Additional remarks phenotype | Mutant/mutation
The mutant expresses a mCherry tagged version of PBANKA_1349200 (PV4) and expresses GFP under the constitutive hsp70 promoter. GFP is fused to the signal peptide of PBANKA_081890 and consequently GFP is localized to the parasitophorous vacuole (see RMgm-1333).
Protein (function)
The protein was selected in a screen for putative PV (parasitophorous vacuole) proteins.
Proteins are targeted to the PV by means of default protein secretion, which is initiated by the recognition and cleavage of an amino-terminal signal peptide (SP). To identify novel PV candidates, we searched the Plasmodium genome database (PlasmoDB) for SP-containing proteins and sequentially removed proteins containing predicted transmembrane domains, export motifs, apicoplast targeting peptides, endoplasmic reticulum (ER) retention signals and GPI anchors, which could potentially redirect the protein to other locations. Twelfe proteins were selected. Five (PV1-5) proteins were shown to have a PV location in blood- and liver-stages.
Phenotype
Expression in blood stages. Evidence for parasitophorous vacuole location.
Expression in liver stages. Evidence for parasitophorous vacuole location.
PV4 is not essential for blood stage development (see RMgm-4536).
Additional information
Five (PV1-5; PBANKA_0919100; PBANKA_1441700; PBANKA_1350500; PBANKA_1349200; PBANKA_0826700) proteins were shown to have a PV location in blood- and liver-stages.
All fve proteins were found to be associated with the parasite periphery and with PV-derived tubules and vesicular structures throughout the entire asexual cycle. Strikingly, a substantial fraction of PV4 was found in the erythrocyte cytoplasm during ring and early trophozoite stages. This exported protein fraction progressively disappeared during parasite growth and was not detected in mature trophozoites or schizonts. During the schizont stage, all five proteins localised around the formed merozoites and co-localised with the hemozoin crystals of the digestive vacuoles. We next investigated whether the PV proteins are indeed associated with the vacuolar lumen rather than the parasite surface. To that end, we imaged ruptured schizonts, which have already disintegrated the iRBC membrane and the PV, thus leading to the loss of all soluble PV contents. Upon merozoite egress all fve proteins localised almost exclusively to the digestive vacuoles. Upon inspection of individual emerging merozoites, PV2-5 were hardly detected, whereas PV1 was found to be associated with a distinct intraparasitic structure, possibly refecting localisation to parasite dense granules, as previously demonstrated for other PV proteins23. In no instance were the PV proteins detected on the surface of emerging merozoites further supporting the notion that they are indeed mostly luminal.
Inoculation of in vitro cultivated hepatoma cells with the transgenic sporozoites revealed that all fve proteins are highly expressed in the liver stage PV. PV5 was not expressed during early pre-erythrocytic growth, but was only present in the PV of more mature parasite stages.
Other mutants |