Mutant/mutation
The mutant expresses the parasitophorous vacuole membrane (PVM) protein EXP1/HEP17 fused to BirA*. The fusion gene is integrated into the silen c/d-ssurRNA gene unit. In addition the mutant expresses mCherry under the HSP70 promoter.

The biotin ligase BirA* was fusedto EXP1, which is a known protein of the PVM in blood and liver stage parasites. With this aim, we generated the plasmid pL0017-CEXP1BirA*-V5, encoding an EXP1-BirA* fusion protein with a C-terminal V5 tag under control of the constitutive P. berghei eef1a promoter. This vector integrates by single-crossover homologous recombination into either the c-ssu-rRNA locus or d-ssurRNA locus of P. berghei and conveys resistance to pyrimethamine.

Protein (function)

Phenotype
To investigate whether this construct is expressed in blood stages and leads to the biotinylation of PVM proteins, we isolated the blood of mice infected with transgenic EXP1-BirA*-V5 parasites by cardiac puncture and cultivated it in an in vitro schizont culture for 18 h in the presence of 150  M biotin. Biotinylated proteins were visualized by immunofluorescence analysis (IFA) using fluorescently labeled streptavidin. As expected, we observed a strong streptavidin signal, which was peripherally located around parasites and co-localized with theV5-taggedEXP1-BirA*fusion protein. We further confirmed this finding by performing Western blot analyses of EXP1BirA*-V5 parasites grown in the presence or absence of biotin and parental WT parasites cultivated in the presence of biotin. We observed a substantial increase in the levels of biotinylated proteins in lysates of EXP1-BirA*-V5 parasites grown in the presence of biotin in comparison to the WT and no-biotin controls, where only very few biotinylated proteins were visible. In conclusion, these data demonstrate that EXP1-BirA*-V5 is active and biotinylates a range of target proteins at the parasite periphery.

Additional information
This parasite was used to biotinylate proteins of the PVM. These we further processed by affinity purification, liquid chromatography-tandem mass spectrometry (LC-MS/MS), and label-free quantitation, leading to a list of 61 known and candidate PVM proteins. Seven proteins were analyzed further during blood and liver stage development. This resulted in the identification of three novel PVM proteins, which were the serine/threonine protein phosphatase UIS2 (PlasmoDB accession no. PBANKA_1328000) and two conserved Plasmodium proteins with unknown functions (PBANKA_0519300 and PBANKA_0509000).

Other mutants