Conformational co-dependence between Plasmodium berghei LCCL proteins promotes complex formation and stability.
Bottom Line: However, GFP-based fluorescence is dramatically reduced without PbLAP1 present, indicating that PbLAP1 and PbLAP3 interact.Moreover, absence of PbLAP1 markedly reduces the half-life of PbLAP3, consistent with a scenario of misfolding.These findings unveil a potential mechanism of conformational interdependence that facilitates assembly and stability of the functional LCCL protein complex.
Affiliation: Department of Pathogen Molecular Biology, London School of Hygiene & Tropical Medicine, London WC1E 7HT, UK.Show MeSH
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Mentions: Validated clones of the double mutant parasite line were assessed by western blot analysis for the expression of the PbLAP3::GFP fusion protein in gametocytes, in comparison with the parental lines. This showed normal expression of the protein in question (Fig. 2A), demonstrating that knockout of PbLAP1 had not adversely affected expression levels of PbLAP3 as is the case for the orthologous proteins in P. falciparum. This result indicated that gametocyte stage co-dependent expression does not occur in P. berghei. Surprisingly, however, GFP fluorescence levels in gametocytes of the double mutant parasite line were dramatically reduced compared to the parental PbLAP3/GFP gametocytes (Fig. 2B), indicating that the absence of PbLAP1 has affected the ability of the PbLAP3::GFP chimera to generate fluorescence. To further investigate the expression and subcellular distribution of PbLAP3::GFP in gametocytes of the double mutant parasite line we performed immunofluorescence using commercially available anti-GFP antibody. Gametocytes were purified as described , fixed for 20 min in 4% paraformaldehyde, washed twice with PBS, blocked and permeabilized for 1 h in PBS supplemented with 0.1% Triton X-100 and 1% BSA. Then the gametocytes were labelled with anti-GFP antibodies conjugated to FITC (ab65180, Abcam, diluted 1 in 1000) for 1 h at room temperature. After a further two PBS washes the gametocyte suspension was examined by confocal microscopy. FITC signal in the double mutant gametocytes was clearly detectable compared to GFP-negative wildtype parasite controls (Fig. 2C), consistent with expression of the PbLAP3::GFP fusion protein in the double mutant. Moreover, the subcellular distribution of FITC signal (Fig. 2C) was comparable to that of GFP signal in the parental PbLAP3/GFP parasite line (Fig. 2B), demonstrating that the subcellular distribution of PbLAP3::GFP had not drastically changed in the absence of PbLAP1. It is therefore unlikely that a change in the protein's localization to a subcellular compartment less favourable for generating GFP fluorescence is responsible for the loss of GFP fluorescence in the double mutant. We postulate that a more likely explanation for the loss of GFP fluorescence is based on a PbLAP1–PbLAP3 molecular interaction: the imposed loss of this interaction in the double mutant parasite line causes a conformational change in PbLAP3 that is adverse to functionality of its GFP tag. Indeed, the folding of amino-terminal fusions with GFP has long been known to influence fluorescence levels of chimeric GFP .
Affiliation: Department of Pathogen Molecular Biology, London School of Hygiene & Tropical Medicine, London WC1E 7HT, UK.