Functional evaluation of Plasmodium export signals in Plasmodium berghei suggests multiple modes of protein export

The erythrocytic stage development of malaria parasites occurs within the parasitophorous vacuole inside the infected-erythrocytes, and requires transport of several parasite-encoded proteins across the parasitophorous vacuole to several locations, including the cytosol and membrane of the infected cell. These proteins are called exported proteins; and a large number of such proteins have been predicted for Plasmodium falciparum based on the presence of an N-terminal motif known as the Plasmodium export element (PEXEL) or vacuolar transport signal (VTS), which has been shown to mediate export. The majority of exported proteins contain one or more transmembrane domains at the C-terminus and one of three types of N-terminus domain architectures. (1) The majority, including the knob-associated histidine rich protein (KAHRP), contain a signal/hydrophobic sequence preceding the PEXEL/VTS motif. (2) Other exported proteins, including the P. berghei variant antigen family bir and the P. falciparum skeleton binding protein-1, do not appear to contain a PEXEL/VTS motif. (3) The P. falciparum erythrocyte membrane protein-1 (PfEMP1) family lacks a signal/hydrophobic sequence before the motif. These different domain architectures suggest the presence of multiple export pathways in malaria parasites. To determine if export pathways are conserved in plasmodia and to develop an experimental system for studying these processes, we investigated export of GFP fused with N- and C-terminus putative export domains in the rodent malaria parasite P. berghei. Export was dependent on specific N- and C-terminal domains. Constructs with a KAHRP-like or bir N-terminus, but not the PfEMP1 N-terminus, exported GFP into the erythrocyte. The C-terminus of a P. falciparum variant antigen rifin prevented GFP export by the KAHRP-like N-terminus. In contrast, GFP chimeras containing KAHRP-like N-termini and the PfEMP1 C-terminus were exported to the surface of erythrocytes. Taken together, these results suggest that proteins with KAHRP-like architecture follow a common export pathway, but that PfEMP1s utilize an alternative pathway. Functional validation of common putative export domains of malaria parasites in P. berghei provides an alternative and simpler system to investigate export mechanisms.