working model of propeptide based trafficking (), the M2AP propeptide appears to function relatively late in the trafficking pathway since M2AP propeptide mutants successfully navigate the ER, Golgi, trans-Golgi network (TGN) and early endosomes but are arrested in the late endosomes (19
) prior to reaching the micronemes (14
). In contrast, trafficking signals within the MIC5 propeptide appear to be used earlier in the secretory system since trafficking defective mutants are arrested or diverted prior to reaching the late endosomes based on immunolocalization within distinct punctate structures and the absence of endosomal proteolytic processing. Unlike MIC5, M2AP has a transmembrane binding partner, MIC2, which provides sufficient targeting information to navigate the early secretory organelles including the ER and Golgi (15
). Thereafter, M2AP appears to guide the complex through the early endosomes to the late endosomes where the M2AP propeptide is required for entry into the micronemes (19
). Since MIC5 does not have a known binding partner, it likely uses its propeptide earlier in the system, thus explaining the diversion of such mutants to an alternative site(s). MIC3 is distinct case since its trafficking to the micronemes is independent of its transmembrane partner MIC8 (12
) and MIC3 propeptide mutants are diverted to the parasitophorous vacuole, presumably via the dense granules ((21
) and ). These findings, together with the extensive interchangeability of microneme propeptides, suggests that propeptides confer a shared trafficking function albeit at several distinct sites within the secretory system, with the site of mutant diversion or retention reflecting the earliest propeptide-dependent step in the pathway.
Figure 9 A working model for propeptide based targeting of M2AP and MIC5 in Toxoplasma. A. Wild-type proM2AP and proMIC5 pass-through the Trans-Golgi Network (TGN), Early Endosomes (EE) and Late Endosomes (LE). proMICs interact with hypothetical sorting receptors (more ...)
Two principal models have been proposed to explain how propeptides facilitate targeting to the regulated secretory pathway of eukaryotic cells. The first model, “sorting by retention” argues that propeptide cleavage triggers a conformational shift in the mature protein, thus inducing aggregation under the mildly acidic conditions of the immature secretory granule (32
). Aggregation promotes retention in the maturing granule whereas proteins not intended for regulated secretion are diverted elsewhere. Much of the data in support of this model comes from the insulin field where it has been shown that processing of proinsulin dramatically alters its structure and that mature insulin is retained better in maturing granules than the proinsulin precursor. The second model, “sorting for entry” involves the use of sorting receptors that guide the selective cargo to the regulated secretory pathway. This model is based in part on the discovery of several putative metazoan sorting receptors in the regulated pathway including carboxypeptidase E (33
) and granins (34
). Also, a recent study showed that sorting of prohormone convertase (PC)1/3 to dense core secretory granules is mediated by a hydrophobic patch within an α-helix near the C-terminus of the protein that helps the protein to associate with the membrane (36
). Interestingly, a leucine residue (L745
) within the hydrophobic patch is critical for this interaction and mutation of this residue abolishes sorting of PC1/3 to dense core secretory vesicles.
Previous work showing that propeptide cleavage is not necessary for microneme targeting (19
) and our current finding that propeptide cleavage is not sufficient for M2AP targeting to the micronemes () is inconsistent with a sorting by retention model. On the other hand, the involvement of a sorting by entry/receptor mechanism in microneme trafficking is attractive for two reasons. First, propeptides reside in the lumen of secretory compartments and do not have direct access to the cytoplasm where much of the sorting machinery exists. Sorting receptors are designed to couple luminal cargo with the cytoplasmic sorting machinery that executes vesicular trafficking. Second, recognition by a sorting receptor could explain the discrete nature of the propeptide sorting element, which we show involves aliphatic residues at or near the N-terminus of the microneme propeptide. Such proximity to the N-terminus may impart solvent and receptor accessibility of these key residues. Finally, engagement of sorting receptors at distinct sites in the secretory pathway could help explain the different fates of microneme propeptide mutants ().
Two of the best-characterized cargo receptors in higher eukaryotes include the mannose-6-phosphate receptor (MPR) (37
) and sortilin (38
), which are involved in the trafficking of lysosomal enzymes from the TGN to the early endosome. The Iuminal domain of sortilin interacts with the cargo, while the cytoplasmic domain interacts with adapter proteins of the vesicular trafficking machinery. Crystal structure analysis of sortilin in association with a cargo protein, neurotensin, has shown that a C-terminal tripeptide motif, Tyr-Ile-Leu, of neurotensin is important for the interaction and that the leucine residue fits into the hydrophobic pocket of the receptor (40
). Vacuolar sorting receptors (VSR) (41
) in plants function in protein trafficking from TGN to vacuole. Studies on VSR-based trafficking of cargo proteins in plants have shown that vacuolar targeting signals reside within propeptides of the cargo and the aliphatic residues isoleucine and leucine are critical for the interaction with VSRs (43
The T. gondii
genome does not encode a MPR but contains a single sortilin gene, TgSortilin (TgME49_09160), and two putative VSRs, TgVSR1 (TgME49_024710) and TgVSR2 (TgME49_112860). Thus, T. gondii
possesses both animal-like and plantlike cargo receptors (45
). A preliminary analysis of these putative cargo receptors revealed that TgSortilin and TgVSR2 are expressed in T. gondii
tachyzoites based on mRNA and protein analysis, but TgVSR1 was not detected (data not shown). TgSortilin and TgVSR2 both reside in late endosomes where they colocalize with VP1. Repeated attempts to disrupt TgSortilin were not successful implying that it is an essential gene. Targeted disruption of TgVSR2 did not affect microneme protein trafficking indicating a distinct or redundant role for this putative cargo receptor. Additional non-canonical sorting receptors might also exist. Further studies will be necessary to determine the role of TgSortilin or other yet-to-be-identified cargo receptors in microneme propeptide sorting.
Since no studies have been performed in other apicomplexan parasites regarding the role of propeptides in microneme protein trafficking, it remains unclear whether these elements play a conserved role in parasites related to T. gondii
. Our finding that the EtMIC5 propeptide can function in microneme protein transport in T. gondii
suggests that functional conservation exists at least within the coccidian branch of the Apicomplexa. In addition to functioning in microneme protein trafficking, propeptides are emerging as important determinants of trafficking to apicomplexan rhoptries (46
) and export of Plasmodium proteins into the cytosol of infected erythrocytes (47
). Although it remains to be determined whether conserved mechanisms are involved, our findings suggest that the key residues necessary for propeptide-based trafficking can be highly discrete and therefore non-recognizable as conserved motifs. Accordingly, determining the molecular basis of propeptide trafficking to the rhoptries or erythrocyte cytosol might also require fine mapping of the residues involved.