The malaria parasite Plasmodium falciparum increases permeability of affected erythrocytes to let in a number of key nutrients important to its growth. The parasite affects the plasmodial surface anion channel (PSAC) via the P. falciparum clag3 gene. Zainabadi (2016) has sought to discover the way in which the clag3 gene affects PSAC function.1
Using cultured P. falciparum, Zainabadi isolated membrane fractions for immunoprecipitation. The parasites used for immunoprecipitation experiments were a recombinant HB3 with a FLAG-tagged CLAG3. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the resulting immunoprecipitate showed CLAG3 immunoprecipitates as a single band of 160 kDa at concentrations greater than or equal to 0.1%. However, at concentrations below 0.1%, Zainabadi found two additional faint bands (150 kDa and 100 kDa), which are consistent with the sizes of two other members of the RhopH complex, RHOPH2 and RHOPH3, which CLAG3 (also known as RHOPH1) has previously been shown to interact with.
To determine whether CLAG3 and its associated bands form a large complex, Zainabadi performed a blue native PAGE analysis on the immunoprecipitate elutes. He found that CLAG3 participates in a ~720 kDa complex at Fos-Choline-12 (FC12) concentrations less than or equal to 0.05% and a ~480 kDa complex at FC12 concentrations greater than or equal to 0.1%. Zainabadi further identified the proteins using an LTQ Orbitrap Velos mass spectrometer (Thermo Scientific) on the bands from the FLAG-tagged parasite and the corresponding gel, identifying the proteins in the 720 kDa complex as CLAG3, RHOPH2 and RHOPH3 and the protein in the 480 kDa complex as CLAG3.
Finally, Zainabadi used the potent PSAC inhibitor MBX2366 to block PSAC activity during extracellular biotin-labeling experiments and then performed reciprocal immunoprecipitation. He biotin-labeled cell membranes before immunoprecipitating with FLAG antibody and then separating with native PAGE, before probing by Western blot for biotin. Only the CLAG3 obtained with 0.5% FC12 (corresponding to the 480 kDa complex) labeled efficiently with biotin.
The data presented by Zainabadi suggests that CLAG3 participates in multiple high–molecular weight complexes, one of which localizes to the erythrocyte surface. Based on this work, the authors are left with more questions about the role of RHOPH2 and RHOPH3. They posit future investigations will help determine their role in PSAC.
1. Zainabadi, K. (2016) “Malaria parasite CLAG3, a protein linked to nutrient channels, participates in high molecular weight membrane-associated complexes in the infected erythrocyte,” PLoS One, 11(6).