CONICET | Buscador de Institutos y Recursos Humanos

At variance with mammals, trypanosomatids have a high percentage of proteins anchored by GPI in their plasma membrane. T. cruzi GPI-anchored virulence factors such as trans-sialidase (TS) and mucins are transported to the membrane by a non-conventional transport involving the contractile vacuole (CV) prior to reach the flagellar pocket and then the plasma membrane. Both proteins are only detectable in CV during the differentiation to trypomastigote. Once on the membrane, they are ordered in mutually exclusive domains, finding mucins in ?lipid rafts-like? areas, whereas TS are not. Also, it is known that each protein has different GPI-lipidic composition. There is only scarce information concerning the signals that determine this arrangement on the plasma membrane or about the role of CV on this novel transport pathway. Here, the involvement of the lipid nature of the GPI anchor in this pathway and in the final membrane assortment were assessed. Recombinant MucII genes were constructed where their native GPI signaling or the TS-GPI signal were inserted and cloned in the inducible expression vector pTcIndex. We found that protein intracellular trafficking was not modified. Using this regulated system with the recombinant MucII genes, we observed that at low levels of expression, both proteins were localized on the membrane with no accumulation in the CV. However, at higher expression levels constant protein accumulation only is observed in the CV from 24h to 96h post-induction and not in the other organelles of the secretory pathway. This suggests that CV could function as a ?bottle-neck?. Then, the membrane arrangement of these proteins was analyzed. We have observed that the TS GPI-signal clearly change the surface protein domain pattern both in size and distribution as compared to the native GPI-signal, thus highlighting the relevance of the GPI anchor in the final destination of surface proteins.