Plasmodium Rab5b is secreted to the cytoplasmic face of the tubovesicular network in infected red blood cells together with N-acylated adenylate kinase 2

Rab5 GTPase regulates membrane trafficking between the plasma membrane and endosomes and harbours a conserved C-terminal isoprenyl modification that is necessary for membrane recruitment. Plasmodium falciparum encodes three Rab5 isotypes, and one of these, Rab5b (PfRab5b), lacks the C-terminal modification but possesses the N-terminal myristoylation motif. PfRab5b was reported to localize to the parasite periphery. However, the trafficking pathway regulated by PfRab5b is unknown.

N-myristoylated Plasmodium Rab5b plays a role that is distinct from that of conventional mammalian Rab5 isotypes. PfRab5b localizes to a compartment close to the ER, translocated to the lumen of the organelle, and co-localizes with PfAK2. PfRab5b and PfAK2 are then transported to the TVN, and PfRab5b localizes on the iRBC cytoplasmic face of TVN. These data demonstrate that PfRab5b is transported from the parasite cytosol to TVN together with N-myristoylated PfAK2 via an uncharacterized membrane-trafficking pathway.

A complementation analysis of Rab5 isotypes was performed in Plasmodium berghei. A constitutively active PfRab5b mutant was expressed under the regulation of a ligand-dependent destabilization domain (DD)-tag system in P. falciparum. The localization of PfRab5b was evaluated after removing the ligand followed by selective permeabilization of the membrane with different detergents. Furthermore, P. falciparum N-terminally myristoylated adenylate kinase 2 (PfAK2) was co-expressed with PfRab5b, and trafficking of PfAK2 to the parasitophorous vacuole membrane was examined by confocal microscopy.

PfRab5b complemented the function of PbRab5b, however, the conventional C-terminally isoprenylated Rab5, PbRab5a or PbRab5c, did not. The constitutively active PfRab5b mutant localized to the cytosol of the parasite and the tubovesicular network (TVN), a region that extends from the parasitophorous vacuole membrane (PVM) in infected red blood cells (iRBCs). By removing the DD-ligand, parasite cytosolic PfRab5b signal disappeared and a punctate structure adjacent to the endoplasmic reticulum (ER) and parasite periphery accumulated. The peripheral PfRab5b was sensitive to extracellular proteolysis after treatment with streptolysin O, which selectively permeabilizes the red blood cell plasma membrane, indicating that PfRab5b localized on the iRBC cytoplasmic face of the TVN. Transport of PfAK2 to the PVM was abrogated by overexpression of PfRab5b, and PfAK2 accumulated in the punctate structure together with PfRab5b.